No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Structures of Asymmetric ClpX Hexamers Reveal Nucleotide-Dependent Motions in a AAA+ Protein-Unfolding Machine
Abstract
ClpX is a AAA+ machine that uses the energy of ATP binding and hydrolysis to unfold native proteins and translocate unfolded polypeptides into the ClpP peptidase. The crystal structures presented here reveal striking asymmetry in ring hexamers of nucleotide-free and nucleotide-bound ClpX. Asymmetry arises from large changes in rotation between the large and small AAA+ domains of individual subunits. These differences prevent nucleotide binding to two subunits, generate a staggered arrangement of ClpX subunits and pore loops around the hexameric ring, and provide a mechanism for coupling conformational changes caused by ATP binding or hydrolysis in one subunit to flexing motions of the entire ring. Our structures explain numerous solution studies of ClpX function, predict mechanisms for pore elasticity during translocation of irregular polypeptides, and suggest how repetitive conformational changes might be coupled to mechanical work during the ATPase cycle of ClpX and related molecular machines.
... Cryo-electron microscopy (cryo-EM) analyses of various AAA proteins in the presence of substrates have revealed that AAA proteins engage substrates ubiquitously by a threading mechanism, in which subunits of AAA proteins assemble in a spiral staircase formation, with each subunit captured interacting with an amino acid residue of an unfolding protein polypeptide or with a DNA nucleotide and progressing in a hand-over-hand fashion. However, the debate over whether AAA proteins carry out ATP hydrolysis via a sequential [3][4][5][6][7][8][9][10][11][12][13][14][15] or a probabilistic mechanism remains [16][17][18][19][20][21] . It is important to note that subunits of various nucleotide states (apo, ADP, and ATP) coexist in the same complex. ...
... Therefore, we decided to use cryo-EM to capture the type of nucleotide bound and the associated conformation of each protomer of mBcs1L while the protein is undergoing active ATP hydrolysis. Similar experiments have been performed with various AAA proteins, and without exception all tested AAA proteins, when substrates were present, were captured with co-existing different nucleotide states (Apo/ADP/ATP) in protomers that arrange in nonplanar formations [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] . ...
... Substrate translocation is thought to go through sequential ATP hydrolysis, as individual subunit transitions from ATP-bound form to extended ADP-bound form, thus moving downwards while pulling the substrate along stepwise. However, this sequential mechanism faces difficulties with the observations from some AAA proteins like RavA and ClpX, which feature two separate seam subunits in cryo-EM structures, leading to the proposed probabilistic mechanism for ATP hydrolysis [16][17][18][19][20] . ...
The human AAA-ATPase Bcs1L translocates the fully assembled Rieske iron-sulfur protein (ISP) precursor across the mitochondrial inner membrane, enabling respiratory Complex III assembly. Exactly how the folded substrate is bound to and released from Bcs1L has been unclear, and there has been ongoing debate as to whether subunits of Bcs1L act in sequence or in unison hydrolyzing ATP when moving the protein cargo. Here, we captured Bcs1L conformations by cryo-EM during active ATP hydrolysis in the presence or absence of ISP substrate. In contrast to the threading mechanism widely employed by AAA proteins in substrate translocation, subunits of Bcs1L alternate uniformly between ATP and ADP conformations without detectable intermediates that have different, co-existing nucleotide states, indicating that the subunits act in concert. We further show that the ISP can be trapped by Bcs1 when its subunits are all in the ADP-bound state, which we propose to be released in the apo form.
... Structures of the ClpP-ATPase complex, however, had been difficult to determine by X-ray diffraction J o u r n a l P r e -p r o o f methods owing to the flexibility of the components' interactions but which underlies their coupled unfoldase and protease functions (84,85). ClpX is inherently more flexible than ClpP, such that its crystal structure was solved only after genetic manipulation to assemble a covalently-linked pseudohexamer lacking the N-terminal zinc-binding domain (ZBD) (86). However, this mutant ClpX structure does not have the same spiral topology as related disaggregases/ATPases and might not be physiologically relevant, limiting inference of the enzyme's precise mechanism (87,88). ...
... Misalignment of asymmetric NmClpX and NmClpP rings causes the substrate translocation channel to twist and constrict at the interface, as is also observed in the structures of LmClpXP1P2 and PAN proteasome (99,101). Unlike the crystal structure of EcClpX pseudohexamer that shows a twofold symmetric dimer of trimers, the cryoEM structure NmClpX shows a shallow, right-handed spiral with pseudo-6-fold symmetry (86). This arrangement causes conserved pore-1 loops lining the substrate channel to form a spiral around the bound peptide ( Figure 2B). ...
... This is easily visualized by looking only at the pore-1 loops of each NmClpX subunit, which harbors the conserved Y153 residue that interacts directly with substrate ( Figure 2B, right). In (86). For instance, in NmClpX Conformation B, the Sensor II arginine (R369, subunit X1) and the Sensor-I arginine finger (R306) from an adjacent clockwise subunit (X2) interact with the β-and γphosphates of ATP ( Figure 3A, left). ...
ClpP is a highly conserved serine protease that is a critical enzyme in maintaining protein homeostasis and is an important drug target in pathogenic bacteria and various cancers. In its functional form, ClpP is a chambered protease composed of two stacked heptameric rings that allow protein degradation to occur internally. ATPase chaperones such as ClpX and ClpA are hexameric ATPases that form larger complexes with ClpP and are responsible for the selection and unfolding of protein substrates prior to their degradation by ClpP. Although individual structures of ClpP and ATPase chaperones have offered mechanistic insights into their function and regulation, their structures together as a complex have only been recently determined to high resolution. Here, we discuss the cryoelectron microscopy structures of ClpP-ATPase complexes and describe findings previously inaccessible from individual Clp structures, including how a hexameric ATPase and a tetradecameric ClpP protease work together in a functional complex. We then discuss the consensus mechanism for substrate unfolding and translocation derived from these structures, consider alternative mechanisms, and present their strengths and limitations. Finally, new insights into the allosteric control of ClpP gained from studies using small molecules and gain or loss-of-function mutations are explored. Overall, this review aims to underscore the multi-layered regulation of ClpP that may present novel ideas for structure-based drug design.
... Each ClpX protomer has two domains, the large and the small domains, that assemble, through the binding of the small domain of one protomer to the large domain of the next one, in an asymmetric hexamer that can bind a maximum of four molecules of ATP and/or ADP (Glynn et al. 2009). Due to its asymmetry and domain-domain orientation the ClpX hexamer can be divided into subunits of two main classes, the "loadable" or L subunits and the "unloadable" or U subunits (Glynn et al. 2009;Baker and Sauer 2012). ...
... Each ClpX protomer has two domains, the large and the small domains, that assemble, through the binding of the small domain of one protomer to the large domain of the next one, in an asymmetric hexamer that can bind a maximum of four molecules of ATP and/or ADP (Glynn et al. 2009). Due to its asymmetry and domain-domain orientation the ClpX hexamer can be divided into subunits of two main classes, the "loadable" or L subunits and the "unloadable" or U subunits (Glynn et al. 2009;Baker and Sauer 2012). In the L subunit, the large and small AAA+ domains of each protomer connect in a nucleotide-binding conformation, corresponding to four subunits of the hexamer. ...
... In the L subunit, the large and small AAA+ domains of each protomer connect in a nucleotide-binding conformation, corresponding to four subunits of the hexamer. Interestingly, on the two U subunits can occur a rotation of 80° between the bound domains of the protomers (large and small AAA+ of each protomer), originating movements as large as 30 Å that destroy the nucleotide-binding site (Glynn et al. 2009;Baker and Sauer 2012). Consequently, the two U subunits, create two flexible sides of the hexamer that allow the adaptation of the pore to different substrate sizes, extending the pore to accommodate larger substrates and then refold and shortening it to contact with smaller substrates (Glynn et al. 2009;Baker and Sauer 2012). ...
We have identified a membrane protein complex of Bacillus subtilis involving an unknown protein, YteJ, and SppA, a membrane protein first described as a signal peptide peptidase and later shown to be also involved in the resistance to antibacterial peptides of the lantibiotic family. Using deletion mutant strains, we showed that both proteins are involved in this resistance. In the ΔsppA strain, the ectopic overexpression of SppA not only restored the resistance, it also induced the formation of elongated cells, a phenotype suppressed by the simultaneous overexpression of YteJ. Furthermore, the expression of truncated versions of YteJ pinpointed the inhibitory role of a specific domain of YteJ. Finally, in vitro biochemical studies showed that SppA protease activity was strongly reduced by the presence of YteJ, supporting the hypothesis of an inhibition by YteJ. Our in vivo and in vitro studies showed that YteJ, via one of its domain, acts as a negative regulator of the protease activity of SppA in this complex. In conclusion, we have shown that SppA/YteJ complex is involved in lantibiotic resistance through the protease activity of SppA, which is regulated by YteJ.
... An example is shown in Figure 2.8 AAA+ ATPases form oligomeric rings, usually hexameric, and the nucleotide binding site sits at the interface between the large and small AAA+ subdomains, with an arginine finger being provided in trans from a neighbouring monomer (Sysoeva, 2017;Wendler et al., 2012). The angle between the large and small subdomains changes during ATP hydrolysis, and this conformational change is often linked to mechanical work (Glynn et al., 2009;Miller and Enemark, 2016). Some AAA+ proteins also have an internal duplication of the AAA+ domain, and form hexamers with two stacked ATPase rings (Puchades et al., 2020;Sysoeva, 2017). ...
... Release of ADP resets the cycle, and the monomer moves back to the top of the spiral ring. D) Cartoon of the 'hand-over-hand' mechanism, with each hand representing a pore loop and coloured as in B). manner, with non-adjacent nucleotide binding sites able to hydrolyse ATP rather than in a strictly sequential rotary manner (Fei et al., 2020;Glynn et al., 2009;Stinson et al., 2013). ...
... There are currently unresolved questions about whether the general around-thering mechanism proposed for clade 3 ATPases is generally applicable to clade 5 ATPases as well (Gates and Martin, 2019). A mechanism for ClpX has been proposed based on biochemical and X-ray crystallography studies in which ATP hydrolysis does not occur sequentially around the hexameric ring but rather occurs stochastically (Glynn et al., 2009;Stinson et al., 2013). One of the recent cryo-EM studies on the ClpXP complex appears to support this mechanism (Fei et al., 2020), but another instead proposes that the clade 3like rotary mechanism applies to ClpX (Ripstein et al., 2020). ...
The field of electron microscopy has undergone rapid transformation in the past few years. It has become one of the most powerful techniques for investigating macromolecular complexes, providing unprecedented structural insights into the fundamental processes of cellular life. In this thesis, I use cryo electron microscopy and negative stain electron microscopy to investigate two biological systems. The first is an enterobacterial protein triad formed of the E. coli proteins LdcI, involved in the acid stress response, and RavA and ViaA, which sensitise E. coli to aminoglycosides. I show that the AAA+ ATPase RavA exists in two distinct conformational states, shedding insight into its ATPase mechanism and revealing unexpected mechanistic similarities to the well-characterised unfoldase ClpX. I also explore the effects of fluorescent protein tags on the structure and function of LdcI, in order to facilitate super-resolution fluorescence microscopy. The second complex is the mitochondrial Complex I assembly complex, which is composed of ACAD9, ECSIT and NDUFAF1 and is involved in the maturation of respiratory Complex I in mitochondria. I investigate the ACAD9-ECSIT-CTD subcomplex using cryo-EM, providing insights into the structure of ACAD9, the location of the ECSIT-CTD binding site and the release of the FAD cofactor of ACAD9 upon ECSIT-CTD binding. Finally, in conjunction with biochemical and biophysical analysis, I place the structural information presented in this thesis in a biological context and lay a platform for future studies of both protein complexes.
... Likewise, the AAA channel is also decorated with five tyrosine residues in the resting state. These tyrosine residues may facilitate substrate engagement and translocation via cation-π or π-π interactions (Glynn et al. 2009). ...
... The pore-1 loops of the RPT subunits feature the conserved '[Tyr/Phe]-[Val/Leu/Ile]-Gly' sequence pattern and arrange into a super-helical staircase. In many ATP-dependent AAA unfoldases, such as ClpX, HslU, LonA, FtsH and PAN (Glynn et al. 2009;Iosefson et al. 2015), their homologous pore-1 loops drive substrate translocation. The pore-2 loops constitute a second super-helical staircase running in the opposite of the pore-1 loop staircase (Fig. 1.22), which may function a 'sensor' for the state of the substrate translocation as these pore-2 loops are directly extended from the Walker B motif and can translate the presence of substrate in the Side views of the pore-1 loops from six RPT subunits decorating the channel, which align along the channel in a spiral staircase formed from RPT1-RPT5, with a backward recession in RPT6 pore-1 loop that is slightly away from the major channel pathway. ...
... The position of RPT6 at the seam of the AAA ring endows it with certain energetic advantage in allosterically triggering the AAA channel opening with little perturbation to the pore loop staircase structure, which is poised to accept an unfolded segment of the substrate without changing the overall ATPase architecture. This mode of coordinated ATP hydrolysis is reminiscent of the nucleotide states in the crystal structure of the ClpX protease, which drive different structural changes in the ATPase ring likely because of the absence of a substrate (Glynn et al. 2009). ...
The 26S proteasome is the most complex ATP-dependent protease machinery, of ~2.5 MDa mass, ubiquitously found in all eukaryotes. It selectively degrades ubiquitin-conjugated proteins and plays fundamentally indispensable roles in regulating almost all major aspects of cellular activities. To serve as the sole terminal “processor” for myriad ubiquitylation pathways, the proteasome evolved exceptional adaptability in dynamically organizing a large network of proteins, including ubiquitin receptors, shuttle factors, deubiquitinases, AAA-ATPase unfoldases, and ubiquitin ligases, to enable substrate selectivity and processing efficiency and to achieve regulation precision of a vast diversity of substrates. The inner working of the 26S proteasome is among the most sophisticated, enigmatic mechanisms of enzyme machinery in eukaryotic cells. Recent breakthroughs in three-dimensional atomic-level visualization of the 26S proteasome dynamics during polyubiquitylated substrate degradation elucidated an extensively detailed picture of its functional mechanisms, owing to progressive methodological advances associated with cryogenic electron microscopy (cryo-EM). Multiple sites of ubiquitin binding in the proteasome revealed a canonical mode of ubiquitin-dependent substrate engagement. The proteasome conformation in the act of substrate deubiquitylation provided insights into how the deubiquitylating activity of RPN11 is enhanced in the holoenzyme and is coupled to substrate translocation. Intriguingly, three principal modes of coordinated ATP hydrolysis in the heterohexameric AAA-ATPase motor were discovered to regulate intermediate functional steps of the proteasome, including ubiquitin-substrate engagement, deubiquitylation, initiation of substrate translocation and processive substrate degradation. The atomic dissection of the innermost working of the 26S proteasome opens up a new era in our understanding of the ubiquitin-proteasome system and has far-reaching implications in health and disease.
... The release of a nucleotide in an AAA-ATPase protein destabilizes the nucleotide-bound conformation, generating large structural arrangements between the small and large AAA subdomains (Glynn et al., 2009). Indeed, the Rpt2 and Rpt6 densities in the S D1 map, the Rpt2 density in the S D2 map, and the Rpt6 density in the S D3 map are of lower quality at slightly lower local resolutions than other Rpt subunits in the same map, indicating that their conformations are destabilized due to nucleotide release ( Figure S6E-G). ...
... The central channel formed by the hexameric ring of the AAA domain of Rpt subunits is narrowed by inward-facing pore loops that were thought to drive the translocation of substrates (Beckwith et al., 2013;Glynn et al., 2009;Zhang et al., 2009a). In the S A state, the pore-1 and pore-2 loops from neighboring subunits were found to pair with each other to constitute the constrictions of the AAA channel . ...
... The constrictions narrowed by the pore loops are substantially reconfigured in the S D2 and S D3 so that the AAA channel is overall widened relative to that in the S D1 state. However, the narrowest constrictions are still too narrow to allow translocation of peptides with large aromatic side chains such as tryptophan in S D1 , S D2 and S D3 (Figure S7), suggesting that conformational flexibility in the pore loops might be necessary to accommodate substrate translocation (Glynn et al., 2009). Indeed, although the pore loops of Rpt6 do not contribute to the constriction of the AAA channel in S D1 , they move in to directly shape the construction of the AAA channel in S D2 . ...
The proteasome is a sophisticated ATP-dependent molecular machine responsible for protein degradation in all eukaryotic cells. It remains elusive how conformational changes of the AAA-ATPase unfoldase in the regulatory particle (RP) control the gating of substrate-translocation channel to the proteolytic chamber of the core particle (CP). Here we report three alternative states of the ATP-γS-bound human proteasome, in which the CP gate is asymmetrically open, visualized by cryo-EM at near-atomic resolutions. Only four nucleotides are stably bound to the AAA-ATPase ring in the open-gate states. Concerted nucleotide exchange gives rise to a back-and-forth wobbling motion of the AAA-ATPase channel, coincident with remarkable transitions of their pore loops between the spiral staircase and saddle-shaped circle topologies. Gate opening in the CP is thus controlled with nucleotide-driven remodeling of the AAA-ATPase unfoldase. These findings demonstrate an elegant mechanism of allosteric coordination among sub-machines within the holoenzyme that is crucial for substrate translocation.
... While some other members of the Hsp100 family have been crystallised in their oligomeric form (Bochtler et al., 2000;Glynn et al., 2009;Wang et al., 2011), the atomic structure of ClpB is known only for the monomer (T. thermophilus). ...
... As mentioned above, we imposed 6-fold symmetry as a first approximation, to simplify the alignment and reconstruction problem. Nevertheless, crystal structures of the AAA+ protein ClpX show that the homo-hexameric assembly can be markedly asymmetric (Glynn et al., 2009;Kon et al., 2012;Stinson et al., 2013). We therefore reanalysed our negative stain EM data without imposing symmetry, in order to study the conformational variability within the hexamer. ...
... Using a gallery of available AAA+ crystal structures we modelled open, closed and intermediate conformations of ClpB AAA-1 and AAA-2. We also created AAA+ dimers of adjacent ClpB subunits based on ClpX pseudo-hexameric crystal structures (Glynn et al., 2009; crosslinked dimers (2), trimers (3), tetramers (4), pentamers (5) and hexamers (6) Stinson et al., 2013; Figure 6-figure supplement 2) that were fitted as rigid bodies into the asymmetric reconstructions. Crystallographic dimers are likely to provide more realistic models of subunit interfaces than can be deduced by fitting individual subunits into low-resolution maps. ...
... In contrast to our present cryo-EM structures, crystal structures of E. coli ClpX DN pseudohexamers (Glynn et al., 2009;Stinson et al., 2013) do not form spirals, bind only four nucleotides, and have conformations incompatible with ClpP and substrate binding. These observations highlight the fact that low-energy conformations observed by any structural method may or may not be relevant to biological function. ...
... It is not clear how many structural changes are involved in the complete reaction cycle for ClpXP or related AAA+ ATPases. For example, crystal structures of ClpX reveal rotations between the large and small AAA+ domains so large that nucleotide cannot bind some subunits (Glynn et al., 2009;Stinson et al., 2013). Although subunits of this type are not present in current cryo-EM structures, they might represent transient functional conformations, as crosslinks engineered to trap these hexamer conformations form in solution and prevent ClpXP degradation but not ATP hydrolysis (Stinson et al., 2013;Stinson et al., 2015). ...
... 443,717 'good' particles were selected for 3D map reconstruction. To generate an initial model for 3D refinement, the crystal structures of a ClpX DN hexamer (PDB 3HWS; Glynn et al., 2009) and a ClpP tetradecamer (PDB code 3MT6; Li et al., 2010) were merged in PyMOL and lowpass filtered to 40 Å . 3D refinement with C 2 symmetry yielded a map with a resolution of~4 Å , but the quality of this map was poor and interpretation of secondary structure elements was impossible. ...
ClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies of the E. coli enzyme that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel, as observed for other AAA+ proteases and protein-remodeling machines. Strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been inferred from these structural features for other AAA+ unfoldases, but biochemical and single-molecule biophysical studies indicate that ClpXP operates by a probabilistic mechanism in which five to eight residues are translocated for each ATP hydrolyzed. We propose structure-based models that could account for the functional results.
... In contrast to our present cryo-EM structures, crystal structures of E. coli ClpX DN pseudohexamers (Glynn et al., 2009;Stinson et al., 2013) do not form spirals, bind only four nucleotides, and have conformations incompatible with ClpP and substrate binding. These observations highlight the fact that low-energy conformations observed by any structural method may or may not be relevant to biological function. ...
... It is not clear how many structural changes are involved in the complete reaction cycle for ClpXP or related AAA+ ATPases. For example, crystal structures of ClpX reveal rotations between the large and small AAA+ domains so large that nucleotide cannot bind some subunits (Glynn et al., 2009;Stinson et al., 2013). Although subunits of this type are not present in current cryo-EM structures, they might represent transient functional conformations, as crosslinks engineered to trap these hexamer conformations form in solution and prevent ClpXP degradation but not ATP hydrolysis (Stinson et al., 2013;Stinson et al., 2015). ...
... 443,717 'good' particles were selected for 3D map reconstruction. To generate an initial model for 3D refinement, the crystal structures of a ClpX DN hexamer (PDB 3HWS; Glynn et al., 2009) and a ClpP tetradecamer (PDB code 3MT6; Li et al., 2010) were merged in PyMOL and lowpass filtered to 40 Å . 3D refinement with C 2 symmetry yielded a map with a resolution of~4 Å , but the quality of this map was poor and interpretation of secondary structure elements was impossible. ...
ClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies of the E. coli enzyme that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel, as observed for other AAA+ proteases and protein-remodeling machines. Strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been inferred from these structural features for other AAA+ unfoldases, but biochemical and single-molecule biophysical studies indicate that ClpXP operates by a probabilistic mechanism in which five to eight residues are translocated for each ATP hydrolyzed. We propose structure-based models that could account for the functional results.
... This second conformation may represent an intermediate state between the "seam up" and "seam down"-oriented RavA spirals inside the LdcI-RavA complex. Moreover, it displays remarkable structural similarity to the approximately two-fold symmetric "dimer of trimers" arrangement of subunits in crystal structures of the extensively studied AAA+ unfoldase ClpX 18,19 and the protein-remodeling AAA+ ATPase PCH2 20 . Consequently, the mechanism of the RavA ATPase cycle may be unexpectedly similar to the meticulously dissected ATP hydrolysis cycle of ClpX 19,[21][22][23] , although the respective families of these two proteins belong to different clades of AAA+ ATPases [24][25][26] . ...
... Viewed from this perspective, the planar double-seam conformation of the RavA hexamer is strikingly reminiscent of the approximately two-fold symmetric "dimer of trimers" arrangement of subunits in hexamers of the AAA+ unfoldase ClpX 18,19 , which belongs to clade 5 AAA+ ATPases and thus lacks the pre-Sensor 2 insertion 25,26 . In crystal structures of ClpX, hexamers are arranged with an approximate two-fold symmetry, and contain four ClpX subunits in a nucleotide loadable (L) and two in unloadable (U) conformation on opposite sides of the hexamer. ...
... In crystal structures of ClpX, hexamers are arranged with an approximate two-fold symmetry, and contain four ClpX subunits in a nucleotide loadable (L) and two in unloadable (U) conformation on opposite sides of the hexamer. In the unloadable ClpX subunits, the small and large AAA+ domains are positioned in an "open" conformation which destroys the nucleotide-binding site 18,19 . The resulting 4L-2U arrangement of ClpX contains a characteristic seam which runs along the hexamer centre. ...
The hexameric MoxR AAA+ ATPase RavA and the decameric lysine decarboxylase LdcI form a 3.3 MDa cage, proposed to assist assembly of specific respiratory complexes in E. coli. Here, we show that inside the LdcI-RavA cage, RavA hexamers adopt an asymmetric spiral conformation in which the nucleotide-free seam is constrained to two opposite orientations. Cryo-EM reconstructions of free RavA reveal two co-existing structural states: an asymmetric spiral, and a flat C2-symmetric closed ring characterised by two nucleotide-free seams. The closed ring RavA state bears close structural similarity to the pseudo two-fold symmetric crystal structure of the AAA+ unfoldase ClpX, suggesting a common ATPase mechanism. Based on these structures, and in light of the current knowledge regarding AAA+ ATPases, we propose different scenarios for the ATP hydrolysis cycle of free RavA and the LdcI-RavA cage-like complex, and extend the comparison to other AAA+ ATPases of clade 7. Jessop, Arragain et al characterise the structure of the E.coli AAA+ ATPase RavA alone and in complex with the acid stress inducible lysine decarboxylase LdcI. Their results reveal two co-existing structural states of RavA and provide insights into the ATPase cycle that might be generalizable to other AAA+ ATPases of clade 7.
... In contrast, interaction with ClpP1−2 has an effect on the overall conformation of ClpX. Whereas the crystal structure of E. coli ClpX shows the ATPase domains in a dimerof-trimers arrangement 33 , our structure shows that upon ClpP1−2 binding, these domains become more regularly arranged and are related by pseudo-six-fold symmetry. Unlike recent substratebound AAA+ structures that show a 'spiral-staircase' arrangement with one 'seam' subunit moderately displaced from the pore [34][35][36] , all neighboring AAA+ domains of ClpX pack closely with each other. ...
... The resolution at the nucleotide pocket is not high enough to visualize nucleotides, but the structure reveals that all six ClpX protomers are in the 'loadable' (L) conformation ( Supplementary Fig. 3). This conformation is in contrast to that of ClpX with the E183Q mutation in its apo state 28,33 , in which two subunits are in the L conformation, and four are in the 'unloadable' (U) conformation ( Supplementary Fig. 3). In the L state, the arrangement of the small and large AAA+ Lower inset, four copies of ZBD dimers (PDB 1OVX) placed into the cryo-EM density at the interface between the ClpX hexamers. ...
... Taken together, tilting of the ClpX ring and stretching of one of the IGF loops is sufficient for the hexameric ClpX to adapt to the seven-fold symmetry of the heptameric ClpP, leaving out one of the binding pockets (Fig. 2g,h). Due to multivalence, this results in strong but flexible binding, which is likely necessary to accommodate the different conformations of ClpX protomers during ATP hydrolysis and substrate processing 12,21,33 . ...
The ClpXP machinery is a two-component protease complex that performs targeted protein degradation in bacteria and mitochondria. The complex consists of the AAA+ chaperone ClpX and the peptidase ClpP. The hexameric ClpX utilizes the energy of ATP binding and hydrolysis to engage, unfold and translocate substrates into the catalytic chamber of tetradecameric ClpP, where they are degraded. Formation of the complex involves a symmetry mismatch, because hexameric AAA+ rings bind axially to the opposing stacked heptameric rings of the tetradecameric ClpP. Here we present the cryo-EM structure of ClpXP from Listeria monocytogenes. We unravel the heptamer-hexamer binding interface and provide novel insight into the ClpX-ClpP cross-talk and activation mechanism. Comparison with available crystal structures of ClpP and ClpX in different states allows us to understand important aspects of the complex mode of action of ClpXP and provides a structural framework for future pharmacological applications.
... Structures in the ClpX reaction cycle are likely to be missing, either before or after a power stroke. Crystal structures reveal rotations between the large and small AAA+ domains so large that nucleotide cannot bind some subunits 22,23 . Although subunits of this type are not present in the current cryo-EM structures, they could represent transient functional conformations, as crosslinks engineered to trap these hexamer conformations form in solution and prevent ClpXP degradation but not ATP hydrolysis 23,46 . ...
... After three iterations of 2D classification, 28,004 particles representing side views were selected for subsequent processing. To generate an initial model for 3D refinement, the crystal structures of a ClpX ∆N hexamer (PDB 3HWS) 22 and a ClpP tetradecamer (PDB code 3MT6) 30 were merged in PyMOL and low-pass filtered to 40 Å. 3D refinement with C 2 symmetry yield a map at a resolution of 6.8 Å. ...
... EM structures were similar to each other (pair-wise Cα RMSD 1.2-2.3 Å) but different than nonspiral crystal structures of ClpX ∆N lacking substrate and ClpP[22][23] . ...
ClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel. Other AAA+ proteases and protein-remodeling machines have similar structures, and strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been inferred from these structural features. However, biochemical and single-molecule biophysical studies indicate that ClpXP operates by a probabilistic mechanism in which 5 to 8 residues are translocated for each ATP hydrolyzed. Thus, our results suggest that structurally similar AAA+ machines operate by a ClpX-like mechanism.
... Structural data on the NB domains of LonA proteases include our structure of EcLon presented here (Fig. 3A), as well as two high-resolution crystal structures of BsLon (PDB ID 3M6A, Fig. 4B) [33] and MtLon (PDB ID 4YPN) [34]. The overall fold of the LonA NB domain is typical of many other AAA + proteins, in particular of the ATPase components of ClpXP (PDB ID 3HTE) [55] and ClpAP proteases (PDB ID 1KSF) [44], as well as both NB domains of ClpB chaperones (PDB IDs 1QVR, 1JBK) [46,56]. ...
... Similarly to other AAA + ATPases, LonA proteases are expected to function as hexamers with a central pore that is lined with axial loops, required for protein unfolding and translocation. Three different pore loops, called 'GYVG' (or pore-1), pore-2 and 'RKH', are usually found in almost all AAA + unfoldases [55,57]. The homologs of GYVG (pore-1) loops are found in all AAA + proteases and chaperones of the ClpB/ Hsp104 family [55,[57][58][59][60]. ...
... Three different pore loops, called 'GYVG' (or pore-1), pore-2 and 'RKH', are usually found in almost all AAA + unfoldases [55,57]. The homologs of GYVG (pore-1) loops are found in all AAA + proteases and chaperones of the ClpB/ Hsp104 family [55,[57][58][59][60]. As currently established [57,61], GYVG loops are involved during all stages of molecular machinery work. ...
LonA proteases and ClpB chaperones are key components of the protein quality control system in bacterial cells. LonA proteases form a unique family of AAA+ proteins due to the presence of an unusual N‐terminal region comprised of two domains: a β‐structured N‐domain and an α‐helical domain, including the coiled‐coil fragment, which is referred to as HI(CC). The arrangement of helices in the HI(CC) domain is reminiscent of the structure of the H1 domain of the first AAA+ module of ClpB chaperones. It has been hypothesized that LonA proteases with a single AAA+ module may also contain a part of another AAA+ module, the full version of which is present in ClpB. Here, we established and tested the structural basis of this hypothesis using the known crystal structures of various fragments of LonA proteases and ClpB chaperones, as well as the newly determined structure of the Escherichia coli LonA fragment (235–584). The similarities and differences in the corresponding domains of LonA proteases and ClpB chaperones were examined in structural terms. The results of our analysis, complemented by the finding of a singular match in the location of the most conserved axial pore‐1 loop between the LonA NB domain and the NB2 domain of ClpB, support our hypothesis that there is a structural and functional relationship between two coil‐coil fragments and implies a similar mechanism of engagement of the pore‐1 loops in the AAA+ modules of LonAs and ClpBs.
... The active, extended form is required for substrate degradation, while an inactive compact state allows peptide product release from the ClpP inner chamber [243][244][245][246]. ClpXP consists of the caseinolytic mitochondrial matrix peptidase chaperone subunit X (ClpX; AAA+ ATPase) and ClpP (a tetradecameric peptidase). ClpX is a hexametric ATP-dependent protein unfoldase and translocase [238]. ClpP is a barrel-like peptidase assembled from two stacked heptameric rings, which enclose a roughly spherical proteolytic chamber [239] ( Figure 4A). ...
... ClpXP consists of the caseinolytic mitochondrial matrix peptidase chaperone subunit X (ClpX; AAA+ ATPase) and ClpP (a tetradecameric peptidase). ClpX is a hexametric ATP-dependent protein unfoldase and translocase [238]. ClpP is a barrel-like peptidase assembled from two stacked heptameric rings, which enclose a roughly spherical proteolytic chamber [239] ( Figure 4A). ...
Breast cancer is the most frequently diagnosed malignancy worldwide and the leading cause of cancer mortality in women. Despite the recent development of new therapeutics including targeted therapies and immunotherapy, triple-negative breast cancer remains an aggressive form of breast cancer, and thus improved treatments are needed. In recent decades, it has become increasingly clear that breast cancers harbor metabolic plasticity that is controlled by mitochondria. A myriad of studies provide evidence that mitochondria are essential to breast cancer progression. Mitochondria in breast cancers are widely reprogrammed to enhance energy production and biosynthesis of macromolecules required for tumor growth. In this review, we will discuss the current understanding of mitochondrial roles in breast cancers and elucidate why mitochondria are a rational therapeutic target. We will then outline the status of the use of mitochondria-targeting drugs in breast cancers, and highlight ClpP agonists as emerging mitochondria-targeting drugs with a unique mechanism of action. We also illustrate possible drug combination strategies and challenges in the future breast cancer clinic.
... 99,100 Indeed, it has been described that the domains of Clp-like systems can unravel to allow the pores to widen to accommodate larger substrates, and then fold back to allow the pores to close again and maintain intimate contacts with smaller substrates. 99,101 This mechanism of pore expansion-contraction via a "snake jaws model" has already been suggested, in which the pore size is controlled by the size of the substrates and the conformation, and structure of the ClpX subunits. 101 Second, it has been reported that the pore loops exhibit large-amplitude fluctuations on the microsecond time frame and change their conformation upon substrate engagement and ATP hydrolysis, indicating that these motions might drive substrate translocation. ...
... 99,101 This mechanism of pore expansion-contraction via a "snake jaws model" has already been suggested, in which the pore size is controlled by the size of the substrates and the conformation, and structure of the ClpX subunits. 101 Second, it has been reported that the pore loops exhibit large-amplitude fluctuations on the microsecond time frame and change their conformation upon substrate engagement and ATP hydrolysis, indicating that these motions might drive substrate translocation. 9 To study the contribution of pore flexibility in the interaction with the substrate, pore size fluctuation with and without substrate was examined throughout the simulation. ...
ClpXP complex is an ATP-dependent mitochondrial matrix protease that binds, unfolds, translocates, and subsequently degrades specific protein substrates. Its mechanisms of operation are still being debated, and several have been proposed, including the sequential translocation of two residues (SC/2R), six residues (SC/6R), and even long-pass probabilistic models. Therefore, it has been suggested to employ biophysical-computational approaches that can determine the kinetics and thermodynamics of the translocation. In this sense, and based on the apparent inconsistency between structural and functional studies, we propose to apply biophysical approaches based on elastic network models (ENM) to study the intrinsic dynamics of the theoretically most probable hydrolysis mechanism. The proposed models ENM suggest that the ClpP region is decisive for the stabilization of the ClpXP complex, contributing to the flexibility of the residues adjacent to the pore, favoring the increase in pore size and, therefore, with the energy of interaction of its residues with a larger portion of the substrate. It is predicted that the complex may undergo a stable configurational change once assembled and that the deformability of the system once assembled is oriented, to increase the rigidity of the domains of each region (ClpP and ClpX) and to gain flexibility of the pore. Our predictions could suggest under the conditions of this study the mechanism of the interaction of the system, of which the substrate passes through the unfolding of the pore in parallel with a folding of the bottleneck. The variations in the distance calculated by molecular dynamics could allow the passage of a substrate with a size equivalent to ∼3 residues. The theoretical behavior of the pore and the stability and energy of binding to the substrate based on ENM models suggest that in this system, there are thermodynamic, structural, and configurational conditions that allow a possible translocation mechanism that is not strictly sequential.
... He found that polypeptide threading is interrupted by pauses off the main translocation pathway, and that ClpX's translocation velocity is force dependent, reaching a maximum of 80 aa s -1 near zero force and vanishing above 20 pN (aa: amino acid). The motor displayed bursts of 1, 2, or 3 nm, suggesting a fundamental step-size of 1 nm per subunit, consistent with high-resolution crystallographic data (Glynn et al., 2009). Binding of ClpP decreases the probability of slippage and enhances the unfolding efficiency of ClpX. ...
... The distribution of burst sizes varies with [ATP] and probably reflects the near-instantaneous coordinated firing of 2, 3, or 4 subunits around the ring. In fact, previous biochemical and structural studies show that at most four subunits in the hexamer can bind ATP at any given time (Glynn et al., 2009;Hersch et al., 2005;Stinson et al., 2013). Consistently, she found that the motor can still function with up to two non-hydrolyzable ATPγS analog nucleotides bound to it. ...
The advent of single-molecule force spectroscopy represents the introduction of forces, torques, and displacements as controlled variables in biochemistry. These methods afford the direct manipulation of individual molecules to interrogate the forces that hold together their structure, the forces and torques that these molecules generate in the course of their biochemical reactions, and the use of force, torque, and displacement as tools to investigate the mechanisms of these reactions. Because of their microscopic nature, the signals detected in these experiments are often dominated by fluctuations, which, in turn, play an important role in the mechanisms that underlie the operation of the molecular machines of the cell. Their direct observation and quantification in single-molecule experiments provide a unique window to investigate those mechanisms, as well as a convenient way to investigate fundamental new fluctuation theorems of statistical mechanics that bridge the equilibrium and non-equilibrium realms of this discipline. In this review we have concentrated on the developments that occurred in our laboratory on the characterization of biopolymers and of molecular machines of the central dogma. Accordingly, some important areas like the study of cytoskeletal motors have not been included. While we adopt at times an anecdotal perspective with the hope of conveying the personal circumstances in which these developments took place, we have made every effort, nonetheless, to include the most important developments that were taking place at the same time in other laboratories.
... These events drive the outward rotation and partial refolding of RPT6 and an iris-like movement in the AAA ring that opens its axial channel for initial substrate insertion into the axial channel. This mode of coordinated ATP hydrolysis was also observed in the crystal structure of the hexameric ClpX protease [144], which drives rather different conformational changes in the ATPase ring compared to those in the 26S proteasome, likely because of the lack of a substrate. this mode is the simultaneous disengagement of at least two adjacent ATPases from the substrate. ...
... Remarkably, three coexisting modes of coordinated ATP hydrolysis were associated with ubiquitin recognition, deubiquitylation, translocation initiation and processive degradation in the substrate-bound human 26S proteasome [11]. Each mode of coordinated ATP hydrolysis was also observed in structural snapshots of various AAA+ ATPases under specific biochemical conditions by studies on the ClpX (Mode 1) [144], ATG3L2 (Mode 2) [133], Cdc48/p97 (Modes 2 and 3) [146,152], Yme1, ClpB, ClpXP, yeast 26S proteasome and bacterial T7 replisome (Mode 3) [118,123,132,147,169,170], suggesting highly conserved dynamic patterns in the structure-function relationships of AAA+ ATPase hexamers. These mechanistic findings, especially the key features and interactions in the high-resolution structures, are expected to facilitate pathological studies of the AAA+ proteases, as well as therapeutic development and drug discovery for regulating proteolysis effects in treating various diseases. ...
Adenosine triphosphatases (ATPases) associated with a variety of cellular activities (AAA+), the hexameric ring-shaped motor complexes located in all ATP-driven proteolytic machines, are involved in many cellular processes. Powered by cycles of ATP binding and hydrolysis, conformational changes in AAA+ ATPases can generate mechanical work that unfolds a substrate protein inside the central axial channel of ATPase ring for degradation. Three-dimensional visualizations of several AAA+ ATPase complexes in the act of substrate processing for protein degradation have been resolved at the atomic level thanks to recent technical advances in cryogenic electron microscopy (cryo-EM). Here, we summarize the resulting advances in structural and biochemical studies of AAA+ proteases in the process of proteolysis reactions, with an emphasis on cryo-EM structural analyses of the 26S proteasome, Cdc48/p97 and FtsH-like mitochondrial proteases. These studies reveal three highly conserved patterns in the structure–function relationship of AAA+ ATPase hexamers that were observed in the human 26S proteasome, thus suggesting common dynamic models of mechanochemical coupling during force generation and substrate translocation.
... It is common that one subunit of the ring is displaced, nucleotide-free and known as the "seam" protomer. Examples of such AAA+ proteins are ClpX, Vps4, LonA [45][46][47] or Hsp104 and ClpB for which opening of the ring is essential for function [48,49]. Asymmetric ring conformations were also observed for the MoxR AAA+ protein RavA [50]. ...
Background
NorQ, a member of the MoxR-class of AAA+ ATPases, and NorD, a protein containing a Von Willebrand Factor Type A (VWA) domain, are essential for non-heme iron (FeB) cofactor insertion into cytochrome c-dependent nitric oxide reductase (cNOR). cNOR catalyzes NO reduction, a key step of bacterial denitrification. This work aimed at elucidating the specific mechanism of NorQD-catalyzed FeB insertion, and the general mechanism of the MoxR/VWA interacting protein families.
Results
We show that NorQ-catalyzed ATP hydrolysis, an intact VWA domain in NorD, and specific surface carboxylates on cNOR are all features required for cNOR activation. Supported by BN-PAGE, low-resolution cryo-EM structures of NorQ and the NorQD complex show that NorQ forms a circular hexamer with a monomer of NorD binding both to the side and to the central pore of the NorQ ring. Guided by AlphaFold predictions, we assign the density that “plugs” the NorQ ring pore to the VWA domain of NorD with a protruding “finger” inserting through the pore and suggest this binding mode to be general for MoxR/VWA couples.
Conclusions
Based on our results, we present a tentative model for the mechanism of NorQD-catalyzed cNOR remodeling and suggest many of its features to be applicable to the whole MoxR/VWA family.
... The ClpX subunit is an asymmetric hexameric ring made of 6 different monomers which occur in 2 different conformations -4 loadable 'L' monomers and 2 unloadable 'U' monomers. Each loadable monomer is capable of loading one ATP, which allows ClpX to bind up to a total of 4 ATPs (Glynn et al., 2009). Each of these monomers are made up of a family-specific N-terminal domain and AAA+ module which consists of a large and a small domain. ...
The homeostasis of extremophiles is one that is a diamond hidden in the rough. The way extremophiles adapt to their extreme environments gives a clue into the true extent of what is possible when it comes to life. The discovery of new extremophiles is ever-expanding and an explosion of knowledge surrounding their successful existence in extreme environments is obviously perceived in scientific literature. The present review paper aims to provide a comprehensive view on the different mechanisms governing the extreme adaptations of extremophiles, along with insights and discussions on what the limits of life can possibly be. The membrane adaptations that are vital for survival are discussed in detail. It was found that there are many alterations in the genetic makeup of such extremophiles when compared to their mesophilic counterparts. Apart from the several proteins involved, the significance of chaperones, efflux systems, DNA repair proteins and a host of other enzymes that adapt to maintain functionality, are enlisted, and explained. A deeper understanding of the underlying mechanisms could have a plethora of applications in the industry. There are cases when certain microbes can withstand extreme doses of antibiotics. Such microbes accumulate numerous genetic elements (or plasmids) that possess genes for multiple drug resistance (MDR). A deeper understanding of such mechanisms helps in the development of potential approaches and therapeutic schemes for treating pathogen-mediated outbreaks. An in-depth analysis of the parameters – radiation, pressure, temperature, pH value and metal resistance – are discussed in this review, and the key to survival in these precarious niches is described.
... Each ATPase domain consists of two subdomains: a βαβ sandwich and an α-helical bundle, called the large and small subdomains, respectively. In the assembled hexamer, both subdomains contact the large subdomain of the clockwise neighboring subunit ( Figure 2), with the small subdomain moving as a rigid body with the neighboring large subunit [151]. ATP binds at the interface between the large and small subdomains and the large subdomain of a neighboring subunit. ...
The AAA-ATPases Pex1 and Pex6 are required for the formation and maintenance of peroxisomes, membrane-bound organelles that harbor enzymes for specialized metabolism. Together, Pex1 and Pex6 form a heterohexameric AAA-ATPase capable of unfolding substrate proteins via processive threading through a central pore. Here, we review the proposed roles for Pex1/Pex6 in peroxisome biogenesis and degradation, discussing how the unfolding of potential substrates contributes to peroxisome homeostasis. We also consider how advances in cryo-EM, computational structure prediction, and mechanisms of related ATPases are improving our understanding of how Pex1/Pex6 converts ATP hydrolysis into mechanical force. Since mutations in PEX1 and PEX6 cause the majority of known cases of peroxisome biogenesis disorders such as Zellweger syndrome, insights into Pex1/Pex6 structure and function are important for understanding peroxisomes in human health and disease.
... Previous analyses on analogous AAA-ATPase systems (including ClpX [45], HslU [46], and PAN [47]) have suggested that a maximum of four subunits can bind nucleotide simultaneously in the functional states, which is mostly consistent with our observation. Moreover, this asymmetric nucleotide occupancy pattern in consecutive subunits is to some extent in line with the recent biochemical analysis of the PAN system [48], but different from the ClpX system in which the two unloaded subunits locate on the opposite positions of the ring [49,50]. ...
The 26S proteasome is an ATP-dependent dynamic 2.5 MDa protease that regulates numerous essential cellular functions through degradation of ubiquitinated substrates. Here we present a near-atomic-resolution cryo-EM map of the S. cerevisiae 26S proteasome in complex with ADP-AlFx. Our biochemical and structural data reveal that the proteasome-ADP-AlFx is in an activated state, displaying a distinct conformational configuration especially in the AAA-ATPase motor region. Noteworthy, this map demonstrates an asymmetric nucleotide binding pattern with four consecutive AAA-ATPase subunits bound with nucleotide. The remaining two subunits, Rpt2 and Rpt6, with empty or only partially occupied nucleotide pocket exhibit pronounced conformational changes in the AAA-ATPase ring, which may represent a collective result of allosteric cooperativity of all the AAA-ATPase subunits responding to ATP hydrolysis. This collective motion of Rpt2 and Rpt6 results in an elevation of their pore loops, which could play an important role in substrate processing of proteasome. Our data also imply that the nucleotide occupancy pattern could be related to the activation status of the complex. Moreover, the HbYX tail insertion may not be sufficient to maintain the gate opening of 20S core particle. Our results provide new insights into the mechanisms of nucleotide driven allosteric cooperativity of the complex and of the substrate processing by the proteasome.
... Out of the total 18 hypermotile strains we isolated, four mutants carried the same Q289* (C865T) mutation (* indicates stop codon) and one mutant carried an L317R (T950G) mutation (Supplementary Figure S3). Thus, these evidences suggested that (Glynn et al., 2009). The 78th valine (V78) in Chain A-F has been highlighted in green (ball and stick). ...
Motility is finely regulated and is crucial to bacterial processes including colonization and biofilm formation. There is a trade-off between motility and growth in bacteria with molecular mechanisms not fully understood. Hypermotile Escherichia coli could be isolated by evolving non-motile cells on soft agar plates. Most of the isolates carried mutations located upstream of the flhDC promoter region, which upregulate the transcriptional expression of the master regulator of the flagellum biosynthesis, FlhDC. Here, we identified that spontaneous mutations in clpX boosted the motility of E. coli largely, inducing several folds of changes in swimming speed. Among the mutations identified, we further elucidated the molecular mechanism underlying the ClpXV78F mutation on the regulation of E. coli motility. We found that the V78F mutation affected ATP binding to ClpX, resulting in the inability of the mutated ClpXP protease to degrade FlhD as indicated by both structure modeling and in vitro protein degradation assays. Moreover, our proteomic data indicated that the ClpXV78F mutation elevated the stability of known ClpXP targets to various degrees with FlhD as one of the most affected. In addition, the specific tag at the C-terminus of FlhD being recognized for ClpXP degradation was identified. Finally, our transcriptome data characterized that the enhanced expression of the motility genes in the ClpXV78F mutations was intrinsically accompanied by the reduced expression of stress resistance genes relating to the reduced fitness of the hypermotile strains. A similar pattern was observed for previously isolated hypermotile E. coli strains showing high expression of flhDC at the transcriptional level. Hence, clpX appears to be a hot locus comparable to the upstream of the flhDC promoter region evolved to boost bacterial motility, and our finding provides insight into the reduced fitness of the hypermotile bacteria.
... The genome of the very simple free-living TACK archaeon Thermogladius calderae (Mardanov et al., 2012), with only 1,414 genes, encodes for a single Hsp60 chaperone and a single PAN-20S protease (Horwitz et al., 2007). The genome of the very simple free-living Aquificae bacterium Desulfurobacterium thermolithotrophum (Göker et al., 2011) with only 1,496 genes, encodes for two chaperones, Hsp20 and Hsp60 and five proteases: ClpAP, ClpXP (Gottesman et al., 1998;Glynn et al., 2009;Zeiler et al., 2013), Lon (Thomas-Wohlever and Lee, 2002), FtsH (Bieniossek et al., 2006) and HslUV (Yoo et al., 1996). By contrast, the genome of the complex ASGARD Heimdallarchaeota archaeon (strain LC_2) (Zaremba-Niedzwiedzka et al., 2017), with 4,485 genes and of the complex Gammaproteobacteria Escherichia coli (Blattner et al., 1997) with 4,391 genes, both encode for five conserved chaperone families: Hsp20, Hsp60, Hsp70, Hsp90, and Hsp100. ...
Life is a non-equilibrium phenomenon. Owing to their high free energy content, the macromolecules of life tend to spontaneously react with ambient oxygen and water and turn into more stable inorganic molecules. A similar thermodynamic picture applies to the complex shapes of proteins: While a polypeptide is emerging unfolded from the ribosome, it may spontaneously acquire secondary structures and collapse into its functional native conformation. The spontaneity of this process is evidence that the free energy of the unstructured state is higher than that of the structured native state. Yet, under stress or because of mutations, complex polypeptides may fail to reach their native conformation and form instead thermodynamically stable aggregates devoid of biological activity. Cells have evolved molecular chaperones to actively counteract the misfolding of stress-labile proteins dictated by equilibrium thermodynamics. HSP60, HSP70 and HSP100 can inject energy from ATP hydrolysis into the forceful unfolding of stable misfolded structures in proteins and convert them into unstable intermediates that can collapse into the native state, even under conditions inauspicious for that state. Aggregates and misfolded proteins may also be forcefully unfolded and degraded by chaperone-gated endo-cellular proteases, and in eukaryotes also by chaperone-mediated autophagy, paving the way for their replacement by new, unaltered functional proteins. The greater energy cost of degrading and replacing a polypeptide, with respect to the cost of its chaperone-mediated repair represents a thermodynamic dilemma: some easily repairable proteins are better to be processed by chaperones, while it can be wasteful to uselessly try recover overly compromised molecules, which should instead be degraded and replaced. Evolution has solved this conundrum by creating a host of unfolding chaperones and degradation machines and by tuning their cellular amounts and activity rates.
... Two HslU hexamers sandwich two HsIV hexamers to form a cylindrical hetero-24mer (Bochtler et al. 2000). ClpX forms a hexamer as well (Glynn et al. 2009) and associates with its conjugate protease ClpP (Wang et al. 1997) to form the ClpXP protease (Gatsogiannis et al. 2019;Fei et al. 2020;Ripstein et al. 2020), which recognizes and degrades proteins that have a degradation signal in a multistep binding and engagement process (Saunders et al. 2020). Some of these degradation signals are added to the C-terminus of an incomplete protein from a stalled ribosome, which directs the incomplete protein to ClpXP for degradation (Flynn et al. 2001). ...
ATPases associated with diverse cellular activities (AAA+ proteins) are a superfamily of proteins found throughout all domains of life. The hallmark of this family is a conserved AAA+ domain responsible for a diverse range of cellular activities. Typically, AAA+ proteins transduce chemical energy from the hydrolysis of ATP into mechanical energy through conformational change, which can drive a variety of biological processes. AAA+ proteins operate in a variety of cellular contexts with diverse functions including disassembly of SNARE proteins, protein quality control, DNA replication, ribosome assembly, and viral replication. This breadth of function illustrates both the importance of AAA+ proteins in health and disease and emphasizes the importance of understanding conserved mechanisms of chemo-mechanical energy transduction. This review is divided into three major portions. First, the core AAA+ fold is presented. Next, the seven different clades of AAA+ proteins and structural details and reclassification pertaining to proteins in each clade are described. Finally, two well-known AAA+ proteins, NSF and its close relative p97, are reviewed in detail.
... The mechanism of substrate processing by the proteasome is complex, but it can be simplified into 3 discrete and sequential modes of substrate processing, as described by Dong et al: Mode 1, Mode 2, and Mode 3. Beginning with substrate binding, Mode 1 describes 2 oppositely positioned ATPase subunits coordinate ATP hydrolysis to initiate substrate binding. This pattern is similar to that seen previously in the nucleotide-binding pattern of a substrate-free proteasome structure and ClpX (99,140). Next, in Mode 2, ATP hydrolysis in adjacent subunits initiates substrate translocation and CP gate opening (discussed further below). ...
Neurodegenerative diseases are characterized by the accumulation of misfolded proteins. This protein aggregation suggests that abnormal proteostasis contributes to aging-related neurodegeneration. A better fundamental understanding of proteins that regulate proteostasis may provide insight into the pathophysiology of neurodegenerative disease and may perhaps reveal novel therapeutic opportunities. The 26S proteasome is the key effector of the ubiquitin-proteasome system responsible for degrading polyubiquitinated proteins. However, additional factors, such as valosin-containing protein (VCP/p97/Cdc48) and C9orf72, play a role in regulation and trafficking of substrates through the normal proteostasis systems of a cell. Nonhuman AAA+ ATPases, such as the disaggregase Hsp104, also provide insights into the biochemical processes that regulate protein aggregation. X-ray crystallography and cryo-electron microscopy (cryo-EM) structures not bound to substrate have provided meaningful information about the 26S proteasome, VCP, and Hsp104. However, recent cryo-EM structures bound to substrate have provided new information about the function and mechanism of these proteostasis factors. Cryo-EM and cryo-electron tomography data combined with biochemical data have also increased the understanding of C9orf72 and its role in maintaining proteostasis. These structural insights provide a foundation for understanding proteostasis mechanisms with near-atomic resolution upon which insights can be gleaned regarding the pathophysiology of neurodegenerative diseases.
... This provides support for the powerstroke model for ClpX, which states that the motion generated is derived from the energy of ATP hydrolysis. The crystal structure of the ClpX translocon was found to have a 1 nm distance between the pore loops of adjacent subunits in different ATP concentration states, indicating a 1-nm firing per hydrolyzing ClpX subunit during polypeptide translocation (29). Indeed, when maximum translocation velocity is divided by the rate of ATP hydrolysis, it gives approximately 1.02 ± 0.03 nm per hydrolyzed ATP. ...
Single-molecule technologies have expanded our ability to detect biological events individually, in contrast to ensemble biophysical technologies, where the result provides averaged information. Recent developments in atomic force microscopy have not only enabled us to distinguish the heterogeneous phenomena of individual molecules, but also allowed us to view up to the resolution of a single covalent bond. Similarly, optical tweezers, due to their versatility and precisions, have emerged as a potent technique to dissect a diverse range of complex biological processes, from nanomechanics of ClpXP protease–dependent degradation to force-dependent processivity of motor proteins. Despite the advantages of optical tweezers, the time scales used in this technology were inconsistent with physiological scenarios, which led to the development of magnetic tweezers, where proteins are covalently linked with the glass surface, which in turn increases the observation window of a single biomolecule from minutes to weeks. Unlike optical tweezers, magnetic tweezers use magnetic fields to impose torque, which makes them convenient for studying DNA topology and topoisomerase functioning. Using modified magnetic tweezers, researchers were able to discover the mechanical role of chaperones, which support their substrate proteins by pulling them during translocation and assist their native folding as a mechanical foldase. In this article, we provide a focused review of many of these new roles of single-molecule technologies, ranging from single bond breaking to complex chaperone machinery, along with the potential to design mechanomedicine, which would be a breakthrough in pharmacological interventions against many diseases.
Expected final online publication date for the Annual Review of Biophysics, Volume 50 is May 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... [117] Therefore, the stabilization of protein is highly significant in life science. [118] Configurational entropy between the structures must be stabilized through hydrophobic interactions and hydrogen bonds. In addition, the contribution of charge-charge interactions, the burial of residues and the formation of ion pairs to pro-tein stability may be equally important. ...
With the rapid development of polymer materials, the simultaneous acquisition of micro‐nano structure and mesoscopic mechanical properties from the material surface with the transverse resolution of nanoscale has become a hot spot in the research of polymer materials. Atomic force microscopy‐based high‐resolution imaging and single‐molecule force spectroscopy techniques have been widely used in various related fields. This review introduces the basic design and working principles of atomic force microscopy as well as single‐molecule force microscopy and their applications in the characterization of polymer surface morphologies, polymer film phase separation, polymer crystallization behavior, polymer chain stretching, protein folding/unfolding, G quadruplex intermolecular and intramolecular interactions, living cell surface proteins, and sugars. The research status of AFM probe modification strategies such as nanomaterial processing, single molecule manipulation and biomolecule etching is reviewed, finally, the limitations and improvement potential of single molecule force spectroscopy based on atomic force microscopy (AFM‐ SMFS) are summarized and discussed, and the new research practice in the field of polymer materials is prospected.
... The recent cryo-EM structure of the Clade 7 ATPase RavA from the MoxR family showed that these hinge-like motions are conserved, but occur instead between the large and small subdomains of neighbouring monomers [53 ]. In addition, similarities between twofold symmetric closed ring states of ClpX [54] and RavA [53 ], and in particular the presence of a nucleotide-free 'double seam', support the idea of a conserved ATPase mechanism between Clades 5 and 7, despite the huge differences in domain architecture. ...
AAA+ ATPases are a diverse protein superfamily which power a vast number of cellular processes, from protein degradation to genome replication and ribosome biogenesis. The latest advances in cryo-EM have resulted in a spectacular increase in the number and quality of AAA+ ATPase structures. This abundance of new information enables closer examination of different types of structural insertions into the conserved core, revealing discrepancies in the current classification of AAA+ modules into clades. Additionally, combined with biochemical data, it has allowed rapid progress in our understanding of structure-functional relationships and provided arguments both in favour and against the existence of a unifying molecular mechanism for the ATPase activity and action on substrates, stimulating further intensive research.
... While the topological organization of the YME1 protomer closely resemble that of FtsH ( Figure 1D), our structure reveals a distinctly different quaternary organization. Instead of a symmetric organization of the AAA+ domains, the YME1 AAA+ domains assemble into a spiral staircase with the ATPases progressively rotated and translated with respect to one another, similar to the organization observed for numerous other AAA+ unfoldases, including the functionally related 26S proteasome ATPase (Glynn 2009, Lander 2012, Matyskiela 2013, Monroe 2017, Ripstein 2017. In this arrangement, a "step" subunit (red in . ...
We present the first atomic model of a substrate-bound inner mitochondrial membrane AAA+ quality control protease, YME1. Our ~3.4 Å cryo-EM structure reveals how the ATPases form a closed spiral staircase encircling an unfolded substrate, directing it toward the flat, symmetric protease ring. Importantly, the structure reveals how three coexisting nucleotide states allosterically induce distinct positioning of tyrosines in the central channel, resulting in substrate engagement and translocation to the negatively charged proteolytic chamber. This tight coordination by a network of conserved residues defines a sequential, around-the-ring ATP hydrolysis cycle that results in step-wise substrate translocation. Furthermore, we identify a hinge-like linker that accommodates the large-scale nucleotide-driven motions of the ATPase spiral independently of the contiguous planar proteolytic base. These results define the first molecular mechanism for a mitochondrial inner membrane AAA+ protease and reveal a translocation mechanism likely conserved for other AAA+ ATPases.
... In the bacteriophage T4 packaging ATPase, the pattern is conserved, but polarity is reversed; the lid subdomain is negatively charged, and the corresponding binding patch is positively charged (Fig. S2). Further, similar complementary charge patches between the lid subdomain and the neighboring subunit are present in ring ATPases residing on other branches of the ASCE evolutionary tree, such as katanin (Zehr et al., 2017), ClpX (Glynn et al., 2009), and Vsp4 (Han et al., 2019). Despite assembling as hexamers rather than pentamers, analogous interactions promote assembly (Fig. S2). ...
Double-stranded DNA viruses package their genomes into pre-assembled protein capsids using virally-encoded ATPase ring motors. While several structures of isolated monomers (subunits) from these motors have been determined, they provide little insight into how subunits within a functional ring coordinate their activities to efficiently generate force and translocate DNA. Here we describe the first atomic-resolution structure of a functional ring form of a viral DNA packaging motor and characterize its atomic-level dynamics via long timescale molecular dynamics simulations. Crystal structures of the pentameric ATPase ring from bacteriophage asccφ28 show that each subunit consists of a canonical N-terminal ASCE ATPase domain connected to a ‘vestigial’ nuclease domain by a small lid subdomain. The lid subdomain closes over the ATPase active site and engages in extensive interactions with a neighboring subunit such that several important catalytic residues are positioned to function in trans. The pore of the ring is lined with several positively charged residues that can interact with DNA. Simulations of the ATPase ring in various nucleotide-bound states provide information about how the motor coordinates sequential nucleotide binding, hydrolysis, and exchange around the ring. Simulations also predict that the ring adopts a helical structure to track DNA, consistent with recent cryo-EM reconstruction of the φ29 packaging ATPase. Based on these results, an atomistic model of viral DNA packaging is proposed wherein DNA translocation is powered by stepwise helical-to-planar ring transitions that are tightly coordinated by ATP binding, hydrolysis, and release.
... In the bacteriophage T4 packaging ATPase, the pattern is conserved, but polarity is reversed; the lid subdomain is negatively charged, and the corresponding binding patch is positively charged (Fig. S2). Further, similar complementary charge patches between the lid subdomain and the neighboring subunit are present in ring ATPases residing on other branches of the ASCE evolutionary tree, such as katanin (Zehr et al., 2017), ClpX (Glynn et al., 2009), and Vsp4 (Han et al., 2019). Despite assembling as hexamers rather than pentamers, analogous interactions promote assembly (Fig. S2). ...
Double-stranded DNA viruses package their genomes into pre-assembled protein capsids using virally-encoded ATPase ring motors. While several structures of isolated monomers (subunits) from these motors have been determined, they provide little insight into how subunits within a functional ring coordinate their activities to efficiently generate force and translocate DNA. Here we describe the first atomic-resolution structure of a functional ring form of a viral DNA packaging motor and characterize its atomic-level dynamics via long timescale molecular dynamics simulations. Crystal structures of the pentameric ATPase ring from bacteriophage asccφ28 show that each subunit consists of a canonical N-terminal ASCE ATPase domain connected to a 'vestigial' nuclease domain by a small lid subdomain. The lid subdomain closes over the ATPase active site and engages in extensive interactions with a neighboring subunit such that several important catalytic residues are positioned to function in trans. The pore of the ring is lined with several positively charged residues that can interact with DNA. Simulations of the ATPase ring in various nucleotide-bound states provide information about how the motor coordinates sequential nucleotide binding, hydrolysis, and exchange around the ring. Simulations also predict that the ring adopts a helical structure to track DNA, consistent with recent cryo-EM reconstruction of the φ29 packaging ATPase. Based on these results, an atomistic model of viral DNA packaging is proposed wherein DNA translocation is powered by stepwise helical-to-planar ring transitions that are tightly coordinated by ATP binding, hydrolysis, and release.
... For NmClpX, Phyre2 (Kelley et al., 2015) was used to perform one-to-one threading onto the previous crystal structure of ClpX from E. coli (PDBID 3HWS chain A) (Glynn et al., 2009). A single chain was then rigidly docked into the X3 position of the 2.9 Å map, and real space refinement and Ab initio model building of regions that poorly fit the density, as well as for regions missing from the homology model was performed in Coot (Emsley and Cowtan, 2004). ...
The ClpXP degradation machine consists of a hexameric AAA+ unfoldase (ClpX) and a pair of heptameric serine protease rings (ClpP) that unfold, translocate, and subsequently degrade client proteins. ClpXP is an important target for drug development against infectious diseases. Although structures are available for isolated ClpX and ClpP rings, it remains unknown how symmetry mismatched ClpX and ClpP work in tandem for processive substrate translocation into the ClpP proteolytic chamber. Here, we present cryo-EM structures of the substrate-bound ClpXP complex from Neisseria meningitidis at 2.3 to 3.3 Å resolution. The structures allow development of a model in which the sequential hydrolysis of ATP is coupled to motions of ClpX loops that lead to directional substrate translocation and ClpX rotation relative to ClpP. Our data add to the growing body of evidence that AAA+ molecular machines generate translocating forces by a common mechanism.
... For NmClpX, Phyre2 (Kelley et al., 2015) was used to perform one-to-one threading onto the previous crystal structure of ClpX from E. coli (PDBID 3HWS chain A) (Glynn et al., 2009). A single chain was then rigidly docked into the X3 position of the 2.9 Å map, and real space refinement and Ab initio model building of regions that poorly fit the density, as well as for regions missing from the homology model was performed in Coot (Emsley and Cowtan, 2004). ...
The ClpXP degradation machine consists of a hexameric AAA+ unfoldase (ClpX) and a pair of heptameric serine protease rings (ClpP) that unfold, translocate, and subsequently degrade client proteins. ClpXP is an important target for drug development against infectious diseases. Although structures are available for isolated ClpX and ClpP rings, it remains unknown how symmetry mismatched ClpX and ClpP work in tandem for processive substrate translocation into the ClpP proteolytic chamber. Here, we present cryo-EM structures of the substrate-bound ClpXP complex from Neisseria meningitidis at 2.3 to 3.3 Å resolution. The structures allow development of a model in which the sequential hydrolysis of ATP is coupled to motions of ClpX loops that lead to directional substrate translocation and ClpX rotation relative to ClpP. Our data add to the growing body of evidence that AAA+ molecular machines generate translocating forces by a common mechanism.
... Core mechanism of ATP-driven activity Numerous biochemical studies of AAA+ proteins indicated that their substrates are threaded through the central pore, which successively imposes a constriction on the substrate polypeptide that eventually forces folded domains to unravel [48][49][50][51][52][53][54] (Fig. 1). Although the key residues required for this activity have long been established, how conformational changes coupled to ATP binding, hydrolysis and product release might drive peptide substrate translocation has been elusive until recently. ...
ATPases associated with diverse cellular activities (AAA+ proteins) are macromolecular machines that convert the chemical energy contained in ATP molecules into powerful mechanical forces to remodel a vast array of cellular substrates, including protein aggregates, macromolecular complexes and polymers. AAA+ proteins have key functionalities encompassing unfolding and disassembly of such substrates in different subcellular localizations and, hence, power a plethora of fundamental cellular processes, including protein quality control, cytoskeleton remodelling and membrane dynamics. Over the past 35 years, many of the key elements required for AAA+ activity have been identified through genetic, biochemical and structural analyses. However, how ATP powers substrate remodelling and whether a shared mechanism underlies the functional diversity of the AAA+ superfamily were uncertain. Advances in cryo-electron microscopy have enabled high-resolution structure determination of AAA+ proteins trapped in the act of processing substrates, revealing a conserved core mechanism of action. It has also become apparent that this common mechanistic principle is structurally adjusted to carry out a diverse array of biological functions. Here, we review how substrate-bound structures of AAA+ proteins have expanded our understanding of ATP-driven protein remodelling. AAA+ proteins are macromolecular machines that remodel a vast array of cellular substrates, including protein aggregates, macromolecular complexes and polymers. Recent advances in cryo-electron microscopy have enabled visualization of them while in action, leading to a better understanding of the mechanisms of engagement and processing of their diverse substrates.
... There are two pore loops, pore loop 1 (PL-I) and pore loop 2 (PL-II), that line the central channel within each AAA domain of Rix7 (Figure 4b). A conserved aromatic-hydrophobic-glycine (most often Y/F-V-G) motif is typically found within the PL-I of AAA unfoldases such as in the regulatory particle of the proteasome or the ClpX component of the ClpXP proteases [97,98]. Even though Rix7 lacks the signature aromatic-hydrophobic-glycine motif within D1, it can engage a substrate throughout the entire central channel, suggesting that Rix7 functions as a molecular unfoldase. ...
AAA-ATPases are molecular engines evolutionarily optimized for the remodeling of proteins and macromolecular assemblies. Three AAA-ATPases are currently known to be involved in the remodeling of the eukaryotic ribosome, a megadalton range ribonucleoprotein complex responsible for the translation of mRNAs into proteins. The correct assembly of the ribosome is performed by a plethora of additional and transiently acting pre-ribosome maturation factors that act in a timely and spatially orchestrated manner. Minimal disorder of the assembly cascade prohibits the formation of functional ribosomes and results in defects in proliferation and growth. Rix7, Rea1, and Drg1, which are well conserved across eukaryotes, are involved in different maturation steps of pre-60S ribosomal particles. These AAA-ATPases provide energy for the efficient removal of specific assembly factors from pre-60S particles after they have fulfilled their function in the maturation cascade. Recent structural and functional insights have provided the first glimpse into the molecular mechanism of target recognition and remodeling by Rix7, Rea1, and Drg1. Here we summarize current knowledge on the AAA-ATPases involved in eukaryotic ribosome biogenesis. We highlight the latest insights into their mechanism of mechano-chemical complex remodeling driven by advanced cryo-EM structures and the use of highly specific AAA inhibitors.
AAA+ ATPases are ubiquitous hexameric unfoldases acting in cellular protein quality control. In complex with proteases, they form protein degradation machinery (the proteasome) in both archaea and eukaryotes. Here, we use solution-state NMR spectroscopy to determine the symmetry properties of the archaeal PAN AAA+ unfoldase and gain insights into its functional mechanism. PAN consists of three folded domains: the coiled-coil (CC), OB and ATPase domains. We find that full-length PAN assembles into a hexamer with C2 symmetry, and that this symmetry extends over the CC, OB and ATPase domains. The NMR data, collected in the absence of substrate, are incompatible with the spiral staircase structure observed in electron-microscopy studies of archaeal PAN in the presence of substrate and in electron-microscopy studies of eukaryotic unfoldases both in the presence and in the absence of substrate. Based on the C2 symmetry revealed by NMR spectroscopy in solution, we propose that archaeal ATPases are flexible enzymes, which can adopt distinct conformations in different conditions. This study reaffirms the importance of studying dynamic systems in solution.
The globally noticeable changing climatic conditions are conducive to pests and pathogens gradation on plants. Additionally, plants must deal with various abiotic stresses amplified by more and more intense industrial pressure. Plants are capable of inducing defense responses against environmental stresses. Among these responses, the regulation of proteolysis and hence the quality and composition of proteins are of fundamental importance. Proteolysis is an essential process required for protein metabolism that employs different types of proteolytic enzymes, for instance, serine, aspartic, metallo- and cysteine peptidases. As unregulated proteolysis could be a serious threat to cells, plants are equipped with a battery of direct and endogenous peptidase inhibitors. Thus, a continuous better understanding of the mechanisms underlying the precise regulation of proteolytic activity and the dynamic changes in the proteome of plants affected by environmental stresses should still be a matter of concern for the international forum of plant stress biologists.
This Research Topic aims to collect and discuss the most recent advances on protein metabolism mechanisms during plant environmental stresses responses using molecular, biochemical, physiological, structural, and systems biology approaches in plants including crop and wild species.
We expect submissions of original research, reviews, and methods, including (but not limited to) investigations on the following subtopics:
1. Novel players involved in the proteolytic-antiproteolytic balance.
2. Synthesis, degradation, and recycling of amino acids and proteins.
3. Roles of proteolytic enzymes and their activators and inhibitors.
4. Protein composition and quality systems: control and regulation.
5. Protein functional status, appropriate folding, post-translational modifications.
6. Hormonal regulation and signal transduction of amino acids and proteins metabolism.
7. Crosstalk between amino acid and protein metabolism and energy, carbohydrate and secondary metabolism, and the carbon-nitrogen budget.
8. Profiling of proteome and post-translationally modified proteome
Please note that a broad spectrum of submissions on protein and amino acid metabolism, profiling of proteome, and post-translationally modified proteome is welcomed, but descriptive manuscripts lacking significant mechanistic and/or physiological insights would be rejected without peer review.
Keywords: abiotic stress, amino acid, biotic stress, phytocystatin, phytohormone, protease, protease activator, protease inhibitor, proteolysis, signal transduction, proteome, protein phosphorylation, protein ubiquitination, protein post-translational modification
NorQ, a member of the MoxR-class of AAA+ ATPases, and NorD, a protein containing a Von Willebrand Factor Type A (VWA) domain, are essential for non-heme iron (FeB) cofactor insertion into cytochrome c-dependent nitric oxide reductase (cNOR). cNOR catalyzes the NO reduction, a key step of bacterial denitrification. This work aimed at elucidating the specific mechanism of NorQD-catalyzed FeB insertion, and the general mechanism of the MoxR/VWA interacting protein families. We show that NorQ-catalyzed ATP hydrolysis, an intact VWA-domain in NorD and specific surface carboxylates on cNOR are all features required for cNOR activation. Supported by BN-PAGE, low-resolution cryo-EM structures of NorQ and the NorQD complex show that NorQ forms a circular hexamer with a monomer of NorD binding both to the side and to the central pore of the NorQ ring. Guided by AlphaFold predictions, we assign the density that ″plugs″ the NorQ ring pore to the VWA domain of NorD with a protruding ″finger″ inserting through the pore, and suggest this binding mode to be general for MoxR/VWA couples. We present a tentative model for the mechanism of NorQD-catalyzed cNOR remodelling and suggest many of its features to be applicable to the whole MoxR/VWA family.
Protein homeostasis is tightly regulated by protein quality control systems such as chaperones and proteases. In cyanobacteria, the ClpXP proteolytic complex is regarded as a representative proteolytic system and consists of a hexameric ATPase ClpX and a tetradecameric peptidase ClpP. However, the functions and molecular mechanisms of ClpX in cyanobacteria remain unclear. This study aimed to decipher the unique contributions and regulatory networks of ClpX in the model cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). We showed that the interruption of clpX led to slower growth, decreased high light tolerance, and impaired photosynthetic cyclic electron transfer. A quantitative proteomic strategy was employed to globally identify ClpX-regulated proteins in Synechocystis cells. In total, we identified 172 differentially expressed proteins (DEPs) upon the interruption of clpX. Functional analysis revealed that these DEPs are involved in diverse biological processes, including glycolysis, nitrogen assimilation, photosynthetic electron transport, ATP-binding cassette (ABC) transporters, and two-component signal transduction. The expression of 24 DEPs was confirmed by parallel reaction monitoring (PRM) analysis. In particular, many hypothetical or unknown proteins were found to be regulated by ClpX, providing new candidates for future functional studies on ClpX. Together, our study provides a comprehensive ClpX-regulated protein network, and the results serve as an important resource for understanding protein quality control systems in cyanobacteria.
AAA+ (ATPases Associated with diverse cellular Activities) proteases unfold substrate proteins by pulling the substrate polypeptide through a narrow pore. To overcome the barrier to unfolding, substrates may require extended association with the ATPase. Failed unfolding attempts can lead to a slip of grip, which may result in substrate dissociation, but how substrate sequence affects slippage is unresolved. Here, we measured single-molecule dwell time using TIRF (Total Internal Reflection Fluorescence) microscopy, scoring time-dependent dissociation of engaged substrates from bacterial AAA+ ATPase unfoldase/translocase ClpX. Substrates comprising a stable domain resistant to unfolding and a C-terminal unstructured tail, tagged with a degron for initiating translocase insertion, were used to determine dwell time in relation to tail length and composition. We found greater tail length promoted substrate retention during futile unfolding. Additionally, we tested two tail compositions known to frustrate unfolding. A poly-glycine tract (polyG) promoted substrate release, but only when adjacent to the folded domain, whereas glycine-alanine repeats (GAr) did not promote release. A high-complexity motif containing polar and charged residues also promoted release. We further investigated the impact of these and related motifs on substrate degradation rates and ATP consumption, using the unfoldase-protease complex ClpXP. Here, substrate domain stability modulates the effects of substrate tail sequences. Although polyG and GAr are both inhibitory for unfolding, they act in different ways. GAr motifs only negatively affected degradation of highly stable substrates, which is accompanied by reduced ClpXP ATPase activity. Together, our results specify substrate characteristics that affect unfolding and degradation by ClpXP.
Simultaneous nanoscale imaging of mRNAs and proteins of the same specimen can provide better information on the translational regulation, molecular trafficking, and molecular interaction of both normal and diseased biological systems. Expansion microscopy (ExM) is an attractive option to achieve such imaging; however, simultaneous ExM imaging of proteins and mRNAs has not been demonstrated. Here, a technique for simultaneous ExM imaging of proteins and mRNAs in cultured cells and tissue slices, which we termed dual-expansion microscopy (dual-ExM), is demonstrated. First, we verified a protocol for the simultaneous labeling of proteins and mRNAs. Second, we combined the simultaneous labeling protocol with ExM to enable the simultaneous ExM imaging of proteins and mRNAs in cultured cells and mouse brain slices and quantitatively study the degree of signal retention after expansion. After expansion, both proteins and mRNAs can be visualized with a resolution beyond the diffraction limit of light in three dimensions. Dual-ExM is a versatile tool to study complex biological systems, such as the brain or tumor microenvironments, at a nanoscale resolution.
ClpXP is an archetypical AAA+ protease, consisting of ClpX and ClpP. ClpX is an ATP-dependent protein unfoldase and polypeptide translocase, whereas ClpP is a self-compartmentalized peptidase. ClpXP is currently the only AAA+ protease for which high-resolution structures exist, the molecular basis of recognition for a protein substrate is understood, extensive biochemical and genetic analysis have been performed, and single-molecule optical trapping has allowed direct visualization of the kinetics of substrate unfolding and translocation. In this review, we discuss our current understanding of ClpXP structure and function, evaluate competing sequential and probabilistic mechanisms of ATP hydrolysis, and highlight open questions for future exploration.
Clp proteases are powerful molecular machines that destroy protein substrates in bacteria, mitochondria, and chloroplasts. These enzymes harness chemical energy from ATP hydrolysis to mechanically unravel and degrade a diverse repertoire of protein targets. Their proteolytic activities contribute to protein quality control, stress responses, and the regulation of specific pathways, including many linked to virulence and pathogenesis. Moreover, Clp proteases have emerged as promising antibacterial targets in drug-resistant pathogens, and as oncotherapeutic targets in human tumors. Although their specific cellular roles and regulatory mechanisms differ among taxa, all Clp proteases share common functional and architectural paradigms. A wealth of biochemical, mechanochemical, and structural studies have established working models for ATP-coupled unfolding and proteolysis. Here, we survey the current state of knowledge in the field and highlight areas of ongoing and future study.
ClpXP in Escherichia coli is a proteasome degrading protein substrates. It consists of one hexamer of ATPase (ClpX) and two heptamers of peptidase (ClpP). The ClpX binds ATP and translocates the substrate protein into the ClpP chamber by binding and hydrolysis of ATP. At single molecular level, ClpX harnesses cycles of power stroke (dwell and burst) to unfold the substrates, then releases the ADP and Pi. Based on the construction and function of ClpXP, especially the recent progress on how ClpX unfold protein substrates, in this mini-review, a currently proposed single ClpX molecular model system detected by optical tweezers, and its prospective for the elucidation of the mechanism of force generation of ClpX in its power stroke and the subunit interaction with each other, were discussed in detail.
The first line of defense in the mitochondrial quality control network involves the stress response from a family of ATP-dependent proteases. We have reported that a solubilized version of the mitochondrial inner membrane ATP-dependent protease YME1L displays nucleotide binding kinetics that are sensitive to the reactive oxygen species hydrogen peroxide under a limiting ATP concentration. Our observations were consistent with an altered YME1L conformational ensemble leading to increased nucleotide binding site accessibility under oxidative stress conditions. To examine this hypothesis further, we report here the results of a comprehensive study of the thermodynamic and kinetic properties underlying the binding of nucleoside di- and triphosphate to the isolated YME1L AAA+ domain (YME1L-AAA+). A combination of fluorescence titrations, molecular dynamics, and stopped-flow fluorescence experiments have demonstrated similarity between nucleotide binding behaviors for YME1L under oxidative conditions and the isolated AAA+ domain. Our data demonstrate that YME1L-AAA+ binds ATP and ADP with affinities equal to ∼30 and 5 μM, respectively, in the absence of Mg2+. We note a negative heterotropic linkage effect between Mg2+ and ATP that arises as the MgCl2 concentration is increased such that the affinity of YME1L-AAA+ for ATP decreases to ∼60 μM in the presence of 10 mM MgCl2. Molecular dynamics methods allow for structural rationalization by revealing condition-dependent conformational populations for YME1L-AAA+. Taken together, these data suggest a preliminary model in which YME1L modulates its affinity for the nucleotide to stabilize against degradation or instability inherent to such stress conditions.
ClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies of the E. coli enzyme that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel, as observed for other AAA+ proteases and protein-remodeling machines. Strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been inferred from these structural features for other AAA+ unfoldases, but biochemical and single-molecule biophysical studies indicate that ClpXP operates by a probabilistic mechanism in which five to eight residues are translocated for each ATP hydrolyzed. We propose structure-based models that could account for the functional results.
Mitochondria control the activity, quality, and lifetime of their proteins with an autonomous system of chaperones, but the signals that direct substrate-chaperone interactions and outcomes are poorly understood. We previously discovered that the mitochondrial AAA+ protein unfoldase ClpX (mtClpX) activates the initiating enzyme for heme biosynthesis, 5-aminolevulinic acid synthase (ALAS), by promoting cofactor incorporation. Here, we ask how mtClpX accomplishes this activation. Using S. cerevisiae proteins, we identified sequence and structural features within ALAS that position mtClpX and provide it with a grip for acting on ALAS. Observation of ALAS undergoing remodeling by mtClpX revealed that unfolding is limited to a region extending from the mtClpX-binding site to the active site. Unfolding along this path is required for mtClpX to gate cofactor binding to ALAS. This targeted unfolding contrasts with the global unfolding canonically executed by ClpX homologs and provides insight into how substrate-chaperone interactions direct the outcome of remodeling.
The ClpXP degradation machine consists of a hexameric AAA+ unfoldase (ClpX) and a pair of heptameric serine protease rings (ClpP) that unfold, translocate, and subsequently degrade client proteins. ClpXP is an important target for drug development against infectious diseases. Although structures are available for isolated ClpX and ClpP rings, it remains unknown how symmetry mismatched ClpX and ClpP work in tandem for processive substrate translocation into the ClpP proteolytic chamber. Here we present cryo-EM structures of the substrate-bound ClpXP complex from Neisseria meningitidis at 2.3 to 3.3 Å resolution. The structures allow development of a model in which the sequential hydrolysis of ATP is coupled to motions of ClpX loops that lead to directional substrate translocation and ClpX rotation relative to ClpP. Our data add to the growing body of evidence that AAA+ molecular machines generate translocating forces by a common mechanism.
The fundamental unit of chromatin, the nucleosome, is an intricate structure that requires histone chaperones for assembly. ATAD2 AAA+ ATPases are a family of histone chaperones that regulate nucleosome density and chromatin dynamics. Here, we demonstrate that the fission yeast ATAD2 homolog, Abo1, deposits histone H3–H4 onto DNA in an ATP-hydrolysis-dependent manner by in vitro reconstitution and single-tethered DNA curtain assays. We present cryo-EM structures of an ATAD2 family ATPase to atomic resolution in three different nucleotide states, revealing unique structural features required for histone loading on DNA, and directly visualize the transitions of Abo1 from an asymmetric spiral (ATP-state) to a symmetric ring (ADP- and apo-states) using high-speed atomic force microscopy (HS-AFM). Furthermore, we find that the acidic pore of ATP-Abo1 binds a peptide substrate which is suggestive of a histone tail. Based on these results, we propose a model whereby Abo1 facilitates H3–H4 loading by utilizing ATP. ATAD2 AAA+ ATPases are a family of histone chaperones that regulate nucleosome density and chromatin dynamics. Here, authors find that the fission yeast ATAD2 homolog Abo1 deposits histone H3–H4 onto DNA in an ATP-hydrolysis-dependent manner, and present the cryo-EM structure of an ATAD2 family ATPase to reveal the structural basis of nucleosome assembly by Abo1.
Most AAA+ remodeling motors denature proteins by pulling on the peptide termini of folded substrates, but it is not well-understood how motors produce grip when resisting a folded domain. Here, at single amino-acid resolution, we identify the determinants of grip by measuring how substrate tail sequences alter the unfolding activity of the unfoldase-protease ClpXP. The seven amino acids abutting a stable substrate domain are key, with residues 2-6 forming a core that contributes most significantly to grip. ClpX grips large hydrophobic and aromatic side chains strongly and small, polar, or charged side chains weakly. Multiple side chains interact with pore loops synergistically to strengthen grip. In combination with recent structures, our results support a mechanism in which unfolding grip is primarily mediated by non-specific van der Waal's interactions between core side chains of the substrate tail and a subset of YVG loops at the top of the ClpX axial pore.
Structural genomics seeks to expand rapidly the number of protein structures in order to extract the maximum amount of information from genomic sequence databases. The advent of several large-scale projects worldwide leads to many new challenges in the ®eld of crystallographic macromolecular structure determination. A novel software package called PHENIX (Python-based Hierarchical ENvironment for Integrated Xtallography) is therefore being developed. This new software will provide the necessary algorithms to proceed from reduced intensity data to a re®ned molecular model and to facilitate structure solution for both the novice and expert crystallographer.
The purpose of this study was to evaluate clinical changes in graft size after treatment with connective tissue autograft in human. 40 premolar teeth in 23 patients having the following mucogingival problemswere selected.
ClpX and ClpA are molecular chaperones that interact with specific proteins and, together with ClpP, activate their ATP-dependent
degradation. The chaperone activity is thought to convert proteins into an extended conformation that can access the sequestered
active sites of ClpP. We now show that ClpX can catalyze unfolding of a green fluorescent protein fused to a ClpX recognition
motif (GFP-SsrA). Unfolding of GFP-SsrA depends on ATP hydrolysis. GFP-SsrA unfolded either by ClpX or by treatment with denaturants
binds to ClpX in the presence of adenosine 5′-O-(3-thiotriphosphate) and is released slowly (t1/2 ≈ 15 min). Unlike ClpA, ClpX cannot trap unfolded proteins in stable complexes unless they also have a high-affinity binding
motif. Addition of ATP or ADP accelerates release (t1/2 ≈ 1 min), consistent with a model in which ATP hydrolysis induces a conformation of ClpX with low affinity for unfolded substrates.
Proteolytically inactive complexes of ClpXP and ClpAP unfold GFP-SsrA and translocate the protein to ClpP, where it remains
unfolded. Complexes of ClpXP with translocated substrate within the ClpP chamber retain the ability to unfold GFP-SsrA. Our
results suggest a bipartite mode of interaction between ClpX and substrates. ClpX preferentially targets motifs exposed in
specific proteins. As the protein is unfolded by ClpX, additional motifs are exposed that facilitate its retention and favor
its translocation to ClpP for degradation.
Publisher Summary X-ray data can be collected with zero-, one-, and two-dimensional detectors, zero-dimensional (single counter) being the simplest and two-dimensional the most efficient in terms of measuring diffracted X-rays in all directions. To analyze the single-crystal diffraction data collected with these detectors, several computer programs have been developed. Two-dimensional detectors and related software are now predominantly used to measure and integrate diffraction from single crystals of biological macromolecules. Macromolecular crystallography is an iterative process. To monitor the progress, the HKL package provides two tools: (1) statistics, both weighted (χ 2 ) and unweighted (R-merge), where the Bayesian reasoning and multicomponent error model helps obtain proper error estimates and (2) visualization of the process, which helps an operator to confirm that the process of data reduction, including the resulting statistics, is correct and allows the evaluation of the problems for which there are no good statistical criteria. Visualization also provides confidence that the point of diminishing returns in data collection and reduction has been reached. At that point, the effort should be directed to solving the structure. The methods presented in the chapter have been applied to solve a large variety of problems, from inorganic molecules with 5 A unit cell to rotavirus of 700 A diameters crystallized in 700 × 1000 × 1400 A cell.
Escherichia coli ClpX, a member of the Clp family of ATPases, has ATP-dependent chaperone activity and is required for specific ATP-dependent proteolytic activities expressed by ClpP. Gel filtration and electron microscopy showed that ClpX subunits (Mr 46, 000) associate to form a six-membered ring (Mr approximately 280, 000) that is stabilized by binding of ATP or nonhydrolyzable analogs of ATP. ClpP, which is composed of two seven-membered rings stacked face-to-face, interacts with the nucleotide-stabilized hexamer of ClpX to form a complex that could be isolated by gel filtration. Electron micrographs of negatively stained ClpXP preparations showed side views of 1:1 and 2:1 ClpXP complexes in which ClpP was flanked on either one or both sides by a ring of ClpX. Thus, as was seen for ClpAP, a symmetry mismatch exists in the bonding interactions between the seven-membered rings of ClpP and the six-membered rings of ClpX. Competition studies showed that ClpA may have a slightly higher affinity (approximately 2-fold) for binding to ClpP. Mixed complexes of ClpA, ClpX, and ClpP with the two ATPases bound simultaneously to opposite faces of a single ClpP molecule were seen by electron microscopy. In the presence of ATP or nonhydrolyzable analogs of ATP, ClpXP had nearly the same activity as ClpAP against oligopeptide substrates (>10,000 min-1/tetradecamer of ClpP). Thus, ClpX and ClpA interactions with ClpP result in structurally analogous complexes and induce similar conformational changes that affect the accessibility and the catalytic efficiency of ClpP active sites.
Interruption of translation in Escherichia coli can lead to the addition of an 11-residue carboxy-terminal peptide tail to the nascent chain. This modification is mediated by SsrA RNA (also called 10Sa RNA and tmRNA) and marks the tagged polypeptide for proteolysis. Degradation in vivo of lambda repressor amino-terminal domain variants bearing this carboxy-terminal SsrA peptide tag is shown here to depend on the cytoplasmic proteases ClpXP and ClpAP. Degradation in vitro of SsrA-tagged substrates was reproduced with purified components and required a substrate with a wild-type SsrA tail, the presence of both ClpP and either ClpA or ClpX, and ATP. Clp-dependent proteolysis accounts for most degradation of SsrA-tagged amino-domain substrates at 32 degrees C, but additional proteases contribute to the degradation of some of these SsrA-tagged substrates at 39 degrees C. The existence of multiple cytoplasmic proteases that function in SsrA quality-control surveillance suggests that the SsrA tag is designed to serve as a relatively promiscuous signal for proteolysis. Having diverse degradation systems able to recognize this tag may increase degradation capacity, permit degradation of a wide variety of different tagged proteins, or allow SsrA-tagged proteins to be degraded under different growth conditions.
Phaser is a program for phasing macromolecular crystal structures by both molecular replacement and experimental phasing methods. The novel phasing algorithms implemented in Phaser have been developed using maximum likelihood and multivariate statistics. For molecular replacement, the new algorithms have proved to be significantly better than traditional methods in discriminating correct solutions from noise, and for single-wavelength anomalous dispersion experimental phasing, the new algorithms, which account for correlations between F
+ and F
−, give better phases (lower mean phase error with respect to the phases given by the refined structure) than those that use mean F and anomalous differences ΔF. One of the design concepts of Phaser was that it be capable of a high degree of automation. To this end, Phaser (written in C++) can be called directly from Python, although it can also be called using traditional CCP4 keyword-style input. Phaser is a platform for future development of improved phasing methods and their release, including source code, to the crystallographic community.
Transposition of phage Mu is catalyzed by an extremely stable transposase-DNA complex. Once recombination is complete, the Escherichia coli ClpX protein, a member of the Clp/Hsp100 chaperone family, initiates disassembly of the complex for phage DNA replication to commence. To understand how the transition between recombination and replication is controlled, we investigated how transposase-DNA complexes are recognized by ClpX. We find that a 10-amino-acid peptide from the carboxy-terminal domain of transposase is required for its recognition by ClpX. This short, positively charged peptide is also sufficient to convert a heterologous protein into a ClpX substrate. The region of transposase that interacts with the transposition activator, MuB protein, is also defined further and found to overlap with that recognized by ClpX. As a consequence, MuB inhibits disassembly of several transposase-DNA complexes that are intermediates in recombination. This ability of MuB to block access to transposase suggests a mechanism for restricting ClpX-mediated remodeling to the proper stage during replicative transposition. We propose that overlap of sequences involved in subunit interactions and those that target a protein for remodeling or destruction may be a useful design for proteins that function in pathways where remodeling or degradation must be regulated.
We have determined the crystal structure of the proteolytic component of the caseinolytic Clp protease (ClpP) from E. coli at 2.3 A resolution using an ab initio phasing procedure that exploits the internal 14-fold symmetry of the oligomer. The structure of a ClpP monomer has a distinct fold that defines a fifth structural family of serine proteases but a conserved catalytic apparatus. The active protease resembles a hollow, solid-walled cylinder composed of two 7-fold symmetric rings stacked back-to-back. Its 14 proteolytic active sites are located within a central, roughly spherical chamber approximately 51 A in diameter. Access to the proteolytic chamber is controlled by two axial pores, each having a minimum diameter of approximately 10 A. From the structural features of ClpP, we suggest a model for its action in degrading proteins.
A new software suite, called Crystallography & NMR System (CNS), has been developed for macromolecular structure determination by X-ray crystallography or solution nuclear magnetic resonance (NMR) spectroscopy. In contrast to existing structure-determination programs, the architecture of CNS is highly flexible, allowing for extension to other structure-determination methods, such as electron microscopy and solid-state NMR spectroscopy. CNS has a hierarchical structure: a high-level hypertext markup language (HTML) user interface, task-oriented user input files, module files, a symbolic structure-determination language (CNS language), and low-level source code. Each layer is accessible to the user. The novice user may just use the HTML interface, while the more advanced user may use any of the other layers. The source code will be distributed, thus source-code modification is possible. The CNS language is sufficiently powerful and flexible that many new algorithms can be easily implemented in the CNS language without changes to the source code. The CNS language allows the user to perform operations on data structures, such as structure factors, electron-density maps, and atomic properties. The power of the CNS language has been demonstrated by the implementation of a comprehensive set of crystallographic procedures for phasing, density modification and refinement. User-friendly task-oriented input files are available for nearly all aspects of macromolecular structure determination by X-ray crystallography and solution NMR.
The number of solved structures of macromolecules that have the same fold and thus exhibit some degree of conformational variability is rapidly increasing. It is consequently advantageous to develop a standardized terminology for describing this variability and automated systems for processing protein structures in different conformations. We have developed such a system as a 'front-end' server to our database of macromolecular motions. Our system attempts to describe a protein motion as a rigid-body rotation of a small 'core' relative to a larger one, using a set of hinges. The motion is placed in a standardized coordinate system so that all statistics between any two motions are directly comparable. We find that while this model can accommodate most protein motions, it cannot accommodate all; the degree to which a motion can be accommodated provides an aid in classifying it. Furthermore, we perform an adiabatic mapping (a restrained interpolation) between every two conformations. This gives some indication of the extent of the energetic barriers that need to be surmounted in the motion, and as a by-product results in a 'morph movie'. We make these movies available over the Web to aid in visualization. Many instances of conformational variability occur between proteins with somewhat different sequences. We can accommodate these differences in a rough fashion, generating an 'evolutionary morph'. Users have already submitted hundreds of examples of protein motions to our server, producing a comprehensive set of statistics. So far the statistics show that the median submitted motion has a rotation of approximately 10 degrees and a maximum Calpha displacement of 17 A. Almost all involve at least one large torsion angle change of >140 degrees. The server is accessible at http://bioinfo.mbb.yale. edu/MolMovDB
ClpX and ClpA are molecular chaperones that interact with specific proteins and, together with ClpP, activate their ATP-dependent degradation. The chaperone activity is thought to convert proteins into an extended conformation that can access the sequestered active sites of ClpP. We now show that ClpX can catalyze unfolding of a green fluorescent protein fused to a ClpX recognition motif (GFP-SsrA). Unfolding of GFP-SsrA depends on ATP hydrolysis. GFP-SsrA unfolded either by ClpX or by treatment with denaturants binds to ClpX in the presence of adenosine 5'-O-(3-thiotriphosphate) and is released slowly (t(1/2) approximately 15 min). Unlike ClpA, ClpX cannot trap unfolded proteins in stable complexes unless they also have a high-affinity binding motif. Addition of ATP or ADP accelerates release (t(1/2) approximately 1 min), consistent with a model in which ATP hydrolysis induces a conformation of ClpX with low affinity for unfolded substrates. Proteolytically inactive complexes of ClpXP and ClpAP unfold GFP-SsrA and translocate the protein to ClpP, where it remains unfolded. Complexes of ClpXP with translocated substrate within the ClpP chamber retain the ability to unfold GFP-SsrA. Our results suggest a bipartite mode of interaction between ClpX and substrates. ClpX preferentially targets motifs exposed in specific proteins. As the protein is unfolded by ClpX, additional motifs are exposed that facilitate its retention and favor its translocation to ClpP for degradation.
Escherichia coli ClpA and ClpX are ATP-dependent protein unfoldases that each interact with the protease, ClpP, to promote specific protein degradation. We have used limited proteolysis and deletion analysis to probe the conformations of ClpA and ClpX and their interactions with ClpP and substrates. ATP gamma S binding stabilized ClpA and ClpX such that that cleavage by lysylendopeptidase C occurred at only two sites. Both proteins were cleaved within in a loop preceding an alpha-helix-rich C-terminal domain. Although the loop varies in size and composition in Clp ATPases, cleavage occurred within and around a conserved triad, IG(F/L). Binding of ClpP blocked this cleavage, and prior cleavage at this site rendered both ClpA and ClpX defective in binding and activating ClpP, suggesting that this site is involved in interactions with ClpP. ClpA was also cut at a site near the junction of the two ATPase domains, whereas the second cleavage site in ClpX lay between its N-terminal and ATPase domains. ClpP did not block cleavage at these other sites. The N-terminal domain of ClpX dissociated upon cleavage, and the remaining ClpXDeltaN remained as a hexamer, associated with ClpP, and expressed ATPase, chaperone, and proteolytic activity. A truncated mutant of ClpA lacking its N-terminal 153 amino acids also formed a hexamer, associated with ClpP, and expressed these activities. We propose that the N-terminal domains of ClpX and ClpA lie on the outside ring surface of the holoenzyme complexes where they contribute to substrate binding or perform a gating function affecting substrate access to other binding sites and that a loop on the opposite face of the ATPase rings stabilizes interactions with ClpP and is involved in promoting ClpP proteolytic activity.
ClpXP is an ATP-dependent protease that denatures native proteins and translocates the denatured polypeptide into an interior peptidase chamber for degradation. To address the mechanism of these processes, Arc repressor variants with dramatically different stabilities and unfolding half-lives varying from months to seconds were targeted to ClpXP by addition of the ssrA degradation tag. Remarkably, ClpXP degraded each variant at a very similar rate and hydrolyzed approximately 150 molecules of ATP for each molecule of substrate degraded. The hyperstable substrates did, however, slow the ClpXP ATPase cycle. These results confirm that ClpXP uses an active mechanism to denature its substrates, probably one that applies mechanical force to the native structure. Furthermore, the data suggest that denaturation is inherently inefficient or that significant levels of ATP hydrolysis are required for other reaction steps. ClpXP degraded disulfide-cross-linked dimers efficiently, even when just one subunit contained an ssrA tag. This result indicates that the pore through which denatured proteins enter the proteolytic chamber must be large enough to accommodate simultaneous passage of two or three polypeptide chains.
ClpX mediates ATP-dependent denaturation of specific target proteins and disassembly of protein complexes. Like other AAA + family members, ClpX contains an alphabeta ATPase domain and an alpha-helical C-terminal domain. ClpX proteins with mutations in the C-terminal domain were constructed and screened for disassembly activity in vivo. Seven mutant enzymes with defective phenotypes were purified and characterized. Three of these proteins (L381K, D382K and Y385A) had low activity in disassembly or unfolding assays in vitro. In contrast to wild-type ClpX, substrate binding to these mutants inhibited ATP hydrolysis instead of increasing it. These mutants appear to be defective in a reaction step that engages bound substrate proteins and is required both for enhancement of ATP hydrolysis and for unfolding/disassembly. Some of these side chains form part of the interface between the C-terminal domain of one ClpX subunit and the ATPase domain of an adjacent subunit in the hexamer and appear to be required for communication between adjacent nucleotide binding sites.
In the ClpXP compartmental protease, ring hexamers of the AAA(+) ClpX ATPase bind, denature and then translocate protein substrates into the degradation chamber of the double-ring ClpP(14) peptidase. A key question is the extent to which functional communication between ClpX and ClpP occurs and is regulated during substrate processing. Here, we show that ClpX-ClpP affinity varies with the protein-processing task of ClpX and with the catalytic engagement of the active sites of ClpP. Functional communication between symmetry-mismatched ClpXP rings depends on the ATPase activity of ClpX and seems to be transmitted through structural changes in its IGF loops, which contact ClpP. A conserved arginine in the sensor II helix of ClpX links the nucleotide state of ClpX to the binding of ClpP and protein substrates. A simple model explains the observed relationships between ATP binding, ATP hydrolysis and functional interactions between ClpX, protein substrates and ClpP.
CCP4mg is a project that aims to provide a general-purpose tool for structural biologists, providing tools for X-ray structure solution, structure comparison and analysis, and publication-quality graphics. The map-fitting tools are available as a stand-alone package, distributed as 'Coot'.
Complex cellular events commonly depend on the activity of molecular "machines" that efficiently couple enzymatic and regulatory functions within a multiprotein assembly. An essential and expanding subset of these assemblies comprises proteins of the ATPases associated with diverse cellular activities (AAA+) family. The defining feature of AAA+ proteins is a structurally conserved ATP-binding module that oligomerizes into active arrays. ATP binding and hydrolysis events at the interface of neighboring subunits drive conformational changes within the AAA+ assembly that direct translocation or remodeling of target substrates. In this review, we describe the critical features of the AAA+ domain, summarize our current knowledge of how this versatile element is incorporated into larger assemblies, and discuss specific adaptations of the AAA+ fold that allow complex molecular manipulations to be carried out for a highly diverse set of macromolecular targets.
The E1 protein of papillomavirus is a hexameric ring helicase belonging to the AAA + family. The mechanism that couples the ATP cycle to DNA translocation has been unclear. Here we present the crystal structure of the E1 hexamer with single-stranded DNA discretely bound within the hexamer channel and nucleotides at the subunit interfaces. This structure demonstrates that only one strand of DNA passes through the hexamer channel and that the DNA-binding hairpins of each subunit form a spiral 'staircase' that sequentially tracks the oligonucleotide backbone. Consecutively grouped ATP, ADP and apo configurations correlate with the height of the hairpin, suggesting a straightforward DNA translocation mechanism. Each subunit sequentially progresses through ATP, ADP and apo states while the associated DNA-binding hairpin travels from the top staircase position to the bottom, escorting one nucleotide of single-stranded DNA through the channel. These events permute sequentially around the ring from one subunit to the next.
Methods developed originally to analyze domain motions from simulation [Proteins 27:425–437, 1997] are adapted and extended for the analysis of X-ray conformers and for proteins with more than two domains. The method can be applied as an automatic procedure to any case where more than one conformation is available. The basis of the methodology is that domains can be recognized from the difference in the parameters governing their quasi-rigid body motion, and in particular their rotation vectors. A clustering algorithm is used to determine clusters of rotation vectors corresponding to main-chain segments that form possible dynamic domains. Domains are accepted for further analysis on the basis of a ratio of interdomain to intradomain fluctuation, and Chasles' theorem is used to determine interdomain screw axes. Finally residues involved in the interdomain motion are identified. The methodology is tested on citrate synthase and the M6I mutant of T4 lysozyme. In both cases new aspects to their conformational change are revealed, as are individual residues intimately involved in their dynamics. For citrate synthase the beta sheet is identified to be part of the hinging mechanism. In the case of T4 lysozyme, one of the four transitions in the pathway from the closed to the open conformation, furnished four dynamic domains rather than the expected two. This result indicates that the number of dynamic domains a protein possesses may not be a constant of the motion. Proteins 30:144–154, 1998. © 1998 Wiley-Liss, Inc.
The CCP4 (Collaborative Computational Project, number 4) program suite is a collection of programs and associated data and subroutine libraries which can be used for macromolecular structure determination by X-ray crystallography. The suite is designed to be flexible, allowing users a number of methods of achieving their aims and so there may be more than one program to cover each function. The programs are written mainly in standard Fortran77. They are from a wide variety of sources but are connected by standard data file formats. The package has been ported to all the major platforms under both Unix and VMS. The suite is distributed by anonymous ftp from Daresbury Laboratory and is widely used throughout the world.
The CCP4 (Collaborative Computational Project, number 4) program suite is a collection of programs and associated data and subroutine libraries which can be used for macromolecular structure determination by X-ray crystallography. The suite is designed to be flexible, allowing users a number of methods of achieving their aims and so there may be more than one program to cover each function. The programs are written mainly in standard Fortran 77. They are from a wide variety of sources but are connected by standard data file formats. The package has been ported to all the major platforms under both Unix and VMS. The suite is distributed by anonymous ftp from Daresbury Laboratory and is widely used throughout the world.
Thesis (Ph. D.)--Yale University, 2001.
The Crystallography and NMR System (CNS) software suite is the result of an international collaborative effort amongst several research groups. It has been developed for macromolecular structure determination by X-ray crystallography or solution nuclear magnetic resonance (NMR) spectroscopy. CNS is available to academic institutions under a licence agreement. The CNS website provides information on the software and its use, an application form for downloading the software, and a bibliography.
In the AAA+ ClpXP protease, ClpX uses repeated cycles of ATP hydrolysis to pull native proteins apart and to translocate the denatured polypeptide into ClpP for degradation. Here, we probe polypeptide features important for translocation. ClpXP degrades diverse synthetic peptide substrates despite major differences in side-chain chirality, size, and polarity. Moreover, translocation occurs without a peptide -NH and with 10 methylenes between successive peptide bonds. Pulling on homopolymeric tracts of glycine, proline, and lysine also allows efficient ClpXP degradation of a stably folded protein. Thus, minimal chemical features of a polypeptide chain are sufficient for translocation and protein unfolding by the ClpX machine. These results suggest that the translocation pore of ClpX is highly elastic, allowing interactions with a wide range of chemical groups, a feature likely to be shared by many AAA+ unfoldases.
Proteolytic AAA+ unfoldases use ATP hydrolysis to power conformational changes that mechanically denature protein substrates and then translocate the polypeptide through a narrow pore into a degradation chamber. We show that a tyrosine residue in a pore loop of the hexameric ClpX unfoldase links ATP hydrolysis to mechanical work by gripping substrates during unfolding and translocation. Removal of the aromatic ring in even a few ClpX subunits results in slippage, frequent failure to denature the substrate and an enormous increase in the energetic cost of substrate unfolding. The tyrosine residue is part of a conserved aromatic-hydrophobic motif, and the effects of mutations in both residues vary with the nucleotide state of the resident subunit. These results support a model in which nucleotide-dependent conformational changes in these pore loops drive substrate translocation and unfolding, with the aromatic ring transmitting force to the polypeptide substrate.
An analysis has been made, from the data which are currently available, of the solvent content of 116 different crystal forms of globular proteins. The fraction of the crystal volume occupied by solvent is most commonly near 43 %, but has been observed to have values from about 27 to 65%. In many cases this range will be sufficiently restrictive to enable the probable number of molecules in the crystallographic asymmetric unit to be determined directly from the molecular weight of the protein and the space group and unit cell dimensions of the crystal.
The HSP100/Clp proteins are a newly discovered family with a great diversity of functions, such as increased tolerance to high temperatures, promotion of proteolysis of specific cellular substrates and regulation of transcription. HSP100/Clp proteins are also synthesized in a variety of specific patterns and, in eukaryotes, are localized to different subcellular compartments. Recent data suggest that a common ability to disassemble higher-order protein structures and aggregates unifies the molecular functions of this diverse family.
Methods developed originally to analyze domain motions from simulation [Proteins 27:425-437, 1997] are adapted and extended for the analysis of X-ray conformers and for proteins with more than two domains. The method can be applied as an automatic procedure to any case where more than one conformation is available. The basis of the methodology is that domains can be recognized from the difference in the parameters governing their quasi-rigid body motion, and in particular their rotation vectors. A clustering algorithm is used to determine clusters of rotation vectors corresponding to main-chain segments that form possible dynamic domains. Domains are accepted for further analysis on the basis of a ratio of interdomain to intradomain fluctuation, and Chasles' theorem is used to determine interdomain screw axes. Finally residues involved in the interdomain motion are identified. The methodology is tested on citrate synthase and the M6I mutant of T4 lysozyme. In both cases new aspects to their conformational change are revealed, as are individual residues intimately involved in their dynamics. For citrate synthase the beta sheet is identified to be part of the hinging mechanism. In the case of T4 lysozyme, one of the four transitions in the pathway from the closed to the open conformation, furnished four dynamic domains rather than the expected two. This result indicates that the number of dynamic domains a protein possesses may not be a constant of the motion.
The degradation of cytoplasmic proteins is an ATP-dependent process. Substrates are targeted to a single soluble protease, the 26S proteasome, in eukaryotes and to a number of unrelated proteases in prokaryotes. A surprising link emerged with the discovery of the ATP-dependent protease HslVU (heat shock locus VU) in Escherichia coli. Its protease component HslV shares approximately 20% sequence similarity and a conserved fold with 20S proteasome beta-subunits. HslU is a member of the Hsp100 (Clp) family of ATPases. Here we report the crystal structures of free HslU and an 820,000 relative molecular mass complex of HslU and HslV-the first structure of a complete set of components of an ATP-dependent protease. HslV and HslU display sixfold symmetry, ruling out mechanisms of protease activation that require a symmetry mismatch between the two components. Instead, there is conformational flexibility and domain motion in HslU and a localized order-disorder transition in HslV. Individual subunits of HslU contain two globular domains in relative orientations that correlate with nucleotide bound and unbound states. They are surprisingly similar to their counterparts in N-ethylmaleimide-sensitive fusion protein, the prototype of an AAA-ATPase. A third, mostly alpha-helical domain in HslU mediates the contact with HslV and may be the structural equivalent of the amino-terminal domains in proteasomal AAA-ATPases.
ClpXP is a protein machine composed of the ClpX ATPase, a member of the Clp/Hsp100 family of remodeling enzymes, and the ClpP peptidase. Here, ClpX and ClpXP are shown to catalyze denaturation of GFP modified with an ssrA degradation tag. ClpX translocates this denatured protein into the proteolytic chamber of ClpP and, when proteolysis is blocked, also catalyzes release of denatured GFP-ssrA from ClpP in a reaction that requires ATP and additional substrate. Kinetic experiments reveal that multiple reaction steps require collaboration between ClpX and ClpP and that denaturation is the rate-determining step in degradation. These insights into the mechanism of ClpXP explain how it executes efficient degradation in a manner that is highly specific for tagged proteins, irrespective of their intrinsic stabilities.
We have determined the crystal structure of an active, hexameric fragment of the gene 4 helicase from bacteriophage T7. The structure reveals how subunit contacts stabilize the hexamer. Deviation from expected six-fold symmetry of the hexamer indicates that the structure is of an intermediate on the catalytic pathway. The structural consequences of the asymmetry suggest a "binding change" mechanism to explain how cooperative binding and hydrolysis of nucleotides are coupled to conformational changes in the ring that most likely accompany duplex unwinding. The structure of a complex with a nonhydrolyzable ATP analog provides additional evidence for this hypothesis, with only four of the six possible nucleotide binding sites being occupied in this conformation of the hexamer. This model suggests a mechanism for DNA translocation.
HslUV is a "prokaryotic proteasome" composed of the HslV protease and the HslU ATPase, a chaperone of the Clp/Hsp100 family. The 3.4 A crystal structure of an HslUV complex is presented here. Two hexameric ATP binding rings of HslU bind intimately to opposite sides of the HslV protease; the HslU "intermediate domains" extend outward from the complex. The solution structure of HslUV, derived from small angle X-ray scattering data under conditions where the complex is assembled and active, agrees with this crystallographic structure. When the complex forms, the carboxy-terminal helices of HslU distend and bind between subunits of HslV, and the apical helices of HslV shift substantially, transmitting a conformational change to the active site region of the protease.
Binding and internalization of a protein substrate by E. coli ClpXP was investigated by electron microscopy. In sideviews of ATP gamma S-stabilized ClpXP complexes, a narrow axial channel was visible in ClpX, surrounded by protrusions on its distal surface. When substrate lambda O protein was added, extra density attached to this surface. Upon addition of ATP, this density disappeared as lambda O was degraded. When ATP was added to proteolytically inactive ClpXP-lambda O complexes, the extra density transferred to the center of ClpP and remained inside ClpP after separation from ClpX. We propose that substrates of ATP-dependent proteases bind to specific sites on the distal surface of the ATPase, and are subsequently unfolded and translocated into the internal chamber of the protease.
The Clp/Hsp100 ATPases are hexameric protein machines that catalyze the unfolding, disassembly and disaggregation of specific protein substrates in bacteria, plants and animals. Many family members also interact with peptidases to form ATP-dependent proteases. In Escherichia coli, for instance, the ClpXP protease is assembled from the ClpX ATPase and the ClpP peptidase. Here, we have used multiple sequence alignments to identify a tripeptide 'IGF' in E. coli ClpX that is essential for ClpP recognition. Mutations in this IGF sequence, which appears to be part of a surface loop, disrupt ClpXP complex formation and prevent protease function but have no effect on other ClpX activities. Homologous tripeptides are found only in a subset of Clp/Hsp100 ATPases and are a good predictor of family members that have a ClpP partner. Mapping of the IGF loop onto a homolog of known structure suggests a model for ClpX-ClpP docking.
The bacterial heat shock locus HslU ATPase and HslV peptidase together form an ATP-dependent HslVU protease. Bacterial HslVU is a homolog of the eukaryotic 26S proteasome. Crystallographic studies of HslVU should provide an understanding of ATP-dependent protein unfolding, translocation, and proteolysis by this and other ATP-dependent proteases.
We present a 3.0 A resolution crystal structure of HslVU with an HslU hexamer bound at one end of an HslV dodecamer. The structure shows that the central pores of the ATPase and peptidase are next to each other and aligned. The central pore of HslU consists of a GYVG motif, which is conserved among protease-associated ATPases. The binding of one HslU hexamer to one end of an HslV dodecamer in the 3.0 A resolution structure opens both HslV central pores and induces asymmetric changes in HslV.
Analysis of nucleotide binding induced conformational changes in the current and previous HslU structures suggests a protein unfolding-coupled translocation mechanism. In this mechanism, unfolded polypeptides are threaded through the aligned pores of the ATPase and peptidase and translocated into the peptidase central chamber.
On the basis of the structure of a HslUV complex, a mechanism of allosteric activation of the HslV protease, wherein binding of the HslU chaperone propagates a conformational change to the active site cleft of the protease, has been proposed. Here, the 3.1 A X-ray crystallographic structure of Haemophilus influenzae HslUV complexed with a vinyl sulfone inhibitor is described. The inhibitor, which reacts to form a covalent linkage to Thr1 of HslV, binds in an "antiparallel beta" manner, with hydrogen-bond interactions between the peptide backbone of the protease and that of the inhibitor, and with two leucinyl side chains of the inhibitor binding in the S1 and S3 specificity pockets of the protease. Comparison of the structure of the HslUV-inhibitor complex with that of HslV without inhibitor and in the absence of HslU reveals that backbone interactions would correctly position a substrate for cleavage in the HslUV complex, but not in the HslV protease alone, corroborating the proposed mechanism of allosteric activation. This activation mechanism differs from that of the eukaryotic proteasome, for which binding of activators opens a gated channel that controls access of substrates to the protease, but does not perturb the active site environment.
ClpX binds substrates bearing specific classes of peptide signals, denatures these proteins, and translocates them through a central pore into ClpP for degradation. ClpX with the V154F po e mutation is severely defective in binding substrates bearing C-motif 1 degradation signals and is also impaired in a subsequent step of substrate engagement. In contrast, this mutant efficiently processes substrates with other classes of recognition signals both in vitro and in vivo. These results demonstrate that the ClpX pore functions in the recognition and catalytic engagement of specific substrates, and that ClpX recognizes different substrate classes in at least two distinct fashions.
The large tumor antigen (LTag) of simian virus 40, an AAA(+) protein, is a hexameric helicase essential for viral DNA replication in eukaryotic cells. LTag functions as an efficient molecular machine powered by ATP binding and hydrolysis for origin DNA melting and replication fork unwinding. To understand how ATP binding and hydrolysis are coupled to conformational changes, we have determined high-resolution structures ( approximately 1.9 A) of LTag hexamers in distinct nucleotide binding states. The structural differences of LTag in various nucleotide states detail the molecular mechanisms of conformational changes triggered by ATP binding/hydrolysis and reveal a potential mechanism of concerted nucleotide binding and hydrolysis. During these conformational changes, the angles and orientations between domains of a monomer alter, creating an "iris"-like motion in the hexamer. Additionally, six unique beta hairpins on the channel surface move longitudinally along the central channel, possibly serving as a motor for pulling DNA into the LTag double hexamer for unwinding.
The SspB adaptor enhances ClpXP degradation by binding the ssrA degradation tag of substrates and the AAA+ ClpX unfoldase. To probe the mechanism of substrate delivery, we engineered a disulfide bond between the ssrA tag and SspB and demonstrated otherwise normal interactions by solving the crystal structure. Although the covalent link prevents adaptor.substrate dissociation, ClpXP degraded GFP-ssrA that was disulfide bonded to the adaptor. Thus, crosslinked substrate must be handed directly from SspB to ClpX. The ssrA tag in the covalent adaptor complex interacted with ClpX.ATPgammaS but not ClpX.ADP, suggesting that handoff occurs in the ATP bound enzyme. By contrast, SspB alone bound ClpX in both nucleotide states. Similar handoff mechanisms will undoubtedly be used by many AAA+ adaptors and enzymes, allowing assembly of delivery complexes in either nucleotide state, engagement of the recognition tag in the ATP state, and application of an unfolding force to the attached protein following hydrolysis.
ATP hydrolysis by AAA+ ClpX hexamers powers protein unfolding and translocation during ClpXP degradation. Although ClpX is a homohexamer, positive and negative allosteric interactions partition six potential nucleotide binding sites into three classes with asymmetric properties. Some sites release ATP rapidly, others release ATP slowly, and at least two sites remain nucleotide free. Recognition of the degradation tag of protein substrates requires ATP binding to one set of sites and ATP or ADP binding to a second set of sites, suggesting a mechanism that allows repeated unfolding attempts without substrate release over multiple ATPase cycles. Our results rule out concerted hydrolysis models involving ClpX(6)*ATP(6) or ClpX(6)*ADP(6) and highlight structures of hexameric AAA+ machines with three or four nucleotides as likely functional states. These studies further emphasize commonalities between distant AAA+ family members, including protein and DNA translocases, helicases, motor proteins, clamp loaders, and other ATP-dependent enzymes.
The AAA+ (ATPases associated with various cellular activities) family is a large and functionally diverse group of enzymes that are able to induce conformational changes in a wide range of substrate proteins. The family's defining feature is a structurally conserved ATPase domain that assembles into oligomeric rings and undergoes conformational changes during cycles of nucleotide binding and hydrolysis. Here, we review the structural organization of AAA+ proteins, the conformational changes they undergo, the range of different reactions they catalyse, and the diseases associated with their dysfunction.
Hexameric ring-shaped ATPases of the AAA + (for ATPases associated with various cellular activities) superfamily power cellular processes in which macromolecular structures and complexes are dismantled or denatured, but the mechanisms used by these machine-like enzymes are poorly understood. By covalently linking active and inactive subunits of the ATPase ClpX to form hexamers, here we show that diverse geometric arrangements can support the enzymatic unfolding of protein substrates and translocation of the denatured polypeptide into the ClpP peptidase for degradation. These studies indicate that the ClpX power stroke is generated by ATP hydrolysis in a single subunit, rule out concerted and strict sequential ATP hydrolysis models, and provide evidence for a probabilistic sequence of nucleotide hydrolysis. This mechanism would allow any ClpX subunit in contact with a translocating polypeptide to hydrolyse ATP to drive substrate spooling into ClpP, and would prevent stalling if one subunit failed to bind or hydrolyse ATP. Energy-dependent machines with highly diverse quaternary architectures and molecular functions could operate by similar asymmetric mechanisms.
The ATP-dependent integral membrane protease FtsH is universally conserved in bacteria. Orthologs exist in chloroplasts and mitochondria, where in humans the loss of a close FtsH-homolog causes a form of spastic paraplegia. FtsH plays a crucial role in quality control by degrading unneeded or damaged membrane proteins, but it also targets soluble signaling factors like σ³² and λ-CII. We report here the crystal structure of a soluble FtsH construct that is functional in caseinolytic and ATPase assays. The molecular architecture of this hexameric molecule consists of two rings where the protease domains possess an all-helical fold and form a flat hexagon that is covered by a toroid built by the AAA domains. The active site of the protease classifies FtsH as an Asp-zincin, contrary to a previous report. The different symmetries of protease and AAA rings suggest a possible translocation mechanism of the target polypeptide chain into the interior of the molecule where the proteolytic sites are located.
• AAA
• protease
• protein degradation
• x-ray
AAA+ proteins form large, ring-shaped complexes, which act as energy-dependent unfoldases of macromolecules. Many crystal structures of proteins in this superfamily have been determined, but mostly in monomeric or non-physiological oligomeric forms. The assembly of ring-shaped complexes from monomer coordinates is, therefore, of considerable interest. We have extracted structural features of complex formation relating to the distance of monomers from the central axis, their relative orientation and the molecular contacts at their interfaces from experimentally determined oligomers and have implemented a semi-automated modeling procedure based on RosettaDock into the iMolTalk server (http://protevo.eb.tuebingen.mpg.de/iMolTalk). As examples of this procedure, we present here models of Apaf-1, MalT and ClpB. We show that the recent EM-based model of the apoptosome is not compatible with the conserved structural features of AAA+ complexes and that the D1 and D2 rings of ClpB are most likely offset by one subunit, in agreement with the structure proposed for ClpA.
In the prokaryotic homolog of the eukaryotic proteasome, HslUV, the "double donut" HslV protease is allosterically activated by HslU, an AAA protein of the Clp/Hsp100 family consisting of three (amino-terminal, carboxy-terminal, and intermediate) domains. The intermediate domains of HslU, which extend like tentacles from the hexameric ring formed by the amino-terminal and carboxy-terminal domains, have been deleted; an asymmetric HslU(DeltaI)(6)HslV(12) complex has been crystallized; and the structure has been solved to 2.5A resolution, revealing an assembly in which a HslU(DeltaI) hexamer binds one end of the HslV dodecamer. The conformation of the protomers of the HslU(DeltaI)-complexed HslV hexamer is similar to that in the symmetric wild-type HslUV complex, while the protomer conformation of the uncomplexed HslV hexamer is similar to that of HslV alone. Reaction in the crystals with a vinyl sulfone inhibitor reveals that the HslU(DeltaI)-complexed HslV hexamer is active, while the uncomplexed HslV hexamer is inactive. These results confirm that HslV can be activated by binding of a hexameric HslU(DeltaI)(6) ring lacking the I domains, that activation is effected through a conformational change in HslV rather than through alteration of the size of the entry channel into the protease catalytic cavity, and that the two HslV(6) rings in the protease dodecamer are activated independently rather than cooperatively.
Hexameric helicases and translocases are required for numerous essential nucleic-acid transactions. To better understand the mechanisms by which these enzymes recognize target substrates and use nucleotide hydrolysis to power molecular movement, we have determined the structure of the Rho transcription termination factor, a hexameric RNA/DNA helicase, with single-stranded RNA bound to the motor domains of the protein. The structure reveals a closed-ring "trimer of dimers" conformation for the hexamer that contains an unanticipated arrangement of conserved loops required for nucleic-acid translocation. RNA extends across a shallow intersubunit channel formed by conserved amino acids required for RNA-stimulated ATP hydrolysis and translocation and directly contacts a conserved lysine, just upstream of the catalytic GKT triad, in the phosphate-binding (P loop) motif of the ATP-binding pocket. The structure explains the molecular effects of numerous mutations and provides new insights into the links between substrate recognition, ATP turnover, and coordinated strand movement.
ClpXP, an ATP-dependent protease, degrades hundreds of different intracellular proteins. ClpX chooses substrates by binding peptide tags, typically displayed at the N or C terminus of the protein to be degraded. Here, we identify a ClpX mutant that displays a 300-fold change in substrate specificity, resulting in decreased degradation of ssrA-tagged substrates but improved degradation of proteins with other classes of degradation signals. The altered-specificity mutation occurs within "RKH" loops, which surround the entrance to the central pore of the ClpX hexamer and are highly conserved in the ClpX subfamily of AAA+ ATPases. These results support a major role for the RKH loops in substrate recognition and suggest that ClpX specificity represents an evolutionary compromise that has optimized degradation of multiple types of substrates rather than any single class.