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Asymmetric Nucleotide Transactions of the HslUV Protease
Abstract
ATP binding and hydrolysis are critical for protein degradation by HslUV, a AAA(+) machine containing one or two HslU(6) ATPases and the HslV(12) peptidase. Although each HslU homohexamer has six potential ATP-binding sites, we show that only three or four ATP molecules bind at saturation and present evidence for three functional subunit classes. These results imply that only a subset of HslU and HslUV crystal structures represents functional enzyme conformations. Our results support an asymmetric mechanism of ATP binding and hydrolysis, and suggest that molecular contacts between HslU and HslV vary dynamically throughout the ATPase cycle. Nucleotide binding controls HslUV assembly and activity. Binding of a single ATP allows HslU to bind HslV, whereas additional ATPs must bind HslU to support substrate recognition and to activate ATP hydrolysis, which powers substrate unfolding and translocation. Thus, a simple thermodynamic hierarchy ensures that substrates bind to functional HslUV complexes, that ATP hydrolysis is efficiently coupled to protein degradation, and that working HslUV does not dissociate, allowing highly processive degradation.
... The 3D reconstruction of the complex with saturating ATP shows a relative orientation of the D1 and D2 rings that resembles the ATPγS state, but the nucleotide binding pockets are less defined, indicative of structurally varying nucleotide states occurring during hydrolysis. The observed asymmetry in D2 ring suggests the presence of different nucleotide states in the hexamer and that Pex1/Pex6 hydrolyzes ATP by a non-concerted mechanism, similar to related ATPases [30,31,54,55]. ...
... One could be the partial inhibition of Pex6 by a hydrolysis-dead Pex1 neighbor, and the other may be related to the total number of ATP-loadable sites in the Pex1/Pex6 hexamer. Several previous studies on related AAA+ enzymes suggest that a closed ring topology allows only four of the six nucleotide-binding sites to be occupied [31,54,55]. Indeed, we observed asymmetry in the nucleotide binding sites in saturating conditions of ATP, ATPγS, and ADP. ...
Pex1 and Pex6 are Type-2 AAA+ ATPases required for the de-novo biogenesis of peroxisomes. Mutations in Pex1 and Pex6 account for the majority of the most severe forms of peroxisome biogenesis disorders in humans. Here we show that the ATP-dependent complex of Pex1 and Pex6 from S. cerevisiae is a heterohexamer with alternating subunits. Within the Pex1/Pex6 complex, only the D2 ATPase ring hydrolyzes ATP, while nucleotide binding in the D1 ring promotes complex assembly. ATP hydrolysis by Pex1 is highly coordinated with that of Pex6. Furthermore, Pex15, the membrane anchor required for Pex1/Pex6 recruitment to peroxisomes inhibits the ATP-hydrolysis activity of Pex1/Pex6.
Copyright © 2015. Published by Elsevier Ltd.
... Crystal structures of other unfoldases such as HslU and Lon show a more symmetric ring structure in which all subunits adopt a similar conformation [35,36]. These symmetric structures may imply a different mechanism of function; however, they seem at odds with existing biochemical data reporting that the hexameric rings of the HslU, PAN, and ClpX unfoldases can only bind four nucleotides at saturation [33,37,38]. It is possible that these structures of HslU and Lon represent nonfunctional conformations or apo states in the absence of nucleotide and/or substrate. ...
... Conformational switching of empty subunits raises the possibility that a subunit's nucleotide affinity or hydrolysis activity may be modulated by the nucleotide occupancy and the state of the hydrolysis cycle elsewhere in the ring. Different classes of subunits with correspondingly different nucleotide affinities have been observed in the hexamers of various ATP-dependent unfoldases, including ClpX [33], PAN [48], and HslU [37]. Thus, it is plausible that a preferred order of nucleotide loading, hydrolysis, and product release steps exists among neighboring subunits as a result of their structurally constrained geometry. ...
ATP-dependent proteases exist in all cells and are crucial regulators of the proteome. These machines consist of a hexameric, ring-shaped motor responsible for engaging, unfolding, and translocating protein substrates into an associated peptidase for degradation. Here, we discuss recent work that has established how the six motor subunits coordinate their ATP-hydrolysis and translocation activities. The closed topology of the ring and the rigidity of subunit/subunit interfaces cause conformational changes within a single subunit to drive motions in other subunits of the hexamer. This structural effect generates allostery between the ATP-binding sites, leading to a preferred order of binding and hydrolysis events among the motor subunits as well as a unique biphasic mechanism of translocation.
... Therefore, we could define the functional effects of substoichiometric ATP binding to PAN in ways that would not have been possible with other AAA ATPases. The presence of different types of nucleotide-binding sites in homohexameric AAA ATPase complexes has been reported previously (Hersch et al., 2005;Singleton et al., 2000;Yakamavich et al., 2008;Zalk and Shoshan-Barmatz, 2003). This binding asymmetry must originate from the binding of a nucleotide to one subunit causing conformational changes in the neighboring ones that then differ structurally from the original ATPbound subunit. ...
... The crystal structure of mutated, linked ClpX also shows a similar dimer of trimers structure (Glynn et al., 2009). Though similar nucleotide exchange reactions have been suggested by others (Hersch et al., 2005;Schumacher et al., 2008;Singleton et al., 2000), albeit without evidence of distinct functional consequences, the crystal structures of some AAA ATPases (e.g., HslU [Bochtler et al., 2000;Sousa et al., 2000;Yakamavich et al., 2008]) revealed seemingly promiscuous binding patterns for ATP analogs or ADP. An unambiguous elucidation of the binding exchange reactions for those ATPases has proven difficult because the number of nucleotides bound per hexamer has rarely been determined to a definite integer value (i.e., prior results could not distinguish between three or four nucleotides per hexamer). ...
In the eukaryotic 26S proteasome, the 20S particle is regulated by six AAA ATPase subunits and, in archaea, by a homologous ring complex, PAN. To clarify the role of ATP in proteolysis, we studied how nucleotides bind to PAN. Although PAN has six identical subunits, it binds ATPs in pairs, and its subunits exhibit three conformational states with high, low, or no affinity for ATP. When PAN binds two ATPγS molecules or two ATPγS plus two ADP molecules, it is maximally active in binding protein substrates, associating with the 20S particle, and promoting 20S gate opening. However, binding of four ATPγS molecules reduces these functions. The 26S proteasome shows similar nucleotide dependence. These findings imply an ordered cyclical mechanism in which two ATPase subunits bind ATP simultaneously and dock into the 20S. These results can explain how these hexameric ATPases interact with and "wobble" on top of the heptameric 20S proteasome.
... substrate recognition (3). Additionally, asymmetric binding and hydrolysis of ATP(s) were demonstrated in vitro for degradation of the substrates in the ClpYQ complex (39). The physiological role and biochemical function in eubacteria or in higher organisms of ClpYQ protease were recently reviewed by Wu et al. (38). ...
... The nucleotide-bound ATP-ClpY molecules are responsible for the recognition of substrates in the ClpYQ complex. It was also later shown that three or four ATPs were required for the ClpYQ complex in the process of recognition, unfolding/translocation, and degradation of the substrate (39). In this study, we demonstrated that the double loops aa 137 to 150 and aa 175 to 209 in domain I of ClpY are essential for association of the natural substrates and that the aa 175 to 209 loop is likely involved in gripping the substrates. ...
In Escherichia coli, ClpYQ (HslUV) is a two-component ATP-dependent protease in which ClpQ is the peptidase subunit and ClpY is the ATPase and the substrate-binding subunit. The ATP-dependent proteolysis is mediated by substrate recognition in the ClpYQ complex. ClpY has three domains, N, I, and C, and these domains are discrete and exhibit different binding preferences. In vivo, ClpYQ targets SulA, RcsA, RpoH, and TraJ molecules. In this study, ClpY was analyzed to identify the molecular determinants required for the binding of its natural protein substrates. Using yeast two-hybrid analysis, we showed that domain I of ClpY contains the residues responsible for recognition of its natural substrates, while domain C is necessary to engage ClpQ. Moreover, the specific residues that lie in the amino acid (aa) 137 to 150 (loop 1) and aa 175 to 209 (loop 2) double loops in domain I of ClpY were shown to be necessary for natural substrate interaction. Additionally, the two-hybrid system, together with random PCR mutagenesis, allowed the isolation of ClpY mutants that displayed a range of binding activities with SulA, including a mutant with no SulA binding trait. Subsequently, via methyl methanesulfonate tests and cpsB::lacZ assays with, e.g., SulA and RcsA as targets, we concluded that aa 175 to 209 of loop 2 are involved in the tethering of natural substrates, and it is likely that both loops, aa 137 to 150 and aa 175 to 209, of ClpY domain I may assist in the delivery of substrates into the inner core for ultimate degradation by ClpQ.
... Still, the resolution in this region did not allow us to accurately distinguish ADP-AlFx from ADP in the occupied pockets. 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.
... ClpYQ also uses only six active sites to support its full catalytic activity, whereas the ClpQ homohexamer has 12 active sites [26]. To ensure high-efficiency processive degradation, the whole process of protein degradation is always accompanied by ATP binding and hydrolysis and the maintenance of an activated ClpYQ complex [27]. ...
Background
Protein degradation systems play crucial roles in all the kingdoms of life. Their natural function is to eliminate proteins that are improperly synthesized, damaged, aggregated, or short-lived, ensuring the timely and accurate regulation of the response to abrupt environmental changes. Thus, proteolysis plays an important role in protein homeostasis, quality control, and the control of regulatory processes, such as adaptation and cell development. Except for the lysosome, ATPases Associated with various cellular Activities (AAA+) ATPase–protease complex is another major protein degradation system in the cell.Methods and ResultsThe AAA+ ATPase–protease complex is a giant energy-dependent protease complex found in almost all kinds of cells, including bacteria, archaea and eukarya. Based on sequence analysis of ClpQ (HslV) and 20S proteasome beta subunits, it was found that bacterial ClpQ possess multiple same highly conserved motifs with 20S proteasome beta subunits of archaea and eukaryote. In this review, we also discussed the structure and functional mechanism, protein degradation signals and pathogenic role of proteasome / Clp protease complex in prokaryotes.Conclusion
Bacterial protein degradation systems play important roles in stress tolerance, protein quality control, DNA protection, transcription and pathogenicity of bacteria. But our current knowledge of the bacterial protease system is incomplete, and further research into the Clp protease complex and associated protein degradation signals will extend our understanding of the metabolism, physiology, reproduction, and pathogenicity of bacteria.
... We determined the first high-resolution structure of the soluble domains of a mitochondrial IM AAA+ protease, providing a structural framework that explains decades of biochemical and genetic investigation of the activity and regulation of this class of unfoldase (Karata 2001, Yamada-Inagawa 2003. Furthermore, our asymmetric substrate-bound YME1 structure containing four ATP, one ADP-like, and one apo-like subunit is in agreement with previous biochemical data describing other unfoldases, including ClpX, PAN, and HslU, which showed a maximum of four ATP molecules and three coexisting functional subunit classes per hexamer (Hersch 2005, Horwitz 2007, Yakamavich 2008. Our model for ATP-driven substrate translocation incorporates and explains a number of previously identified mechanistic features from a wide variety of AAA+ ATPases, including direct interactions of the aromatic pore-loop residues with highly diverse substrates (Schlieker 2004, Siddiqui 2004, Hinnerwisch 2005, Park 2005, Nyquist and Martin 2014. ...
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.
... ITC experiments using ClpB-K212A, which is deficient in nucleotide binding in AAA-1, allowed us to determine a binding stoichiometry of 3.7 ± 0.3 ADP in AAA-2 of the mutant hexamer ( Figure 6-figure supplement 3). The same ADP binding stoichiometry was also found for the repressed and hyperactive variants ( for the AAA+ proteins MCM, ClpX and HslU (Moreau et al., 2007;Yakamavich et al., 2008). The AAA-1 ring is more asymmetric than AAA-2 and it is not easily interpretable by fitting crystallographic dimer models. ...
... 13,21,22 HslUV is a slow ATPase (~70 min −1 AAA+ 6 −1 ) but can unfold stable substrates as well as ClpAP or ClpXP depending on the location of the degradation marker. 22,25 Finally, FtsH, which is only membrane integrated and growth essential among E. coli AAA+ proteases, is a slow ATPase (50-100 min −1 AAA+ 6 −1 ) and cannot actively unfold globular proteins. 22,26 The total ATP costs required to degrade a single copy of substrate are widely varied (0.2-6.6 ATP/ residue) depending on the stability of substrate and the type of AAA+ protease. ...
ATPases associated with diverse cellular activities (AAA+) proteases utilize ATP hydrolysis to actively unfold native or misfolded proteins and translocate them into a protease chamber for degradation. This basic mechanism yields diverse cellular consequences, including the removal of misfolded proteins, control of regulatory circuits, and remodeling of protein conformation. Among various bacterial AAA+ proteases, FtsH is only membrane‐integrated and plays a key role in membrane protein quality control. Previously, we have shown that FtsH has substantial unfoldase activity for degrading membrane proteins overcoming a dual energetic burden of substrate unfolding and membrane dislocation. Here, we asked how efficiently FtsH utilizes ATP hydrolysis to degrade membrane proteins. To answer this question, we measured degradation rates of the model membrane substrate GlpG at various ATP hydrolysis rates in the lipid bilayers. We find that the dependence of degradation rates on ATP hydrolysis rates is highly nonlinear: (i) FtsH cannot degrade GlpG until it reaches a threshold ATP hydrolysis rate; (ii) after exceeding the threshold, the degradation rates steeply increase and saturate at the ATP hydrolysis rates far below the maxima. During the steep increase, FtsH efficiently utilizes ATP hydrolysis for degradation, consuming only 40–60% of the total ATP cost measured at the maximal ATP hydrolysis rates. This behavior does not fundamentally change against water‐soluble substrates as well as upon addition of the macromolecular crowding agent Ficoll 70. The Hill analysis shows that the nonlinearity stems from coupling of three to five ATP hydrolysis events to degradation, which represents unique cooperativity compared to other AAA+ proteases including ClpXP, HslUV, Lon, and proteasomes.
... HslU forms a hexameric ring that binds in the presence of ATP to one or both ends of the protease, resulting into the formation of the HslVU ATP-dependent protease. Substrate recognition is most likely performed by the HslU regulator that then unfolds and translocates the captured protein into the internal proteolytic chamber of HslV, by an ATP-dependent mechanism [6,[8][9][10]. ...
HslVU is an ATP-dependent proteolytic complex present in certain bacteria and in the mitochondrion of some primordial eukaryotes, including deadly parasites such as Leishmania. It is formed by the dodecameric protease HslV and the hexameric ATPase HslU, which binds via the C-terminal end of its subunits to HslV and activates it by a yet unclear allosteric mechanism. We undertook the characterization of HslV from Leishmania major (LmHslV), a trypanosomatid that expresses two isoforms for HslU, LmHslU1 and LmHslU2. Using a novel and sensitive peptide substrate, we found that LmHslV can be activated by peptides derived from the C-termini of both LmHslU1 and LmHslU2. Truncations, Ala- and D-scans of the C-terminal dodecapeptide of LmHslU2 (LmC12-U2) showed that five out of the six C-terminal residues of LmHslU2 are essential for binding to and activating HslV. Peptide cyclisation with a lactam bridge allowed shortening of the peptide without loss of potency. Finally, we found that dodecapeptides derived from HslU of other parasites and bacteria are able to activate LmHslV with similar or even higher efficiency. Importantly, using electron microscopy approaches, we observed that the activation of LmHslV was accompanied by a large conformational remodeling, which represents a yet unidentified layer of control of HslV activation.
... The ClpQ dodecamer has 12 potential active sites, maximally six of which are sufficient for the degradation of the substrates (Lee et al., 2009). In addition, the asymmetric hydrolysis of three or four ATPs in ClpY hexamer are required for the binding/ unfolding/translocation and degradation of substrates by ClpQ (Yakamavich et al., 2008). The pore I site (90∼93 aa; GYVG) and the pore II site (265∼269 aa;GESSG) in the N domain of ClpY are in the center of its hexameric circle (Wang et al., 2001aZolkiewski, 2006). ...
In Escherichia coli, ClpYQ (HslUV) is a two-component ATP-dependent protease, in which ClpQ is the peptidase subunit and ClpY is the ATPase and unfoldase. ClpY functions to recognize protein substrates, and denature and translocate the unfolded polypeptides into the proteolytic site of ClpQ for degradation. However, it is not clear how the natural substrates are recognized by the ClpYQ protease and the mechanism by which the substrates are selected, unfolded and translocated by ClpY into the interior site of ClpQ hexamers. Both Lon and ClpYQ proteases can degrade SulA, a cell division inhibitor, in bacterial cells. In this study, using yeast two-hybrid and in vivo degradation analyses, we first demonstrated that the C-terminal internal hydrophobic region (139th∼149th aa) of SulA is necessary for binding and degradation by ClpYQ. A conserved region, GFIMRP, between 142th and 147th residues of SulA, were identified among various Gram-negative bacteria. By using MBP-SulA(F143Y) (phenylalanine substituted with tyrosine) as a substrate, our results showed that this conserved residue of SulA is necessary for recognition and degradation by ClpYQ. Supporting these data, MBP-SulA(F143Y), MBP-SulA(F143N) (phenylalanine substituted with asparagine) led to a longer half-life with ClpYQ protease in vivo. In contrast, MBP-SulA(F143D) and MBP-SulA(F143S) both have shorter half-lives. Therefore, in the E. coli ClpYQ protease complex, ClpY recognizes the C-terminal region of SulA, and F143 of SulA plays an important role for the recognition and degradation by ClpYQ protease.
... Many AAA+ proteases exhibit moderate positive cooperativity in ATP hydrolysis with Hill coefficients (n H,ATP ) of 1.4-2.0 [24][25][26][27] . In contrast, FtsH lacked cooperativity (n H,ATP = 0.9-1.0) in all tested lipid environments, implying, either negligible coupling among the AAA+ subunits or a 1:1 binding stoichiometry between ATP and FtsH hexamer. ...
ATP-dependent protein degradation mediated by AAA+ proteases is one of the major cellular pathways for protein quality control and regulation of functional networks. While a majority of studies of protein degradation have focused on water-soluble proteins, it is not well understood how membrane proteins with abnormal conformation are selectively degraded. The knowledge gap stems from the lack of an in vitro system in which detailed molecular mechanisms can be studied as well as difficulties in studying membrane protein folding in lipid bilayers. To quantitatively define the folding-degradation relationship of membrane proteins, we reconstituted the degradation using the conserved membrane-integrated AAA+ protease FtsH as a model degradation machine and the stable helical-bundle membrane protein GlpG as a model substrate in the lipid bilayer environment. We demonstrate that FtsH possesses a substantial ability to actively unfold GlpG, and the degradation significantly depends on the stability and hydrophobicity near the degradation marker. We find that FtsH hydrolyzes 380‒550 ATP molecules to degrade one copy of GlpG. Remarkably, FtsH overcomes the dual-energetic burden of substrate unfolding and membrane dislocation with the ATP cost comparable to that for water-soluble substrates by robust ClpAP/XP proteases. The physical principles elucidated in this study provide general insights into membrane protein degradation mediated by ATP-dependent proteolytic systems.
... Our high-resolution structure of the soluble do- mains of a mitochondrial IM AAA+ protease provides a structural framework that explains decades of biochemical and genetic investiga- tion of the activity and regulation of this class of unfoldase (36,52). Furthermore, our asym- metric substrate-bound YME1 structure contain- ing four ATP, one ADP-like, and one apo-like subunits is in agreement with previous bio- chemical data describing other unfoldases, in- cluding ClpX, PAN, and HslU, which showed a maximum of four ATP molecules and three co- existing functional subunit classes per hexamer (40,53,54). Our model for ATP-driven substrate translocation incorporates and explains a number of previously identified mechanistic features from a wide variety of AAA+ ATPases, including direct interactions of the aromatic pore loop residues with highly diverse substrates (29,30,34,55,56). ...
Feeding a protease step by step
Proteins that degrade damaged or misfolded mitochondrial proteins are essential for mitochondrial function. A key player is the hexameric protease YME1, in which each subunit is anchored in the inner mitochondrial membrane by a helix and has an adenosine triphosphatase (ATPase) domain and a protease domain in the intermembrane space. Puchades et al. report a high-resolution structure that shows that the ATPase domains form an asymmetric spiral staircase that stacks above a planar protease ring. Conserved tyrosine residues in the central pore of the spiral staircase interact with a substrate peptide. The ATP hydrolysis cycle is sequential and coordinated with changes in the position of the tyrosine residues that result in stepwise translocation of the substrate into the protease chamber.
Science , this issue p. eaao0464
... Still, the resolution in this region did not allow us to accurately distinguish ADP-AlFx from ADP in the occupied pockets. 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]. ...
... Still, the resolution in this region did not allow us to accurately distinguish ADP-AlFx from ADP in the occupied pockets. 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.Cell Research advance online publication 20 January 2017; doi:10.1038/cr.2017.12.
... Three main mechanisms have been proposed for coupling nucleotide turnover with substrate translocation in hexameric NTPases, involving either stochastic, concerted or rotary firing 15 41,42 . The concerted model envisions six NTPase subunits that simultaneously bind, hydrolyse or release the nucleotide. ...
The Cdc45-MCM-GINS (CMG) helicase unwinds DNA during the elongation step of eukaryotic genome duplication and this process depends on the MCM ATPase function. Whether CMG translocation occurs on single- or double-stranded DNA and how ATP hydrolysis drives DNA unwinding remain open questions. Here we use cryo-electron microscopy to describe two subnanometre resolution structures of the CMG helicase trapped on a DNA fork. In the predominant state, the ring-shaped C-terminal ATPase of MCM is compact and contacts single-stranded DNA, via a set of pre-sensor 1 hairpins that spiral around the translocation substrate. In the second state, the ATPase module is relaxed and apparently substrate free, while DNA intimately contacts the downstream amino-terminal tier of the MCM motor ring. These results, supported by single-molecule FRET measurements, lead us to suggest a replication fork unwinding mechanism whereby the N-terminal and AAA+ tiers of the MCM work in concert to translocate on single-stranded DNA.
... As a model for intra-ring communication, Cordova et al. [35] proposed that stochastic firing of one ClpX subunit triggers a coordinated chain of ATP hydrolysis or release events in the remaining subunits to generate additional power strokes. The total number of such events, which may proceed sequentially or stochastically through interaction with neighboring subunits, is limited by the asymmetric ring loading with up to 4 nucleotides under saturating conditions [63,64]. In our simulations, the upper bound of the length of translocation steps, Δ S 3 l, is consistent with the description of the ClpY allosteric cycle to comprise six two-subunit moves. ...
Clp ATPases are powerful ring shaped nanomachines which participate in the degradation pathway of the protein quality control system, coupling the energy from ATP hydrolysis to threading substrate proteins (SP) through their narrow central pore. Repetitive cycles of sequential intra-ring ATP hydrolysis events induce axial excursions of diaphragm-forming central pore loops that effect the application of mechanical forces onto SPs to promote unfolding and translocation. We perform Langevin dynamics simulations of a coarse-grained model of the ClpY ATPase-SP system to elucidate the molecular details of unfolding and translocation of an α/β model protein. We contrast this mechanism with our previous studies which used an all-α SP. We find conserved aspects of unfolding and translocation mechanisms by allosteric ClpY, including unfolding initiated at the tagged C-terminus and translocation via a power stroke mechanism. Topology-specific aspects include the time scales, the rate limiting steps in the degradation pathway, the effect of force directionality, and the translocase efficacy. Mechanisms of ClpY-assisted unfolding and translocation are distinct from those resulting from non-allosteric mechanical pulling. Bulk unfolding simulations, which mimic Atomic Force Microscopy-type pulling, reveal multiple unfolding pathways initiated at the C-terminus, N-terminus, or simultaneously from both termini. In a non-allosteric ClpY ATPase pore, mechanical pulling with constant velocity yields larger effective forces for SP unfolding, while pulling with constant force results in simultaneous unfolding and translocation.
... Homohexameric ClpX is an asymmetric assembly as only four out of six nucleotide binding sites are occupied (Hersch et al., 2005;Glynn et al., 2009) (Figure 2A). The AAA+ proteins PAN and HslU also only bind four ATP molecules under saturating nucleotide concentrations, indicating that the existence of loaded and empty nucleotide binding sites is a common feature of this protein family (Yakamavich et al., 2008;Smith et al., 2011). ...
Unicellular and sessile organisms are particularly exposed to environmental stress such as heat shock causing accumulation and aggregation of misfolded protein species. To counteract protein aggregation, bacteria, fungi, and plants encode a bi-chaperone system composed of ATP-dependent Hsp70 and hexameric Hsp100 (ClpB/Hsp104) chaperones, which rescue aggregated proteins and provide thermotolerance to cells. The partners act in a hierarchic manner with Hsp70 chaperones coating first the surface of protein aggregates and next recruiting Hsp100 through direct physical interaction. Hsp100 proteins bind to the ATPase domain of Hsp70 via their unique M-domain. This extra domain functions as a molecular toggle allosterically controlling ATPase and threading activities of Hsp100. Interactions between neighboring M-domains and the ATPase ring keep Hsp100 in a repressed state exhibiting low ATP turnover. Breakage of intermolecular M-domain interactions and dissociation of M-domains from the ATPase ring relieves repression and allows for Hsp70 interaction. Hsp70 binding in turn stabilizes Hsp100 in the activated state and primes Hsp100 ATPase domains for high activity upon substrate interaction. Hsp70 thereby couples Hsp100 substrate binding and motor activation. Hsp100 activation presumably relies on increased subunit cooperation leading to high ATP turnover and threading power. This Hsp70-mediated activity control of Hsp100 is crucial for cell viability as permanently activated Hsp100 variants are toxic. Hsp100 activation requires simultaneous binding of multiple Hsp70 partners, restricting high Hsp100 activity to the surface of protein aggregates and ensuring Hsp100 substrate specificity.
... Strikingly, however, some crystal structures showed symmetric binding of six nucleotides to each HslU hexamer, whereas others show asymmetric binding to only three or four subunits of the hexamer. Solution studies revealed that a maximum of three or four nucleotides bind the hexamer (Yakamavich et al., 2008), suggesting that the asymmetric structures are more physiologically relevant. Such asymmetry would also be consistent with structural and biochemical results for ClpX (Hersch et al., 2005;Glynn et al., 2009), whose AAA+ module shares ~50% sequence homology with that of HslU. ...
Protein degradation is a central component of all biological processes. The proteome must constantly change in response to environmental stimuli. As a result, protein synthesis and regulated proteolysis are vital to cell survival. In Escherichia coli, the protease HslUV is one of five ATP-dependent proteases that shoulder the major burden of intracellular protein degradation. Although ample data exist for describing the structural architecture of the HslUV protease, very little is known about its mechanisms of function. I took a two-pronged approach to understand the functional principles that govern this protease. My first goal was to understand the rules of substrate recognition. To do so, I performed a variety of experiments on two model proteins: Arc repressor and [chi]cIN repressor. I found that both substrates had common requirements for HslUV degradation, suggesting a conserved mode of recognition by this protease. Mutagenesis of either substrate terminus affected binding and degradation kinetics. While degron mutations generally affect only enzyme-substrate binding properties in other bacterial proteases, the changes described here often affected the maximal rate of HslUV degradation. Moreover, specific occlusion of either the N-terminus or C-terminus of these substrates resulted in a substantial defect in degradation. A synergistic inhibitory effect was observed for the simultaneous masking of both termini. These results suggested a mechanism of tethering prior to engagement for degradation of HslUV substrates. I then sought to define the regions of HslU that were important for recognition and found that two segments, the GYVG pore loop and the intermediate (I) domain, played crucial roles. Investigation of mutants altered at these sites supported a mechanism of tethering of the substrate C-terminus to the I domain and engagement of the substrate N-terminus in the pore. I showed that degradation of an Arc substrate proceeds processively from the N-terminus towards the Cterminus, lending further support to this idea. Interestingly, I also discovered that the I domain plays a very important role in ATP hydrolysis by HslU and coordinates substrate recognition and stimulation of ATP turnover. This trait appears to be unique for HslU and is not a property of the accessory domains of other AAA+ protein unfolding machines.
... The F441Y mutant has partially reduced ATPase activity ATPase and protease activities are tightly connected in twocomponent ATP-dependent proteases. As already reported, the HslVU complex shows approximately 3-fold higher ATPase activity than HslU alone [40]. Moreover, the inactive deletion mutant of the catalytic threonine residue of HslV is known to increase ATPase activity even further than active wild-type HslV [23]. ...
The ATP-dependent HslVU complexes are found in all three biological kingdoms. A single HslV protease exists in each species of prokaryotes, archaea, and eukaryotes, but two HslUs (HslU1 and HslU2) are present in the mitochondria of eukaryotes. Previously, a tyrosine residue at the C-terminal tail of HslU2 has been identified as a key determinant of HslV activation in Trypanosoma brucei and a phenylalanine at the equivalent position to E. coli HslU is found in T. brucei HslU1. Unexpectedly, we found that an F441Y mutation in HslU enhanced the peptidase and caseinolytic activity of HslV in E. coli but it showed partially reduced ATPase and SulA degradation activity. Previously, only the C-terminal tail of HslU has been the focus of HslV activation studies. However, the Pro315 residue interacting with Phe441 in free HslU has also been found to be critical for HslV activation. Hence, our current biochemical analyses explore the importance of the loop region just before Pro315 for HslVU complex functionality. The proline and phenylalanine pair in prokaryotic HslU was replaced with the threonine and tyrosine pair from the functional eukaryotic HslU2. Sequence comparisons between multiple HslUs from three different biological kingdoms in combination with biochemical analysis of E. coli mutants have uncovered important new insights into the molecular evolutionary pathway of HslU.
... Crystal structures of ClpX and other related bacterial protease ATPase caps show that the rings can be asymmetric, so that some subunits are in conformations that are not bound to nucleotides 130,142 . The structures are consistent with biochemical studies of many AAA+ proteases, which show that only a maximum of four nucleotides are bound within the ring at one time 98,[144][145][146][147] . It is therefore unlikely that the conformational changes that drive the mechanical unravelling of substrates happen in parallel in all subunits 142,147 . ...
The ubiquitin proteasome system (UPS) is the main ATP-dependent protein degradation pathway in the cytosol and nucleus of eukaryotic cells. At its centre is the 26S proteasome, which degrades regulatory proteins and misfolded or damaged proteins. In a major breakthrough, several groups have determined high-resolution structures of the entire 26S proteasome particle in different nucleotide conditions and with and without substrate using cryo-electron microscopy combined with other techniques. These structures provide some surprising insights into the functional mechanism of the proteasome and will give invaluable guidance for genetic and biochemical studies of this key regulatory system.
... 14,18,19 Experimental studies of ClpX constructs with varied assemblies of active and inactive subunits of ClpX have suggested that ATP hydrolysis proceeds through a random sequential mechanism. 19 In addition, under saturating conditions, ClpX (as does ClpY) binds only 3-4 nucleotides per hexamer 18,20 and crystal structures have revealed asymmetric nucleotide binding. 8,9,16,21 In accord with experimental results, computer simulations of ClpY-mediated unfolding and translocation of a four-helix bundle substrate exclude the functional viability of a concerted allosteric mechanism. ...
Essential protein quality control includes mechanisms of substrate protein (SP) unfolding and translocation performed by powerful ring-shaped AAA+ (ATPases associated with various cellular activities) nanomachines. These SP remodeling actions are effected by mechanical forces imparted by AAA+ loops that protrude into the central channel. Sequential intra-ring allosteric motions, which underlie repetitive SP-loop interactions, have been proposed to comprise clockwise (CW), counterclockwise (CCW), or random (R) conformational transitions of individual AAA+ subunits. To probe the effect of these allosteric mechanisms on unfoldase and translocase functions, we perform Langevin dynamics simulations of a coarse-grained model of an all-alpha SP processed by the single-ring ClpY ATPase or by the double-ring p97 ATPase. We find that, in all three allosteric mechanisms, the SP undergoes conformational transitions along a common set of pathways, which reveals that the active work provided by the ClpY machine involves single loop-SP interactions. Nevertheless, the rates and yields of SP unfolding and translocation are controlled by mechanism-dependent loop-SP binding events, as illustrated by faster timescales of SP processing in CW allostery compared with CCW and R allostery. The distinct efficacy of allosteric mechanisms is due to the asymmetric collaboration of adjacent subunits, which involves CW-biased structural motions of AAA+ loops and results in CW-compatible torque applied onto the SP. Additional simulations of mutant ClpY rings, which render a subset of subunits catalytically-defective or reduce their SP binding affinity, reveal that subunit-based conformational transitions play the major role in SP remodeling. Based on these results we predict that the minimally functional AAA+ ring includes three active subunits, only two of which are adjacent.
... 1d and 6c-iv, see the discussion below). Such ranges of nucleotide occupancy have been observed in related structures such as AAA + unfoldases (ClpX, HslU, and FtsH [52][53][54][55][56]). Joly et al. have shown that the ATPase activity of PspF 1-275 was significantly reduced under ATP saturating conditions, suggesting that inhibition may occur as a result of increasing nucleotide binding site occupancy [27]. ...
... In previous studies of nucleotide binding to ClpX hexamers, evidence was presented for tight, weak, and empty sites with an approximate ratio of four binding to two nonbinding subunits (Hersch et al., 2005). Subsequent studies with the hexameric HslU and PAN AAA+ unfoldases revealed similar nucleotidebinding categories and ratios (Yakamavich et al., 2008;Smith et al., 2011). Our current studies support multiple classes of nucleotide-binding sites and suggest a basic ring pattern of [weak-empty-tight-weak-empty-tight] sites, with the proviso that empty sites may bind nucleotide transiently and very weakly. ...
ClpX, a AAA+ ring homohexamer, uses the energy of ATP binding and hydrolysis to power conformational changes that unfold and translocate target proteins into the ClpP peptidase for degradation. In multiple crystal structures, some ClpX subunits adopt nucleotide-loadable conformations, others adopt unloadable conformations, and each conformational class exhibits substantial variability. Using mutagenesis of individual subunits in covalently tethered hexamers together with fluorescence methods to assay the conformations and nucleotide-binding properties of these subunits, we demonstrate that dynamic interconversion between loadable and unloadable conformations is required to couple ATP hydrolysis by ClpX to mechanical work. ATP binding to different classes of subunits initially drives staged allosteric changes, which set the conformation of the ring to allow hydrolysis and linked mechanical steps. Subunit switching between loadable and unloadable conformations subsequently isomerizes or resets the configuration of the nucleotide-loaded ring and is required for mechanical function.
... By contrast, TonLonB exhibited a binding of 3 ATPcS per hexamer with a higher affinity (2 mM) [19]. In fact, a maximum occupancy of four nucleotides to the six available ATP/ADP binding sites in the hexameric ring of AAA + modules were observed from solution studies242526, as well as in some crystal structures [19,27,28]. As proposed previously, such unequal binding of nucleotides may be necessary for the ATPase cycle to drive conformational changes in the hexameric AAA + ring to generate mechanical power for substrate unfolding and trans- location [1]. ...
Lon proteases are a family of ATP-dependent proteases involved in protein quality control, with a unique proteolytic domain and an AAA(+) (ATPases associated with various cellular activities) module accommodated within a single polypeptide chain. They were classified into two types as either the ubiquitous soluble LonA or membrane-inserted archaeal LonB. In addition to the energy-dependent forms, a number of medically and ecologically important groups of bacteria encode a third type of Lon-like proteins in which the conserved proteolytic domain is fused to a large N-terminal fragment lacking canonical AAA(+) motifs. Here we showed that these Lon-like proteases formed a clade distinct from LonA and LonB. Characterization of one such Lon-like protease from Meiothermus taiwanensis indicated that it formed a hexameric assembly with a hollow chamber similar to LonA/B. The enzyme was devoid of ATPase activity but retained an ability to bind symmetrically six nucleotides per hexamer; accordingly, structure-based alignment suggested possible existence of a non-functional AAA-like domain. The enzyme degraded unstructured or unfolded protein and peptide substrates, but not well-folded proteins, in ATP-independent manner. These results highlight a new type of Lon proteases that may be involved in breakdown of excessive damage or unfolded proteins during stress conditions without consumption of energy.
... The visualization of fully symmetrized-particles in which all subunits occupy similar conformations and undergo uniform, en bloc transitions between nucleotide binding events has at times been taken as evidence for the concerted model [20,21]. Other analyses, particularly for some protein unfoldases, have shown that individual NTPase subunits can in certain circumstances bind nucleotide and function independently of each other, suggesting a lack of enforced firing order [22,23]. ...
Ring-shaped, oligomeric translocases are multisubunit enzymes that couple the hydrolysis of Nucleoside TriPhosphates (NTPs) to directed movement along extended biopolymer substrates. These motors help unwind nucleic acid duplexes, unfold protein chains, and shepherd nucleic acids between cellular and/or viral compartments. Substrates are translocated through a central pore formed by a circular array of catalytic subunits. Cycles of nucleotide binding, hydrolysis, and product release help reposition translocation loops in the pore to direct movement. How NTP turnover allosterically induces these conformational changes, and the extent of mechanistic divergence between motor families, remain outstanding problems. This review examines the current models for ring-translocase function and highlights the fundamental gaps remaining in our understanding of these molecular machines.
... Thus, four subunits might build the functional cooperative unit that binds and process the substrate, if one of the protein rings contains active nucleotide binding sites. A similar number of active subunits has been reported for another two hexameric unfoldases, ClpX [29] and HslU [30]. Although they both could bind six ATPs in solution, they appear to bind a maximum of four nucleotide molecules. ...
ClpB is a member of the AAA+ superfamily that forms a ring-shaped homohexamer. Each protomer contains two nucleotide binding domains arranged in two rings that hydrolyze ATP. We extend here previous studies on ClpB nucleotide utilization requirements by using an experimental approach that maximizes random incorporation of different subunits into the protein hexamer. Incorporation of one subunit unable to bind or hydrolyze ATP knocks down the chaperone activity, while the wt hexamer can accommodate two mutant subunits that hydrolyze ATP in only one protein ring. Four subunits seem to build the functional cooperative unit, provided that one of the protein rings contains active nucleotide binding sites.
2020 The Author(s) At low temperatures, protein degradation by the AAA+ HslUV protease is very slow. New crystal structures reveal that residues in the intermediate domain of the HslU6 unfoldase can plug its axial channel, blocking productive substrate binding and subsequent unfolding, translocation, and degradation by the HslV12 peptidase. Biochemical experiments with wild-type and mutant enzymes support a model in which heat-induced melting of this autoinhibitory plug activates HslUV proteolysis.
A continuous FeMo cofactor supply for nitrogenase maturation is ensured in Azotobacter vinelandii by developing a cage-like molybdenum storage protein (MoSto) capable to store ca. 120 molybdate molecules (
MoO
4
2
-
) as discrete polyoxometalate (POM) clusters. To gain mechanistic insight into this process, MoSto was characterized by Mo and ATP/ADP content, structural, and kinetic analysis. We defined three functionally relevant states specified by the presence of both ATP/ADP and POM clusters (MoStofunct ), of only ATP/ADP (MoStobasal ) and of neither ATP/ADP nor POM clusters (MoStozero ), respectively. POM clusters are only produced when ATP is hydrolyzed to ADP and phosphate. Vmax was ca. 13 μmolphosphate ·min-1 ·mg-1 and Km for molybdate and ATP/Mg2+ in the low micromolar range. ATP hydrolysis presumably proceeds at subunit α, inferred from a highly occupied α-ATP/Mg2+ and a weaker occupied β-ATP/no Mg2+ -binding site found in the MoStofunct structure. Several findings indicate that POM cluster storage is separated into a rapid ATP hydrolysis-dependent molybdate transport across the protein cage wall and a slow molybdate assembly induced by combined auto-catalytic and protein-driven processes. The cage interior, the location of the POM cluster depot, is locked in all three states and thus not rapidly accessible for molybdate from the outside. Based on Vmax , the entire Mo storage process should be completed in less than 10 s but requires, according to the molybdate content analysis, ca. 15 min. Long-time incubation of MoStobasal with nonphysiological high molybdate amounts implicates an equilibrium in and outside the cage and POM cluster self-formation without ATP hydrolysis. DATABASES: The crystal structures MoSto in the MoSto-F6, MoSto-F7, MoStobasal , MoStozero , and MoSto-F1vitro states were deposited to PDB under the accession numbers PDB 6GU5, 6GUJ, 6GWB, 6GWV, and 6GX4.
As the endpoint for the ubiquitin-proteasome system, the 26S proteasome is the principal proteolytic machine responsible for regulated protein degradation in eukaryotic cells. The proteasome's cellular functions range from general protein homeostasis and stress response to the control of vital processes such as cell division and signal transduction. To reliably process all the proteins presented to it in the complex cellular environment, the proteasome must combine high promiscuity with exceptional substrate selectivity. Recent structural and biochemical studies have shed new light on the many steps involved in proteasomal substrate processing, including recognition, deubiquitination, and ATP-driven translocation and unfolding. In addition, these studies revealed a complex conformational landscape that ensures proper substrate selection before the proteasome commits to processive degradation. These advances in our understanding of the proteasome's intricate machinery set the stage for future studies on how the proteasome functions as a major regulator of the eukaryotic proteome. Expected final online publication date for the Annual Review of Biochemistry Volume 87 is June 20, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
The hexameric AAA ATPase Vps4 drives membrane fission by remodeling and disassembling ESCRT-III filaments. Building upon our earlier 4.3 Å resolution cryo-EM structure (Monroe, Han et al. 2017), we now report a 3.2 Å structure of Vps4 bound to an ESCRT-III peptide substrate. The new structure reveals that the peptide approximates a b-strand conformation whose helical symmetry matches that of the five Vps4 subunits it contacts directly. Adjacent Vps4 subunits make equivalent interactions with successive substrate dipeptides through two distinct classes of side chain binding pockets formed primarily by Vps4 pore loop 1. These pockets accommodate a wide range of residues, while main chain hydrogen bonds may help dictate substrate-binding orientation. The structure supports a 'conveyor belt' model of translocation in which ATP binding allows a Vps4 subunit to join the growing end of the helix and engage the substrate, while hydrolysis and release promotes helix disassembly and substrate release at the lagging end.
The HslUV proteolytic machine consists of HslV, a double-ring self-compartmentalized peptidase, and one or two AAA+ HslU ring hexamers that hydrolyze ATP to power the unfolding of protein substrates and their translocation into the proteolytic chamber of HslV. Here, we use genetic-tethering and disulfide-bonding strategies to construct HslU pseudohexamers containing mixtures of ATPase active and inactive subunits at defined positions in the hexameric ring. Genetic tethering impairs HslV binding and degradation, even for pseudohexamers with six active subunits, but disulfide-linked pseudohexamers do not have these defects, indicating that the peptide tether interferes with HslV interactions. Importantly, pseudohexamers containing different patterns of hydrolytically active and inactive subunits retain the ability to unfold protein substrates and/or collaborate with HslV in their degradation, supporting a model in which ATP hydrolysis and linked mechanical function in the HslU ring operate by a probabilistic mechanism.
EC number
3.4.25.2
Recommended name
HslU-HslV peptidase
Synonyms
AAA+ HslUV protease <4> [36]
ATP-dependent protease <4> [49]
ATP-dependent protease hslV
ClpQ
ClpYQ <1,2,4> [32,36,43,47]
ClpYQ complex <4> [49]
ClpYQ protease <4> [35,47]
CodW <3> [41]
CodW-CodX <3> [31]
HslU ATPase <4> [33]
HslU chaperone <4> [37]
HslUV <2,4> [33,34,36,43,45,47]
HslUV complex <4> [37,49]
HslUV protease <4> [35,45,47]
HslUV protease-chaperone complex <2> [34]
HslV peptidase <4> [33,37]
HslV protease <2,4,6> [37,40,48]
HslV-HslU <4> [31]
HslVU ATP-dependent protease <4> [48]
HslVU protease <4> [39]
PfHslUV <5> [44]
T01.006 (Merops-ID)
heat shock protein hslV
hslVU <4> (<4> ATP-dependent protease consisting of two heat shock proteins, the HslU ATPase and HslV peptidase [38,46]) [38,42,46]
CAS registry number
178303-43-0
AAA+ proteases are universal barrel-like and ATP-fueled machines preventing the accumulation of aberrant proteins and regulating the proteome according to the cellular demand. They are characterized by two separate operating units, the ATPase and peptidase domains. ATP-dependent unfolding and translocation of a substrate into the proteolytic chamber is followed by ATP-independent degradation. This review addresses the structure and function of bacterial AAA+ proteases with a focus on the ATP-driven mechanisms and the coordinated movements in the complex mainly based on the knowledge of ClpXP. We conclude by discussing strategies how novel protease substrates can be trapped by mutated AAA+ protease variants. This article is protected by copyright. All rights reserved.
The Escherichia coli ATP dependent proteases play several important roles in protein quality control and they use the energy from ATP hydrolysis to recognize and degrade the abnormal proteins. ClpYQ protease, one of the ATP dependent proteases, is a two component complex. ClpY recognizes, unfolds and translocates the natural substrates into the catalytic core site of ClpQ for the degradation. The cell division inhibitor, SulA, induced during an SOS response, prevented a cell division. However, both Lon and ClpYQ proteases were capable of degrading it and that could restore the normal cell growth. In this study, through the in vitro assays, the 141 to 150 residues of SulA were shown necessary for interaction with ClpY. There is a conserved region between 142 to 147 residues, G142F143I, 44M145R146P147, in SulA, from Gram-negative bacteria. SulA∗ I144N point mutant, with a substitution of Asn for Ile, was further used for detection of its degradation with ClpYQ protease. MBP(maltose-binding-protein)-SulA∗I144N has a longer half-life than that of MBP-SulA in the presence of ClpQ and ClpY. Moreover, SulA∗I144N mutant has no inhibitory activity on cell division; this mutant has an effect on the function of SulA. Our results suggested that the conserved hydrophobic region in SulA is necessary for its inhibitory activity as well as for an association with ClpY for its degradation by ClpYQ protease.
The concept of molecular machines in biology has transformed the medical field in a profound way. Many essential processes that occur in the cell, including transcription, translation, protein folding and protein degradation, are all carried out by molecular machines. This volume focuses on important molecular machines whose architecture is known and whose functional principles have been established by tools of biophysical imaging (X-ray crystallography and cryo-electron microscopy) and fluorescence probing (single-molecule FRET). This edited volume includes contributions from prominent scientists and researchers who understand and have explored the structure and functions of these machines. This book is essential for students and professionals in the medical field who want to learn more about molecular machines.
As the first ATP-dependent protease to be identified, Lon holds a special place in the history of cellular biology. In fact, the concept of ATP-dependent protein degradation was established through the findings that led to the discovery of Lon. Therefore, this chapter begins with a historical perspective, describing the milestones that led to the discovery of Lon and ATP-dependent proteolysis, starting from the early findings in the 1960s until the demonstration of Lon's ATP-dependent proteolytic activity in vitro, in 1981. Most of our knowledge on Lon derives from studies of the Escherichia coli Lon ortholog, and, therefore, most of this chapter relates to this particular enzyme. Nonetheless, Lon is not only found in most bacterial species, it is also found in Archaea and in the mitochondrion and chloroplast of eukaryotic cells. Therefore many of the conclusions gained from studies on the E. coli enzyme are relevant to Lon proteases in other organisms. Lon, more than any other bacterial or organellar protease, is associated with the degradation of misfolded proteins and protein quality control. In addition, Lon also degrades many regulatory proteins that are natively folded, thus it also plays a prominent role in regulation of physiological processes. Throughout the years, many Lon substrates have been identified, confirming its role in the regulation of diverse cellular processes, including cell division, DNA replication, differentiation, and adaptation to stress conditions. Some examples of these functions are described and discussed here, as is the role of Lon in the degradation of misfolded proteins and in protein quality control. Finally, this chapter deals with the exquisite sensitivity of protein degradation inside a cell. How can a protease distinguish so many substrates from cellular proteins that should not be degraded? Can the specificity of a protease be regulated according to the physiological needs of a cell? This chapter thus broadly discusses the substrate specificity of Lon and its allosteric regulation.
In Escherichia coli, ClpYQ (HslUV) is a two-component ATP-dependent protease composed of ClpY (HslU), an ATPase with unfolding activity, and ClpQ (HslV), a peptidase. In the ClpYQ proteolytic complex, the hexameric rings of ClpY (HslU) are responsible for protein recognition, unfolding, and translocation into the proteolytic inner chamber of the dodecameric ClpQ (HslV). Each of the three domains, N, I, and C, in ClpY has its own distinct activity. The double loops (amino acids [aa] 137 to 150 and 175 to 209) in domain I of ClpY are necessary for initial recognition/tethering of natural substrates such as SulA, a cell division inhibitor protein. The highly conserved sequence GYVG (aa 90 to 93) pore I site, along with the GESSG pore II site (aa 265 to 269), contribute to the central pore of ClpY in domain N. These two central loops of ClpY are in the center of its hexameric ring in which the energy of ATP hydrolysis allows substrate translocation and then degradation by ClpQ. However, no data have been obtained to determine the effect of the central loops on substrate binding or as part of the processivity of the ClpYQ complex. Thus, we probed the features of ClpY important for substrate engagement and protease processivity via random PCR or site-specific mutagenesis. In yeast two-hybrid analysis and pulldown assays, using isolated ClpY mutants and the pore I or pore II site of ClpY, each was examined for its influence on the adjoining structural regions of the substrates. The pore I site is essential for the translocation of the engaged substrates. Our in vivo study of the ClpY mutants also revealed that an ATP-binding site in domain N, separate from its role in polypeptide (ClpY) oligomerization, is required for complex formation with ClpQ. Additionally, we found that the tyrosine residue at position 408 in ClpY is critical for stabilization of hexamer formation between subunits. Therefore, our studies suggest that stepwise activities of the ClpYQ protease are necessary to facilitate the processive degradation of its natural substrates.
ClpB is a hexameric chaperone that solubilizes and reactivates protein aggregates in cooperation with the Hsp70/DnaK chaperone system. Each of the identical protein monomers contains two nucleotide binding domains (NBD), whose ATPase activity must be coupled to exert on the substrate the mechanical work required for its reactivation. However, how communication between these sites occurs is at present poorly understood. We have studied herein the affinity of each of the NBDs for nucleotides in WT ClpB and protein variants in which one or both sites are mutated to selectively impair nucleotide binding or hydrolysis. Our data show that the affinity of NBD2 for nucleotides (K(d) = 3-7 μm) is significantly higher than that of NBD1. Interestingly, the affinity of NBD1 depends on nucleotide binding to NBD2. Binding of ATP, but not ADP, to NBD2 increases the affinity of NBD1 (the K(d) decreases from ≈160-300 to 50-60 μm) for the corresponding nucleotide. Moreover, filling of the NBD2 ring with ATP allows the cooperative binding of this nucleotide and substrates to the NBD1 ring. Data also suggest that a minimum of four subunits cooperate to bind and reactivate two different aggregated protein substrates.
AAA+ family proteolytic machines (ClpXP, ClpAP, ClpCP, HslUV, Lon, FtsH, PAN/20S, and the 26S proteasome) perform protein quality control and are used in regulatory circuits in all cells. These machines contain a compartmental protease, with active sites sequestered in an interior chamber, and a hexameric ring of AAA+ ATPases. Substrate proteins are tethered to the ring, either directly or via adaptor proteins. An unstructured region of the substrate is engaged in the axial pore of the AAA+ ring, and cycles of ATP binding/hydrolysis drive conformational changes that create pulses of pulling that denature the substrate and translocate the unfolded polypeptide through the pore and into the degradation chamber. Here, we review our current understanding of the molecular mechanisms of substrate recognition, adaptor function, and ATP-fueled unfolding and translocation. The unfolding activities of these and related AAA+ machines can also be used to disassemble or remodel macromolecular complexes and to resolubilize aggregates.
Isothermal titration calorimetry (ITC) is a fast, accurate and label-free method for measuring the thermodynamics and binding affinities of molecular associations in solution. Because the method will measure any reaction that results in a heat change, it is applicable to many different fields of research from biomolecular science, to drug design and materials engineering, and can be used to measure binding events between essentially any type of biological or chemical ligand. ITC is the only method that can directly measure binding energetics including Gibbs free energy, enthalpy, entropy and heat capacity changes. Not only binding thermodynamics but also catalytic reactions, conformational rearrangements, changes in protonation and molecular dissociations can be readily quantified by performing only a small number of ITC experiments. In this review, we highlight some of the particularly interesting reports from 2008 employing ITC, with a particular focus on protein interactions with other proteins, nucleic acids, lipids and drugs. As is tradition in these reviews we have not attempted a comprehensive analysis of all 500 papers using ITC, but emphasize those reports that particularly captured our interest and that included more thorough discussions we consider exemplify the power of the technique and might serve to inspire other users.
The hexameric membrane-spanning ATP-dependent metalloprotease FtsH is universally conserved in eubacteria, mitochondria, and chloroplasts, where it fulfills key functions in quality control and signaling. As a member of the self-compartmentalizing ATPases associated with various cellular activities (AAA+ proteases), FtsH converts the chemical energy stored in ATP via conformational rearrangements into a mechanical force that is used for substrate unfolding and translocation into the proteolytic chamber. The crystal structure of the ADP state of Thermotoga maritima FtsH showed a hexameric assembly consisting of a 6-fold symmetric protease disk and a 2-fold symmetric AAA ring. The 2.6 A resolution structure of the cytosolic region of apo-FtsH presented here reveals a new arrangement where the ATPase ring shows perfect 6-fold symmetry with the crucial pore residues lining an open circular entrance. Triggered by this conformational change, a substrate-binding edge beta strand appears within the proteolytic domain. Comparison of the apo- and ADP-bound structure visualizes an inward movement of the aromatic pore residues and generates a model of substrate translocation by AAA+ proteases. Furthermore, we demonstrate that mutation of a conserved glycine in the linker region inactivates FtsH.
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.
Hexameric ring-shaped AAA+ molecular motors have a key function of active translocation of a macromolecular chain through the central pore. By performing multiscale molecular dynamics (MD) simulations, we revealed that HslU, a AAA+ motor in a bacterial homologue of eukaryotic proteasome, translocates its substrate polypeptide via paddling mechanism during ATP-driven cyclic conformational changes. First, fully atomistic MD simulations showed that the HslU pore grips the threaded signal peptide by the highly conserved Tyr-91 and Val-92 firmly in the closed form and loosely in the open form of the HslU. The grip depended on the substrate sequence. These features were fed into a coarse-grained MD, and conformational transitions of HslU upon ATP cycles were simulated. The simulations exhibited stochastic unidirectional translocation of a polypeptide. This unidirectional translocation is attributed to paddling motions of Tyr-91s between the open and the closed forms: downward motions of Tyr-91s with gripping the substrate and upward motions with slipping on it. The paddling motions were caused by the difference between the characteristic time scales of the pore-radius change and the up-down displacements of Tyr-91s. Computational experiments on mutations at the pore and the substrate were in accord with several experiments.
HslVU is a bacterial ATP-dependent protease distantly related to eukaryotic proteasomes consisting of hexameric HslU ATPase and dodecameric HslV protease. As a homolog of the 20 S proteasome beta-subunits, HslV also uses the N-terminal threonine as the active site residue. However, unlike the proteasome that has only 6 active sites among the 14 beta-subunits, HslV has 12 active sites that could potentially contribute to proteolytic activity. Here, by using a series of HslV dodecamers containing different numbers of active sites, we demonstrate that like the proteasome, HslV with only approximately 6 active sites is sufficient to support full catalytic activity. However, a further reduction of the number of active sites leads to a proportional decrease in activity. Using proteasome inhibitors, we also demonstrate that substrate-mediated stabilization of the HslV-HslU interaction remains unchanged until the number of the active sites is decreased to approximately 6 but is gradually compromised upon further reduction. These results with a mathematical model suggest HslVU utilizes no more than 6 active sites at any given time, presumably because of the action of HslU. These results also suggest that each ATP-bound HslU subunit activates one HslV subunit and that substrate bound to the HslV active site stimulates the HslU ATPase activity by stabilizing the HslV-HslU interaction. We propose this mechanism plays an important role in supporting complete degradation of substrates while preventing wasteful ATP hydrolysis in the resting state by controlling the interaction between HslV and HslU through the catalytic engagement of the proteolytic active sites.
Clp proteases are the most widespread energy-dependent proteases in bacteria. Their two-component architecture of protease core and ATPase rings results in an inventory of several Clp protease complexes that often coexist. Here, we present insights into Clp protease function, from their assembly to substrate recruitment and processing, and how this is coupled to the expense of energy.
The bacterial AAA+ chaperone ClpB provides thermotolerance by disaggregating aggregated proteins in collaboration with the DnaK chaperone system. Like many other AAA+ proteins, ClpB is believed to act as a biological motor converting the chemical energy of ATP into molecular motion. ClpB has two ATPase domains, NBD1 and NBD2, on one polypeptide chain. The functional unit of ClpB is a homohexameric ring, with a total of 12 potential nucleotide binding sites. Previously, two separate constructs, one each containing NBD1 or NBD2, have been shown to form a functional complex with chaperone activity when mixed. Here we aimed to elucidate the nucleotide binding properties of the ClpB complex using pre-steady state kinetics and fluorescent nucleotides. For this purpose, we first disassembled the complex and characterized in detail the binding kinetics of a construct comprising NBD2 and the C-terminal domain of ClpB. The monomeric construct bound nucleotides very tightly. ADP bound 2 orders of magnitude more tightly than ATP; this difference in binding affinity resulted almost exclusively from different dissociation rate constants. The nucleotide binding properties of NBD2 changed when this construct was complemented with a construct comprising NBD1 and the middle domain. Our approach shows how complex formation can influence the binding properties of the individual domains and allows us to assign nucleotide binding features of this highly complex, multimeric enzyme to specific domains.
HslVU is an ATP-dependent protease in bacteria consisting of HslV dodecamer and HslU hexamer. Upon ATP binding, HslU ATPase allosterically activates the catalytic function of HslV protease by 1-2 orders of magnitude. However, relatively little is known about the role of HslV in the control of HslU function. Here we describe the involvement of the N-terminal Thr active sites (Thr-1) of HslV in the communication between HslV and HslU. Binding of proteasome inhibitors to Thr-1 led to a dramatic increase in the interaction between HslV and HslU with a marked increase in ATP hydrolysis by HslU. Moreover, carbobenzoxy-leucyl-leucyl-leucinal (MG132) could bind to Thr-1 of free HslV, and this binding induced a tight interaction between HslV and HslU with the activation of HslU ATPase, suggesting that substrate-bound HslV can allosterically regulate HslU function. Unexpectedly, the deletion of Thr-1 also caused a dramatic increase in the affinity between HslV and HslU even in the absence of ATP. Furthermore, the increase in the number of the Thr-1 deletion mutant subunit in place of HslV subunit in a dodecamer led to a proportional increase in the affinity between HslV and HslU with gradual activation of HslU ATPase. Although the molecular mechanism elucidating how the Thr-1 deletion influences the interaction between HslV and HslU remains unknown, these results suggest an additional allosteric mechanism for the control of HslU function by HslV. Taken together, our findings indicate a critical involvement of Thr-1 of HslV in the reciprocal control of HslU function and, thus, for their communication.
HslVU is an ATP-dependent prokaryotic protease complex. Despite
detailed crystal and molecular structure determinations of free HslV
and HslU, the mechanism of ATP-dependent peptide and protein hydrolysis
remained unclear, mainly because the productive complex of HslV and
HslU could not be unambiguously identified from the crystal data. In
the crystalline complex, the I domains of HslU interact with HslV.
Observations based on electron microscopy data were interpreted in the
light of the crystal structure to indicate an alternative mode of
association with the intermediate domains away from HslV. By generation
and analysis of two dozen HslU mutants, we find that the amidolytic and
caseinolytic activities of HslVU are quite robust to mutations on both
alternative docking surfaces on HslU. In contrast, HslVU activity
against the maltose-binding protein-SulA fusion protein depends on the
presence of the I domain and is also sensitive to mutations in the
N-terminal and C-terminal domains of HslU. Mutational studies around
the hexameric pore of HslU seem to show that it is involved in the
recognition/translocation of maltose-binding protein-SulA but not of
chromogenic small substrates and casein. ATP-binding site mutations,
among other things, confirm the essential role of the “sensor
arginine” (R393) and the “arginine finger” (R325) in the
ATPase action of HslU and demonstrate an important role for E321.
Additionally, we report a better refined structure of the HslVU complex
crystallized along with resorufin-labeled casein.
The hslVU operon in Escherichia coli encodes two heat shock proteins, HslV, a 19-kDa protein homologous to β-type subunits of the 20 S proteasomes, and HslU,
a 50-kDa protein related to the ATPase ClpX. We have recently shown that HslV and HslU can function together as a novel ATP-dependent
protease, the HslVU protease. We have now purified both proteins to apparent homogeneity from extracts of E. coli carrying the hslVU operon on a multicopy plasmid. HslU by itself cleaved ATP, and pure HslV is a weak peptidase degrading certain hydrophobic
peptides. HslU dramatically stimulated peptide hydrolysis by HslV when ATP is present. With a 1:4 molar ratio of HslV to HslU,
approximately a 200-fold increase in peptide hydrolysis was observed. HslV stimulated the ATPase activity of HslU 2–4-fold,
but had little influence on the affinity of HslU to ATP. The nonhydrolyzable ATP analog, β,γ-methylene-ATP, did not support
peptide hydrolysis. Other nucleotides (CTP, dATP) that were slowly hydrolyzed by HslU allowed some peptide hydrolysis. Therefore,
ATP cleavage appears essential for the HslV activity. Upon gel filtration on a Sephacryl S-300 column, HslV behaved as a 250-kDa
oligomer (i.e. 12–14 subunits), and HslU behaved as a 100-kDa protein (i.e. a dimer) in the absence of ATP, but as a 450-kDa multimer (8–10 subunits) in its presence. Therefore ATP appears necessary
for oligomerization of HslU. Thus the HslVU protease appears to be a two-component protease in which HslV harbors the peptidase
activity, while HslU provides an essential ATPase activity.
HslVU is a new two-component protease in Escherichia coli composed of the proteasome-related peptidase HslIV and the ATPase HsIU. We have used electron microscopy and image analysis to examine the structural organization of HslV and HslU homo-oligomers and the active HslVU enzyme. Electron micrographs of HslV reveal ring-shaped particles, and averaging of top views reveal six-fold rotational symmetry, in contrast to other beta-type proteasome subunits, which form rings with seven-fold symmetry. Side views of HslV show two rings stacked together, thus, HslV behaves as dodecamer. The ATPase HslU forms ring-shaped particles in the presence of ATP, AMP-PNP or ADP, suggesting that nucleotide binding, but not hydrolysis, is required for oligomerization. Subunit crosslinking, STEM mass estimation, and analysis of HslU top views indicate that HslU exists both as hexameric and heptameric rings. With AMP-PNP present, maximal proteolytic activity is observed with a molar ratio of HslU to HslV subunits of 1:1, and negative staining electron microscopy shows that HslV and HsIU form cylindrical four-ring structures in which the HsIV dodecamer is flanked at each end by a HslU ring.
We have isolated a new type of ATP-dependent protease from Escherichia coli. It is the product of the heat-shock locus hslVU that encodes two proteins: HslV, a 19-kDa protein similar to proteasome beta subunits, and HslU, a 50-kDa protein related to the ATPase ClpX. In the presence of ATP, the protease hydrolyzes rapidly the fluorogenic peptide Z-Gly-Gly-Leu-AMC and very slowly certain other chymotrypsin substrates. This activity increased 10-fold in E. coli expressing heat-shock proteins constitutively and 100-fold in cells expressing HslV and HslU from a high copy plasmid. Although HslV and HslU could be coimmunoprecipitated from cell extracts of both strains with an anti-HslV antibody, these two components were readily separated by various types of chromatography. ATP stimulated peptidase activity up to 150-fold, whereas other nucleoside triphosphates, a nonhydrolyzable ATP analog, ADP, or AMP had no effect. Peptidase activity was blocked by the anti-HslV antibody and by several types of inhibitors of the eukaryotic proteasome (a threonine protease) but not by inhibitors of other classes of proteases. Unlike eukaryotic proteasomes, the HslVU protease lacked tryptic-like and peptidyl-glutamyl-peptidase activities. Electron micrographs reveal ring-shaped particles similar to en face images of the 20S proteasome or the ClpAP protease. Thus, HslV and HslU appear to form a complex in which ATP hydrolysis by HslU is essential for peptide hydrolysis by the proteasome-like component HslV.
ClpQ (HslV) is a homolog of the beta-subunits of the 20S proteasome. In E. coli, it is expressed from an operon that also encodes ClpY (HslU), an ATPase homologous to the protease chaperone, ClpX. ClpQ (subunit Mr 19,000) and ClpY (subunit Mr 49,000) were purified separately as oligomeric proteins with molecular weights of approximately 220,000 and approximately 350,000, respectively, estimated by gel filtration. Mixtures of ClpY and ClpQ displayed ATP-dependent proteolytic activity against casein, and a complex of the two proteins was isolated by gel filtration in the presence of ATP. Image processing of negatively stained electron micrographs revealed strong six-fold rotational symmetry for both ClpY and ClpQ, suggesting that the subunits of both proteins are arranged in hexagonal rings. The molecular weight of ClpQ combined with its symmetry is consistent with a double hexameric ring, whereas the data on ClpY suggest only one such ring. The symmetry mismatch previously observed between hexameric ClpA and heptameric ClpP in the related ClpAP protease is apparently not reproduced in the symmetry-matched ClpYQ system.
Heat shock locus V (HslV; also called ClpQ) is the proteolytic core of the ATP-dependent protease HslVU in Escherichia coli. It has sequence similarity with the beta-type subunits of the eukaryotic and archaebacterial proteasomes. Unlike these particles, which display 72-point symmetry, it is a dimer of hexamers with 62-point symmetry. The crystal structure of HslV at 3.8-A resolution, determined by isomorphous replacement and symmetry averaging, shows that in spite of the different symmetry of the particle, the fold and the contacts between subunits are conserved. A tripeptide aldehyde inhibitor, acetyl-Leu-Leu-norleucinal, binds to the N-terminal threonine residue of HslV, probably as a hemiacetal, relating HslV also functionally to the proteasomes of archaea and eukaryotes.
HslVU is a new Escherichia coli ATP-dependent protease composed of two multimeric complexes: the HslU ATPase and the HslV peptidase. Prior studies indicated that HslVU requires ATP hydrolysis for the cleavage of peptides and proteins. We show here that ATP concentrations that activate hydrolysis of benzyloxycarbonyl-Gly-Gly-Leu-7-amido-4-methylcoumarin are 50-100 fold lower than those necessary for degradation of proteins (e.g. casein). Also, the nonhydrolyzable analogs of ATP, 5'-adenylyl beta, gamma-imidodiphosphate (AMP-PNP) and adenosine 5'-(alpha, beta-methylene)triphosphate, can support peptide hydrolysis, but only after an initial time lag not seen with ATP. This delay decreased at higher temperatures and with higher HslU or HslV concentrations and was eliminated by preincubation of HslU and HslV together. Thus, ATP hydrolysis accelerates the association of HslU and HslV, which occurs slowly with the nonhydrolyzable analog. The addition of KCl stimulated 4-6-fold the peptidase activity with AMP-PNP present and eliminated the time lag, but KCl had no stimulatory effect with ATP. NH4+ and Cs+ had similar effects as K+, but Na+ and Li+ were ineffective. AMP-PNP by itself supported hydrolysis of casein and other polypeptides only 20% as well as ATP, but in the presence of K+, Cs+, or NH4+, AMP-PNP activated casein degradation even better than ATP, although it was not hydrolyzed. In addition, MgCl2, MnCl2, and CaCl2 allowed some peptidase and caseinase activity in the absence of any nucleotide. However, Mn2+ and Ca2+, unlike Mg2+, abolished ATP hydrolysis and prevented further activation by ATP or AMP-PNP. These findings indicate that ATP binding to a high affinity site triggers the formation of an active state capable of peptide cleavage, although ATP hydrolysis facilitates this process. Rapid degradation of proteins requires a distinct state of the enzyme, which is normally reached through ATP hydrolysis at low affinity sites. However, AMP-PNP binding together with K+ can induce a form of HslVU that degrades proteins without energy consumption.
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.
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.
The structure of the Haemophilus influenzae HslV protease of the HslUV 'prokaryotic proteasome' has been solved by molecular replacement and refined with data to 1.9 A resolution. The protease is a 'double donut' of hexameric rings; two alternative sets of intermolecular interactions between protomers in the rings result in 'quasi-equivalent' packing within the assembly. Anomalous scattering data from crystals with potassium present in the mother liquor reveal a K(+) ion bound with octahedral coordination near the active-site Thr1 residue. The site also binds Na(+) ions and is likely to bind Mg(2+), suggesting that monovalent and divalent metal ions may influence the catalytic activity of the protease.
The bacterial HslVU ATP-dependent protease is a homolog of the eukaryotic 26 S proteasome. HslU ATPase forms a hexameric ring,
and HslV peptidase is a dodecamer consisting of two stacked hexameric rings. In HslVU complex, the HslU and HslV central pores
are aligned, and the proteolytic active sites are sequestered in an internal chamber of HslV, with access to this chamber
restricted to small axial pores. Here we show that the C-terminal tails of HslU play a critical role in the interaction with
and activation of HslV peptidase. A synthetic tail peptide of 10 amino acids could replace HslU in supporting the HslV-mediated
hydrolysis of unfolded polypeptide substrates such as α-casein, as well as of small peptides, suggesting that the HslU C terminus
is involved in the opening of the HslV pore for substrate entry. Moreover, deletion of 7 amino acids from the C terminus prevented
the ability of HslU to form an HslVU complex with HslV. In addition, deletion of the C-terminal 10 residues prevented the
formation of an HslU hexamer, indicating that the C terminus is required for HslU oligomerization. These results suggest that
the HslU C-terminal tails act as a molecular switch for the assembly of HslVU complex and the activation of HslV peptidase.
HslVU is a bacterial homolog of the proteasome, where HslV is the protease that is activated by HslU, an ATPase and chaperone. Structures of singly and doubly capped HslVU particles have been reported, and different binding modes have been observed. Even among HslVU structures with I-domains distal to HslV, no consensus mode of activation has emerged. A feature in the Haemophilus influenzae HslVU structure, insertion of the C termini of HslU into pockets in HslV, was not seen in all other structures of the enzyme. Here we report site-directed mutagenesis, peptide activation, and fluorescence experiments that strongly support the functional relevance of the C terminus insertion mechanism: we find that mutations in HslV that disrupt the interaction with the C termini of HslU invariably lead to inactive enzyme. Conversely, synthetic peptides derived from the C terminus of HslU bind to HslV with 10(-5) M affinity and can functionally replace full HslU particles for both peptide and casein degradation but fail to support degradation of a folded substrate. Thus, the data can be taken as evidence for separate substrate unfoldase and protease stimulation activities in HslU. Enhanced HslV proteolysis could be due to the opening of a gated channel or allosteric activation of the active sites. To distinguish between these possibilities, we have mutated a series of residues that line the entrance channel into the HslV particle. Our mutational and fluorescence experiments demonstrate that allosteric activation of the catalytic sites is required in HslV, but they do not exclude the possibility of channel opening taking place as well. The present data support the conclusion that the H. influenzae structure with I-domains distal to HslV captures the active species and point to significant differences in the activation mechanism of HslV, ClpP, and the proteasome.
Machines of protein destruction-including energy-dependent proteases and disassembly chaperones of the AAA(+) ATPase family-function in all kingdoms of life to sculpt the cellular proteome, ensuring that unnecessary and dangerous proteins are eliminated and biological responses to environmental change are rapidly and properly regulated. Exciting progress has been made in understanding how AAA(+) machines recognize specific proteins as targets and then carry out ATP-dependent dismantling of the tertiary and/or quaternary structure of these molecules during the processes of protein degradation and the disassembly of macromolecular complexes.
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.
Many eubacteria contain an ATP-dependent protease complex, which is built by multiple copies of the HslV and HslU proteins and is therefore called HslVU. HslU proteins are AAA + ATPases, while HslV proteins are proteases that show highly significant similarity to beta subunits of proteasomes. Therefore, the HslVU complex has been envisaged as a precursor or ancestral type of proteasome. Here we show that species of most of the main eukaryotic lineages have HslU and HslV genes very similar to those found in proteobacteria. We have detected them in amoebozoa, plantae, chromoalveolata, rhizaria, and excavata species. Phylogenetic analyses suggest that these genes have been obtained by endosymbiosis from the proteobacterial ancestor that gave rise to eukaryotic mitochondria. The products encoded by these eukaryotic genes adopt, according to modeling based on the known crystal structures of prokaryotic HslU and HslV proteins, conformations that are compatible with their being fully active, suggesting that functional HslVU complexes may be present in many eukaryotic species.
ClpX, a heat shock protein 100 chaperone, which acts as the regulatory subunit of the ATP-dependent ClpXP protease, is responsible
for intracellular protein remodeling and degradation. To provide a structural basis for a better understanding of the function
of the Clp ATPase family, the crystal structures of Helicobacter pylori ClpX, lacking an N-terminal Cys cluster region complexed with ADP, was determined. The overall structure of ClpX is similar
to that of heat shock locus U (HslU), consisting of two subdomains, with ADP bound at the subdomain interface. The crystal
structure of ClpX reveals that a conserved tripeptide (LGF) is located on the tip of ClpP binding loop extending from the
N-terminal subdomain. A hexameric model of ClpX suggests that six tripeptides make hydrophobic contacts with the hydrophobic
clefts of the ClpP heptmer asymmetrically. In addition, the nucleotide binding environment provides the structural explanation
for the hexameric assembly and the modulation of ATPase activity.
The bacterial heat shock locus ATPase HslU is an AAA(+) protein that has structures known in many nucleotide-free and -bound states. Nucleotide is required for the formation of the biologically active HslU hexameric assembly. The hexameric HslU ATPase binds the dodecameric HslV peptidase and forms an ATP-dependent HslVU protease.
We have characterized four distinct HslU conformational states, going sequentially from open to closed: the empty, SO(4), ATP, and ADP states. The nucleotide binds at a cleft formed by an alpha/beta domain and an alpha-helical domain in HslU. The four HslU states differ by a rotation of the alpha-helical domain. This classification leads to a correction of nucleotide identity in one structure and reveals the ATP hydrolysis-dependent structural changes in the HslVU complex, including a ring rotation and a conformational change of the HslU C terminus. This leads to an amended protein unfolding-coupled translocation mechanism.
The observed nucleotide-dependent conformational changes in HslU and their governing principles provide a framework for the mechanistic understanding of other AAA(+) proteins.
HslVU in Escherichia coli a new two-component ATP-dependent protease composed of two heat-shock proteins, the HslU ATPase and the HslV peptidase which is related to proteasome β-type subunits. Here we show that the reconstituted HslVU enzyme degrades not only certain hydrophobic peptides but also various polypeptides, including insulin B-chain, casein, and carboxymethylated lactalbumin. Maximal proteolytic activity was obtained with a 1:2 molar ratio of HslV (a 250-kDa complex) to HslU (a 450-kDa complex). By itself, HslV could slowly hydrolyze these polypeptides, but its activity was stimulated 20-fold by HslU in the presence of ATP. The ATPase activity of HslU was stimulated up to 50% by the protein substrates, but not by nonhydrolyzed proteins, and this stimulation further increased 2–3-fold in the presence of HslV. Concentrations of insulin B-chain that maximally stimulated the ATPase allowed maximal rates of the B-chain hydrolysis. Furthermore, addition of increasing amounts of ADP or N-ethylmaleimide reduced ATP and protein or peptide hydrolysis in parallel. Thus, HslVU is a protein-activated ATPase as well as an ATP-dependent proteinase, and these processes appear linked.
Surprisingly, the protein and peptide substrates do not compete with each other for hydrolysis. Lactacystin strongly inhibits protein degradation, but has little effect on peptide hydrolysis, while the peptide aldehydes are potent inhibitors of hydrolysis of small peptides, but have little effect on proteins. Thus, the functional requirements for ATP-dependent hydrolysis of peptides and proteins appear different.
HslVU is an ATP-dependent protease consisting of two multimeric components: the HslU ATPase and the HslV peptidase. To gain an insight into the role of ATP hydrolysis in protein breakdown, we determined the insulin B-chain-degrading activity and assembly of HslVU in the presence of ATP and its nonhydrolyzable analogs. While beta,gamma-methylene-ATP could not support the proteolytic activity, beta,gamma-imido-ATP supported it to an extent less than 10% of that seen with ATP. Surprisingly, however, HslVU degraded insulin B-chain even more rapidly in the presence of ATPgammaS than with ATP. Furthermore, the ability of ATP and its analogs in supporting the proteolytic activity was closely correlated with their ability in supporting the oligomerization of HslU and the formation of the HslVU complex. However, ADP, which is capable of supporting the HslU oligomerization, could not support the HslVU complex formation or the proteolytic activity, suggesting that the conformation of the ADP-bound HslU oligomer is different from that of ATP-bound form. Thus, it appears that ATP-binding, but not its hydrolysis, is essential for assembly and proteolytic activity of HslVU.
Using a combination of computer methods for iterative database searches and multiple sequence alignment, we show that protein sequences related to the AAA family of ATPases are far more prevalent than reported previously. Among these are regulatory components of Lon and Clp proteases, proteins involved in DNA replication, recombination, and restriction (including subunits of the origin recognition complex, replication factor C proteins, MCM DNA-licensing factors and the bacterial DnaA, RuvB, and McrB proteins), prokaryotic NtrC-related transcription regulators, the Bacillus sporulation protein SpoVJ, Mg2+, and Co2+ chelatases, the Halobacterium GvpN gas vesicle synthesis protein, dynein motor proteins, TorsinA, and Rubisco activase. Alignment of these sequences, in light of the structures of the clamp loader delta' subunit of Escherichia coli DNA polymerase III and the hexamerization component of N-ethylmaleimide-sensitive fusion protein, provides structural and mechanistic insights into these proteins, collectively designated the AAA+ class. Whole-genome analysis indicates that this class is ancient and has undergone considerable functional divergence prior to the emergence of the major divisions of life. These proteins often perform chaperone-like functions that assist in the assembly, operation, or disassembly of protein complexes. The hexameric architecture often associated with this class can provide a hole through which DNA or RNA can be thread; this may be important for assembly or remodeling of DNA-protein complexes.
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.
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.
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.
The structure of the Haemophilus influenzae HslU protein, a molecular chaperone of the Clp/Hsp100 family, has been solved to 2.3 A by molecular replacement using a model of the homologous Escherichia coli protein. The crystals in which the structure was solved have an unusual twinning, or one-dimensional disorder, in which each successive crystal-packing layer is displaced laterally relative to the one below it. A model for the twinning and an algorithm for detwinning the data are described. It is known from other work that when the HslU hexamer binds its cognate protease HslV, the carboxy-terminal helices of HslU protomers distend and bind between HslV subunits. Comparison of HslU alone with its structure in the HslUV complex reveals several conserved amino-acid residues whose side-chain interactions differ between the two structures, suggesting that they may be part of a conformational switch that facilitates the release of the HslU carboxy-terminal helices when HslV binds.
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.
Heat-shock locus VU (HslVU) is an ATP-dependent proteolytic system and a prokaryotic homolog of the proteasome. It consists of HslV, the protease, and HslU, the ATPase and chaperone. We have cloned, sequenced and expressed both protein components from the hyperthermophile Thermotoga maritima. T. maritima HslU hydrolyzes a variety of nucleotides in a temperature-dependent manner, with the optimum lying between 75 and 80 degrees C. It is also nucleotide-unspecific for activation of HslV against amidolytic and caseinolytic activity. The Escherichia coli and T. maritima HslU proteins mutually stimulate HslV proteins from both sources, suggesting a conserved activation mechanism. The crystal structure of T. maritima HslV was determined and refined to 2.1-A resolution. The structure of the dodecameric enzyme is well conserved compared to those from E. coli and Haemophilus influenzae. A comparison of known HslV structures confirms the presence of a cation-binding site, although its exact role in the proteolytic mechanism of HslV remains unclear. Amongst factors responsible for the thermostability of T. maritima HslV, extensive ionic interactions/salt-bridge networks, which occur specifically in the T. maritima enzyme in comparison to its mesophilic counterparts, seem to play an important role.
The HslUV protease-chaperone complex degrades specific protein substrates in an ATP-dependent reaction. Current models propose that the HslU chaperone, a AAA protein of the Clp/Hsp100 family, binds and unfolds substrates and translocates the polypeptide into the catalytic cavity of the HslV protease. These processes are being characterized using substrates that are targeted to HslUV with a carboxy-terminal fusion of the natural substrate SulA or the carboxy-terminal 11 amino acid residues thereof. In a tandem fusion of green fluorescent protein with SulA, HslUV degrades the SulA moiety but not green fluorescent protein. Wild type and mutant Arc repressor variants are degraded; over a range of substrate stabilities, the specific rate of degradation and its dependence on substrate stability is similar to that of ClpXP. For a hyperstable Arc variant having an intermolecular disulfide bond, the rate of degradation by HslUV is an order of magnitude slower than by ClpXP. Similarity in degradation rates for a subset of substrates by HslUV and ClpXP suggests a similarity in mechanism of the apparent rate-limiting steps of unfolding and translocation by the chaperone components HslU and ClpX. The fall-off in degradation by HslUV for the more stable substrates that are degraded by ClpXP is consistent with the two systems acting on different spectra of biological substrates.
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.
ATP-dependent protein degradation is controlled principally by substrate recognition. The AAA+ HslU ATPase is thought to bind protein substrates, denature them, and translocate the unfolded polypeptide into the HslV peptidase. The lack of well-behaved high-affinity substrates for HslUV (ClpYQ) has hampered understanding of the rules and mechanism of substrate engagement. We show that HslUV efficiently degrades Arc repressor, especially at heat-shock temperatures. Degradation depends on sequences near the N terminus of Arc. Fusion protein and peptide-binding experiments demonstrate that this sequence is a degradation tag that binds directly to HslU. Strong binding of this tag to the enzyme requires ATP and Mg(2+). Furthermore, fusion of this sequence to a protein with marked mechanical stability leads to complete degradation. Thus, these experiments demonstrate that HslUV is a powerful protein unfoldase and that initial substrate engagement by the HslU ATPase must occur after ATP binding.
The HslVU complex is a bacterial two-component ATP-dependent protease, consisting of HslU chaperone and HslV peptidase. Investigation of protein-protein interactions using SPR in Escherichia coli HslVU and the protein substrates demonstrates that HslU and HslV have moderate affinity (Kd = 1 microM) for each other. However, the affinity of HslU for HslV fivefold increased (Kd approximately 0.2 microM) after binding with the MBP approximately SulA protein indicating the formation of a "ternary complex" of HslV-HslU-MBP approximately SulA. The molecular interaction studies also revealed that HslU strongly binds to MBP approximately SulA with 10(-9) M affinity but does not associate with nonstructured casein. Conversely, HslV does not interact with the MBP-SulA whereas it strongly binds with casein (Kd = 0.2 microM) requiring an intact active site of HslV. These findings provide evidence for "substrate-induced" stable HslVU complex formation. Presumably, the binding of HslU to MBP approximately SulA stimulates a conformational change in HslU to a high-affinity form for HslV.
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.
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.
'Chambered proteases', including the eukaryotic 26S proteasome, use the energy of ATP to drive the unfolding and translocation of a polypeptide substrate into a chamber of sequestered proteolytic active sites. These proteases have diverse functions and are found in all three kingdoms of life. Understanding chambered proteases requires answers to two questions — how do these remarkable machines select the correct target proteins and how do they bring about the processive degradation of these molecules?
ClpB is a ring-shaped molecular chaperone that has the remarkable ability to disaggregate stress-damaged proteins. Here we present the electron cryomicroscopy reconstruction of an ATP-activated ClpB trap mutant, along with reconstructions of ClpB in the AMPPNP, ADP, and in the nucleotide-free state. We show that motif 2 of the ClpB M domain is positioned between the D1-large domains of neighboring subunits and could facilitate a concerted, ATP-driven conformational change in the AAA-1 ring. We further demonstrate biochemically that ATP is essential for high-affinity substrate binding to ClpB and cannot be substituted with AMPPNP. Our structures show that in the ATP-activated state, the D1 loops are stabilized at the central pore, providing the structural basis for high-affinity substrate binding. Taken together, our results support a mechanism by which ClpB captures substrates on the upper surface of the AAA-1 ring before threading them through the ClpB hexamer in an ATP hydrolysis-driven step.
Transcription initiation by the sigma54 form of bacterial RNA polymerase requires hydrolysis of ATP by an enhancer binding protein (EBP). We present SAS-based solution structures of the ATPase domain of the EBP NtrC1 from Aquifex aeolicus in different nucleotide states. Structures of apo protein and that bound to AMPPNP or ADP-BeF(x) (ground-state mimics), ADP-AlF(x) (a transition-state mimic), or ADP (product) show substantial changes in the position of the GAFTGA loops that contact polymerase, particularly upon conversion from the apo state to the ADP-BeF(x) state, and from the ADP-AlF(x) state to the ADP state. Binding of the ATP analogs stabilizes the oligomeric form of the ATPase and its binding to sigma54, with ADP-AlF(x) having the largest effect. These data indicate that ATP binding promotes a conformational change that stabilizes complexes between EBPs and sigma54, while subsequent hydrolysis and phosphate release drive the conformational change needed to open the polymerase/promoter complex.
In the ClpXP proteolytic machine, ClpX uses the energy of ATP hydrolysis to unfold protein substrates and translocate them through a central pore and into the degradation chamber of ClpP. Here, we demonstrate a bipartite system of ClpX-ClpP interactions that serves multiple functional roles. High-affinity contacts between six loops near the periphery of the hexameric ClpX ring and a ClpP ring establish correct positioning and increase degradation activity but are insensitive to nucleotide state. These static peripheral interactions maintain a stable ClpXP complex, while other parts of this machine change conformation hundreds of times per minute. By contrast, relatively weak axial contacts between loops at the bottom of the ClpX central channel and N-terminal loops of ClpP vary dynamically with the nucleotide state of individual ClpX subunits, control ATP-hydrolysis rates, and facilitate efficient protein unfolding. Thus, discrete static and dynamic interactions mediate binding and communication between ClpX and ClpP.
HslVU is an ATP-dependent protease from Escherichia coli and known to degrade SulA, a cell division inhibitor, both in vivo and in vitro, like the ATP-dependent protease Lon. In this study, the cleavage specificity of HslVU toward SulA was investigated. The enzyme was shown to produce 58 peptides with various sizes (3-31 residues), not following the 'molecular ruler' model. Cleavage occurred at 39 peptide bonds preferentially after Leu in an ATP-dependent manner and in a processive fashion. Interestingly, the central and C-terminal regions of SulA, which are known to be important for the function of SulA, such as inhibition of cell division and molecular interaction with certain other proteins, were shown to be preferentially cleaved by HslVU, as well as by Lon, despite the fact that the peptide bond specificities of the two enzymes were distinct from each other.