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Protein Binding and Disruption by Clp/Hsp100 Chaperones
Abstract
Clp/Hsp100 chaperones work with other cellular chaperones and proteases to control the quality and amounts of many intracellular proteins. They employ an ATP-dependent protein unfoldase activity to solubilize protein aggregates or to target specific classes of proteins for degradation. The structural complexity of Clp/Hsp100 proteins combined with the complexity of the interactions with their macromolecular substrates presents a considerable challenge to understanding the mechanisms by which they recognize and unfold substrates and deliver them to downstream enzymes. Fortunately, high-resolution structural data is now available for several of the chaperones and their functional partners, which together with mutational data on the chaperones and their substrates has provided a glimmer of light at the end of the Clp/Hsp100 tunnel.
... Walker A forms the floor of the nucleotidebinding pocket and binds the ATP phosphate. Walker B positions cations to bind metals that play a role in ATP catalysis [14,16,17,19]. To date, 12 Clp regulatory subunits with diverse cellular functions have been identified across various bacterial species (Table 1). ...
... The NBDs in Class I both consist of Domain 1 (D1) and Domain 2 (D2) however, the amino acid sequence of each domain differs. The difference in the amino acid sequence in the NBDs suggest that gene fusion, rather than gene duplication, may be the route through which members of class I evolved [14,18]. The NBDs contain canonical Walker A and Walker B which are essential for the breakdown of ATP. ...
... Walker A forms the floor of the nucleotide-binding pocket and binds the ATP phosphate. Walker B positions cations to bind metals that play a role in ATP catalysis [14,16,17,19]. Table 1. ...
Caseinolytic proteins (Clp), which are present in both prokaryotes and eukaryotes, play a major role in cell protein quality control and survival of bacteria in harsh environmental conditions. Recently, a member of this protein family, ClpK was identified in a pathogenic strain of Klebsiella pneumoniae which was responsible for nosocomial infections. ClpK is linked to the thermal stress survival of this pathogen. The genome wide analysis of Clp proteins in Klebsiella spp. indicates that ClpK is present in only 34% of the investigated strains. This suggests that the uptake of the clpk gene is selective and may only be taken up by a pathogen that needs to survive harsh environmental conditions. In silico analyses and molecular dynamic simulations show that ClpK is mainly α-helical and is highly dynamic. ClpK was successfully expressed and purified to homogeneity using affinity and anion exchange chromatography. Biophysical characterization of ClpK showed that it is predominantly alpha-helical, and this is in agreement with in silico analysis of the protein structure. Furthermore, the purified protein is biologically active and hydrolyses ATP in a concentration- dependent manner.
... They are responsible in resolubilizing stress-damaged proteins from aggregates (Hung & Masison, 2006, Mogk & Bukau, 2004, and require other chaperones like the Hsp70 system to act along side in a cooperative manner to fold them into their native state (Motohashi et al., 1999, Glover & Lindquist, 1998. ClpA, ClpC and ClpX disaggregate proteins by degrading them along with the help of the protease ClpP (Maurizi & Xia, 2004, Kress et al., 2007, Martin et al., 2007. ...
... Structurally, all Hsp100 chaperones contain a conserved core, referred to as the AAA module, which consists of two nucleotide binding subdomains (NBD) (Maurizi & Xia, 2004). ...
... ClpP are peptidases that break down misfolded polypeptides and prevent further aggregation. Image taken fromMaurizi & Xia, 2004. ...
... The zinc-binding domain is present in ClpX and ClpK. The role of this domain in unknown in ClpK, however it is positioned in the N-terminal domain of ClpX and plays a role in substrate recognition [16,57,61]. Therefore, essentially compounds targeting the zinc-binding domain could inhibit substrate recognition and would theoretically lead to the build-up of damaged proteins in the cell. ...
... (b) Members of the ClpA subfamily contain the tripeptide for ClpP interaction and therefore redirect proteins which cannot be unfolded and reactivated to ClpP for degradation. Adapted from Maurizi and Xia[61]. ...
Bacterial antibiotic resistance is rapidly growing globally and poses a severe health threat as the number of multidrug resistant (MDR) and extensively drug-resistant (XDR) bacteria increases. The observed resistance is partially due to natural evolution and to a large extent is attributed to antibiotic misuse and overuse. As the rate of antibiotic resistance increases, it is crucial to develop new drugs to address the emergence of MDR and XDR pathogens. A variety of strategies are employed to address issues pertaining to bacterial antibiotic resistance and these strategies include: (1) the anti-virulence approach, which ultimately targets virulence factors instead of killing the bacterium, (2) employing antimicrobial peptides that target key proteins for bacterial survival and, (3) phage therapy, which uses bacteriophages to treat infectious diseases. In this review, we take a renewed look at a group of ESKAPE pathogens which are known to cause nosocomial infections and are able to escape the bactericidal actions of antibiotics by reducing the efficacy of several known antibiotics. We discuss previously observed escape mechanisms and new possible therapeutic measures to combat these pathogens and further suggest caseinolytic proteins (Clp) as possible therapeutic targets to combat ESKAPE pathogens. These proteins have displayed unmatched significance in bacterial growth, viability and virulence upon chronic infection and under stressful conditions. Furthermore, several studies have showed promising results with targeting Clp proteins in bacterial species, such as Mycobacterium tuberculosis, Staphylococcus aureus and Bacillus subtilis.
... HSP101 is a member of the type I HSP100 ClpB/AAAϩ-ATPases, which canonically form ring-shaped hexamers with a central channel. Related ClpB proteins are characterized by a unique N-terminal domain that binds substrate proteins, either directly or through adapter proteins, and two conserved AAAϩ domains that bind and hydrolyze ATP (35). In addition, they contain a unique coiled-coil middle domain that regulates ATPase and unfoldase activities (36)(37)(38) and a short C-terminal domain, which is essential for hexamerization (39,40). ...
... It has been proposed that HSP101 threads cargo directly into the PTEX channel via its ATPase activity and is required for unfolding cargo (21,39). Most HSP100/ClpB family members engage with cochaperones to unfold proteins to disassemble and disaggregate proteins (35,37,(45)(46)(47). Whether HSP101 works independently of a cochaperone remains to be tested. ...
The Plasmodium parasites that cause malaria export hundreds of proteins into their host red blood cell (RBC). These exported proteins drastically alter the structural and functional properties of the RBC and play critical roles in parasite virulence and survival. To access the RBC cytoplasm, parasite proteins must pass through the Plasmodium translocon of exported proteins (PTEX) located at the membrane interfacing the parasite and host cell. Our data provide evidence that HSP101, a component of PTEX, serves to unfold protein cargo requiring translocation. We also reveal that addition of a transmembrane domain to soluble cargo influences its ability to be translocated by parasites in which the HSP101 motor and unfolding activities have become uncoupled. Therefore, we propose that proteins with transmembrane domains use an alternative unfolding pathway prior to PTEX to facilitate export.
... Gene knockout and conditional knockdown experiments in either P. falciparum or the rodent malaria species, P. berghei, have revealed only HSP101, PTEX150 and EXP2 are essential for protein export and parasite survival in the blood-stages and therefore comprise the core PTEX complex (Matthews et al., 2013;Matz, Matuschewski & Kooij, 2013;Beck, Muralidharan, Oksman & Goldberg, 2014;Garten et al., 2018). HSP101 is a member of the ClpB/HSP100 protein family (de Koning-Ward et al., 2009), which can be distinguished by their unique N-terminal domain, two AAA + nucleotidebinding domains (NBD1 and 2) that bind and hydrolyse ATP, a unique coiled-coil middle domain and a C-terminal domain that is critical for hexamerisation (Maurizi & Xia, 2004;Mackay, Helsen, Tkach & Glover, 2008;Hodson, Marshall & Burston, 2012). Based on these properties, HSP101 is predicted to serve as the PTEX motor, utilising ATP to unfold proteins and drive protein translocation through its central cavity and the channel spanning the PVM (de Koning-Ward et al., 2009). ...
... With the exception of HSP70-x, which is only present in P. falciparum and other members of the Plasmodium subgenus Laverania, no HSP70 chaperones that could potentially co-operate with HSP101 in other Plasmodium species have been identified in the PV. Thus, HSP101 appears distinct from other ClpB/HSP100 family members present in prokaryotes and other eukaryotes, which cooperate with DnaJ/HSP70 co-chaperones to disassemble and disaggregate proteins (Maurizi & Xia, 2004;Snider, Thibault & Houry, 2008;Deville et al., 2017;Gates et al., 2017). As Blue-Native PAGE suggests PTEX may not exist as a static translocon (Bullen et al., 2012;Elsworth et al., 2016), cargo may even be recognized by dissociated HSP101, after which HSP101 bound to cargo assembles into the PTEX complex. ...
Plasmodium parasites that cause the disease malaria have developed an elaborate trafficking pathway to facilitate the export of hundreds of effector proteins into their host cell, the erythrocyte. In this review, we outline how certain effector proteins contribute to parasite survival, virulence and immune evasion. We also highlight how parasite proteins destined for export are recognised at the endoplasmic reticulum to facilitate entry into the export pathway and how the effector proteins are able to transverse the bounding parasitophorous vaculoar membrane (PVM) via the Plasmodium translocon of exported proteins to gain access to the host cell. Some of the gaps in our understanding of the export pathway are also presented. Finally, we examine the degree of conservation of some of the key components of the Plasmodium export pathway in closely related apicomplexan parasites, which may provide insight into how the diverse apicomplexan parasites have adapted to survival pressures encountered within their respective host cells.
... Chaperones of this family are members of the large AAA ATPase superfamily of enzymes that catalyze mechanical processes such as locomotion, unwinding, disassembly and unfolding of other macromolecules (Maurizi and Xia, 2004). Hsp100/Clp chaperones are divided into two classes. ...
... All members of this family have a conserved structural corethe AAA module that consists of 2 subdomains -a large α/β domain consisting of five-stranded parallel β-sheets, flanked by α-helices and connected by a mobile linker to a smaller helical C-terminal domain. ATP binds between the two domains in a crevice containing Walker A and B motifs (the catalytic residues for ATP hydrolysis) and sensors 1 and 2 which respond to the nucleotide state (Maurizi and Xia, 2004). ClpA and ClpB have two AAA modules in tandem (NBD1 and NBD2) linked to a large helical N-domain, as well as a ClpP-binding loop in the C-terminal module. ...
Abiotic stresses with a dehydration component (drought, salt, and freezing) involve, as a common feature, increased numbers of inactive proteins – denatured, aggregated or oxidatively damaged. Maintaining proteins in their functional conformation, preventing aggregation of non-native proteins, refolding of denatured proteins to their native conformation and removal of non-functional and potentially harmful polypeptides are all vital for cell survival under dehydration stress. To achieve this, plants respond to drought by synthesis of protective proteins such as dehydrins and chaperones and by degradation of irreversibly damaged proteins by proteases. Here we review the important cellular functions of dehydrins, chaperones, proteases and protease inhibitors, together with their role in the response to drought, that make them potential biochemical markers for assessing drought tolerance.
... Chaperones of this family are members of the large AAA ATPase superfamily of enzymes that catalyze mechanical processes such as locomotion, unwinding, disassembly and unfolding of other macromolecules (Maurizi and Xia, 2004). Hsp100/Clp chaperones are divided into two classes. ...
... All members of this family have a conserved structural corethe AAA module that consists of 2 subdomains -a large α/β domain consisting of five-stranded parallel β-sheets, flanked by α-helices and connected by a mobile linker to a smaller helical C-terminal domain. ATP binds between the two domains in a crevice containing Walker A and B motifs (the catalytic residues for ATP hydrolysis) and sensors 1 and 2 which respond to the nucleotide state (Maurizi and Xia, 2004). ClpA and ClpB have two AAA modules in tandem (NBD1 and NBD2) linked to a large helical N-domain, as well as a ClpP-binding loop in the C-terminal module. ...
Abiotic stresses with a dehydration component (drought, salt, and freezing) involve, as a common feature, increased numbers of inactive proteins – denatured, aggregated or oxidatively damaged. Maintaining proteins in their functional conformation, preventing aggregation of non-native proteins, refolding of denatured proteins to their native conformation and removal of non-functional and potentially harmful polypeptides are all vital for cell survival under dehydration stress. To achieve this, plants respond to drought by synthesis of protective proteins such as dehydrins and chaperones and by degradation of irreversibly damaged proteins by proteases. Here we review the important cellular functions of dehydrins, chaperones, proteases and protease inhibitors, together with their role in the response to drought, that make them potential biochemical markers for assessing drought tolerance.
... Chaperones of this family are members of the large AAA ATPase superfamily of enzymes that catalyze mechanical processes such as locomotion, unwinding, disassembly and unfolding of other macromolecules (Maurizi and Xia, 2004). Hsp100/Clp chaperones are divided into two classes. ...
... All members of this family have a conserved structural corethe AAA module that consists of 2 subdomains -a large α/β domain consisting of five-stranded parallel β-sheets, flanked by α-helices and connected by a mobile linker to a smaller helical C-terminal domain. ATP binds between the two domains in a crevice containing Walker A and B motifs (the catalytic residues for ATP hydrolysis) and sensors 1 and 2 which respond to the nucleotide state (Maurizi and Xia, 2004). ClpA and ClpB have two AAA modules in tandem (NBD1 and NBD2) linked to a large helical N-domain, as well as a ClpP-binding loop in the C-terminal module. ...
Abiotic stresses with a dehydration component (drought, salt, and freezing) involve, as a common feature, increased numbers of inactive proteins – denatured, aggregated or oxidatively damaged. Maintaining proteins in their functional conformation, preventing aggregation of non-native proteins, refolding of denatured proteins to their native conformation and removal of non-functional and potentially harmful polypeptides are all vital for cell survival under dehydration stress. To achieve this, plants respond to drought by synthesis of protective proteins such as dehydrins and chaperones and by degradation of irreversibly damaged proteins by proteases. Here we review the important cellular functions of dehydrins, chaperones, proteases and protease inhibitors, together with their role in the response to drought, that make them potential biochemical markers for assessing drought tolerance.
... The Clp chaperones participate in the disaggregation of improperly folded and damaged proteins, facilitation of DNA transposition, selective coordination of substrate degradation, regulation of the inheritance of prion-like factors, and modulation of gene expression [1,4,[7][8][9]. The Clp/Hsp100 family members have been grouped under Class I and II on the basis of the presence of two or one highly conserved nucleotide-binding domains (NBD) [10,11]. The Class 1 proteins, Clp A-E and L, all have two distinct NBDs or AAA+ modules, whereas Class II proteins, Clp X and Y, have only a single AAA+ module [10,11]. ...
... The Clp/Hsp100 family members have been grouped under Class I and II on the basis of the presence of two or one highly conserved nucleotide-binding domains (NBD) [10,11]. The Class 1 proteins, Clp A-E and L, all have two distinct NBDs or AAA+ modules, whereas Class II proteins, Clp X and Y, have only a single AAA+ module [10,11]. Clp A, X and C associate with the oligomeric peptidase, ClpP to form an ATP-dependent protease [12][13][14]. ...
ClpX is a general stress protein which belongs to the heat shock protein, Clp/Hsp100 family of molecular chaperones. ClpX, in association with ClpP degrades proteins in an ATP dependent manner. Some members of the Clp family have been shown to be involved in the pathogenesis of many bacteria. The Helicobacter pylori genome demonstrates the presence of ClpX along with ClpA, ClpB and ClpP, the other members of the caseinolytic protease family. H. pylori ClpX is a 386 amino acid long protein. In this study, we have over-expressed H. pylori ClpX in E. coli, purified the recombinant protein to homogeneity and functionally characterized it. The recombinant H. pylori ClpX showed an inherent ATPase activity and prevented the heat induced aggregation of a model protein in vitro. The chaperonic activity of H. pylori ClpX was dependent on ATP hydrolysis and involved hydrophobic interaction with the substrate protein. Biophysical studies reveal the secondary structure tolerance of ClpX at various temperatures and in the presence of guanidine hydrochloride. The study demonstrates that H. pylori ClpX manifests chaperonic activity in the absence of any adaptor protein.
... CLPP is, together with CLPX, a component of ClpXP, an ATP-dependent serine protease that catalyzes unfolding and degradation of misfolded mitochondrial proteins (33). CLPP is highly conserved between bacteria and eukaryota. ...
... In bacteria, ClpXP is a barrel-shaped oligomer composed of a double heptameric ring of ClpP bordered by hexameric rings of ClpX or ClpA, with a proteasome-like overall architecture (34)(35)(36). ClpX and ClpA present the substrates in an unfolded conformation to the ClpP protease (33). Human CLPP, identified through its homology to the Escherichia coli ClpP (37), contains an N-terminal mitochondria targeting sequence (37). ...
A core challenge in cancer immunotherapy is to understand the basis for efficacious vaccine responses in human patients. In previous work we identified a melanoma patient who displayed a low-level antivaccine cytolytic T-cell (CTL) response in blood with tumor regression after vaccination with melanoma antigens (MAGE). Using a genetic approach including T-cell receptor β (TCRβ) cDNA libraries, we found very few antivaccine CTLs in regressing metastases. However, a far greater number of TCRβ sequences were found with several of these corresponding to CTL clones specific for nonvaccine tumor antigens, suggesting that antigen spreading was occurring in regressing metastases. In this study, we found another TCR belonging to tumor-specific CTL enriched in regressing metastases and detectable in blood only after vaccination. We used the TCRβ sequence to detect and clone the desired T cells from tumor-infiltrating lymphocytes isolated from the patient. This CD8 clone specifically lysed autologous melanoma cells and displayed HLA-A2 restriction. Its target antigen was identified as the mitochondrial enzyme caseinolytic protease. The target antigen gene was mutated in the tumor, resulting in production of a neoantigen. Melanoma cell lysis by the CTL was increased by IFN-γ treatment due to preferential processing of the antigenic peptide by the immunoproteasome. These results argue that tumor rejection effectors in the patient were indeed CTL responding to nonvaccine tumor-specific antigens, further supporting our hypothesis. Among such antigens, the mutated antigen we found is the only antigen against which no T cells could be detected before vaccination. We propose that antigen spreading of an antitumor T-cell response to truly tumor-specific antigens contributes decisively to tumor regression.
... In cooperation with the Hsp70/DnaK system the sHsp-bound proteins can be released and refolded into their native states (Veinger et al., 1998; Haslbeck 2002). The Hsp100/Clp family of chaperones dissolve protein aggregates and disassemble protein structures (Maurizi and Di 2004; Weibezahn et al., 2004). These proteins are divided into two subfamilies with distinct enzymatic functions. ...
... The ClpB subfamily displays protein disaggregating activity that is used in co-operation with Hsp70/DnaK systems (Ben-Zvi and Goloubinoff 2001) whereas members of the ClpA subfamily have unfolding activity and act primarily in cooperation with proteases, such as ClpP and ClpQ, to catalyze proteolysis (Horwich et al., 1999; Weibezahn et al., 2004) but may act as molecular chaperone independently of the proteases as well (Wickner et al., 1994; Wawrzynow et al., 1996). Hsp100 proteins belong to the AAA + superfamily (ATPase associated proteins with various cellular activities), which is a ubiquitous family of ATP-dependent proteins (Schirmer et al., 1996; Maurizi and Di 2004). Morever, Hsp100 proteins assemble into hexameric or heptameric rings (Beuron et al., 1998; Ortega et al., 2000; Sousa et al., 2000) reminiscent to GroEL but exhibit no sequence similarity to chaperonins (Schirmer et al., 1996; Horwich et al., 1999). ...
... ClpB is a large protein forming hexameric structures. ClpB proteins form a family of ATPases that disaggregate and solubilise aggregated proteins [41][42][43][44]. Similar to other prokaryotic ClpBs, ClpB of Mtb is composed of an N-terminal domain (NTD) and two nucleotide-binding domains (NBD1 and NBD2), which form a ring-shaped hexamer ( Figure 3). ...
Tuberculosis (TB) is the leading global cause of death f rom an infectious bacterial agent. Therefore, limiting its epidemic spread is a pressing global health priority. The chaperone-like protein HtpG of M. tuberculosis (Mtb) is a large dimeric and multi-domain protein with a key role in Mtb pathogenesis and promising antigenic properties. This dual role, likely associated with the ability of Heat Shock proteins to act both intra- and extra-cellularly, makes HtpG highly exploitable both for drug and vaccine development. This review aims to gather the latest updates in HtpG structure and biological function, with HtpG operating in conjunction with a large number of chaperone molecules of Mtb. Altogether, these molecules help Mtb recovery after exposure to host-like stress by assisting the whole path of protein folding rescue, from the solubilisation of aggregated proteins to their refolding. Also, we highlight the role of structural biology in the development of safer and more effective subunit antigens. The larger availability of structural information on Mtb antigens and a better understanding of the host immune response to TB infection will aid the acceleration of TB vaccine development.
... The latter two explanations both deserve consideration. Our study did not cover the other E. coli chaperone systems such as TF (3,83), small heat shock proteins (70,84), the ClpB disaggregase (85)(86)(87), or HtpG-a foldase that operates with the DnaK system (88, 89)-all of which might play important roles in refolding certain clients. Nevertheless, several additional lines of evidence support the view that the 105 chaperone-nonrefolders fold cotranslationally. ...
The journey by which proteins navigate their energy landscapes to their native structures is complex, involving (and sometimes requiring) many cellular factors and processes operating in partnership with a given polypeptide chain’s intrinsic energy landscape. The cytosolic environment and its complement of chaperones play critical roles in granting many proteins safe passage to their native states; however, it is challenging to interrogate the folding process for large numbers of proteins in a complex background with most biophysical techniques. Hence, most chaperone-assisted protein refolding studies are conducted in defined buffers on single purified clients. Here, we develop a limited proteolysis–mass spectrometry approach paired with an isotope-labeling strategy to globally monitor the structures of refolding Escherichia coli proteins in the cytosolic medium and with the chaperones, GroEL/ES (Hsp60) and DnaK/DnaJ/GrpE (Hsp70/40). GroEL can refold the majority (85%) of the E. coli proteins for which we have data and is particularly important for restoring acidic proteins and proteins with high molecular weight, trends that come to light because our assay measures the structural outcome of the refolding process itself, rather than binding or aggregation. For the most part, DnaK and GroEL refold a similar set of proteins, supporting the view that despite their vastly different structures, these two chaperones unfold misfolded states, as one mechanism in common. Finally, we identify a cohort of proteins that are intransigent to being refolded with either chaperone. We suggest that these proteins may fold most efficiently cotranslationally, and then remain kinetically trapped in their native conformations.
... Plants, bacteria, and fungi rescue aggregated proteins using a powerful ClpB/DnaK bi-chaperone system (Mogk et al., 2015). ClpB proteins form a family of AAA + ATPases that disaggregate and solubilize aggregated proteins (Doyle and Wickner, 2009;Lee et al., 2004;Maurizi and Xia, 2004;Shorter and Southworth, 2019). These disaggregases are traditionally called ClpB in bacteria, and the analogous enzyme in eukaryotes is called Hsp100. ...
The M. tuberculosis (Mtb) ClpB is a protein disaggregase that helps to rejuvenate the bacterial cell. DnaK is a protein foldase that can function alone, but it can also bind to the ClpB hexamer to physically couple protein disaggregation with protein refolding, although the molecular mechanism is not well understood. Here, we report the cryo-EM analysis of the Mtb ClpB-DnaK bi-chaperone in the presence of ATPγS and a protein substrate. We observe three ClpB conformations in the presence of DnaK, identify a conserved TGIP loop linking the oligonucleotide/oligosaccharide-binding domain and the nucleotide-binding domain that is important for ClpB function, derive the interface between the regulatory middle domain of the ClpB and the DnaK nucleotide-binding domain, and find that DnaK binding stabilizes, but does not bend or tilt, the ClpB middle domain. We propose a model for the synergistic actions of aggregate dissolution and refolding by the Mtb ClpB-DnaK bi-chaperone system.
... For proteins detected only in Nps1* or in AC, the missing values were replaced with 1/10 of the lowest value in the dataset, and the heat maps were generated with normalized log2 abundances. Carotenogenic proteins that are aggregated or misfolded can be folded into their active conformation by Clp B protease system in the presence of ATP together with Hsp 70 and DNAJ proteins (Maurizi and Xia, 2004). Alternatively, the above misfolded or aggregated proteins can be degraded by Clp C/Clp protease system or facilitated for refolding (Nishimura and van Wijk, 2015;Pulido et al., 2016;D'Andrea et al., 2018). ...
Phototropins, the UVA‐blue light photoreceptors, endow plants to detect the direction of light and optimize photosynthesis by regulating chloroplasts positioning and stomatal gas exchange. Little is known about their functions in other developmental responses. A tomato Non‐phototropic seedling1 (Nps1) mutant, bearing an Arg495His substitution in the vicinity of LOV2 domain in phototropin1, dominant‐negatively blocks phototropin1 responses. The fruits of Nps1 mutant were enriched in carotenoids, particularly lycopene, than its parent, Ailsa Craig. Contrarily, CRISPR/CAS9‐edited loss of function phototropin1 mutants displayed subdued carotenoids than the parent. The enrichment of carotenoids in Nps1 fruits is genetically linked with the mutation and exerted in a dominant‐negative fashion. Nps1 also altered volatile profiles with high levels of lycopene‐derived 6‐methyl 5‐hepten2‐one. The transcript levels of several MEP and carotenogenesis pathways genes were upregulated in Nps1. Nps1 fruits showed altered hormonal profiles with subdued ethylene emission and reduced respiration. Proteome profiles showed a causal link between higher carotenogenesis and increased levels of protein protection machinery, which may stabilize proteins contributing to MEP and carotenogenesis pathways. The enhancement of carotenoid content by Nps1 in a dominant‐negative fashion offers a potential tool for high lycopene‐bearing hybrid tomatoes.
... Four types of motifs were found in all MdSMXLs with similar distributions (Fig. 5a) and these were subsequently identified using the SMART program. Both motif 1 and motif 2 were Clp-N domains that could act as protein binding sites (Maurizi and Xia 2004). Motif 3 was a P-loop NTPase domain, which is characterized by a conserved nucleotide phosphate-binding motif (Aravind et al. 2004). ...
Strigolactones (SLs) are a recently discovered type of plant hormone that controls various developmental processes. The DWARF53 (D53) protein in rice and the SMAX1-LIKE (SMXL) family in Arabidopsis repress SL signaling. In this study, bioinformatics analyses were performed, and 236 SMXL proteins were identified in 28 sequenced plants. A phylogenetic analysis indicated that all potential SMXL proteins could be divided into three groups and that the SMXL proteins may have originated in Bryophytes. An analysis of the SMXL chromosomal locations suggested that gene duplication events at different times led to expansion of the SMXL family members in Angiospermae. Subsequently, the gene structure and protein modeling of MdSMXLs showed that they are highly conserved. The expression patterns of MdSMXLs indicated that they were expressed in different organs of apple (stems, roots, leaves, flowers, and fruits) at varying levels and that MdSMXLs may participate in the SL signaling pathway and the response to abiotic stress. This study provides a valuable foundation for additional investigations into the function of the SMXL gene family in plants.
... 39,40 N-domains can contact substrates directly 41,42 or via adaptor proteins. 43 The hexameric association state and ATPase-dependent chaperone activity of the apicoplastic ClpB1 have been demonstrated in vitro; however nothing is known about its roles in vivo; it might be involved in maintaining a functional apicoplastic proteome during red blood cell infection. 44 To aid design novel anti-malarials using computational tools and combinatorial screening of libraries, knowledge of the three-dimensional structure of the targeted component is a prerequisite. ...
Caseinolytic chaperones and proteases (Clp) belong to the AAA+ protein superfamily and are part of the protein quality control machinery in cells. The eukaryotic parasite Plasmodium falciparum, the causative agent of malaria, has evolved an elaborate network of Clp proteins including two distinct ClpB ATPases. ClpB1 and ClpB2 are involved in different aspects of parasitic proteostasis. ClpB1 is present in the apicoplast, a parasite-specific and plastid-like organelle hosting various metabolic pathways necessary for parasite growth. ClpB2 localizes to the parasitophorous vacuole membrane where it drives protein export as core subunit of a parasite-derived protein secretion complex, the Plasmodium Translocon of Exported proteins (PTEX); this process is central to parasite virulence and survival in the human host. The functional associations of these two chaperones with parasite-specific metabolism and protein secretion make them prime drug targets. ClpB proteins function as unfoldases and disaggregases and share a common architecture consisting of four domains - a variable N-terminal domain that binds different protein substrates, followed by two highly conserved catalytic ATPase domains, and a C-terminal domain. Here, we report and compare the first crystal structures of the N terminal domains of ClpB1 and ClpB2 from Plasmodium and analyze their molecular surfaces. Solution scattering analysis of the N domain of ClpB2 shows that the average solution conformation is similar to the crystalline structure. These structures represent the first step towards the characterization of these two malarial chaperones and the reconstitution of the entire PTEX to aid structure-based design of novel anti-malarial drugs. This article is protected by copyright. All rights reserved.
© 2015 The Protein Society.
... Hsp104/ClpB belongs to the Hsp100/Clp superfamily of ATPase associated with various cellular activities (AAA+) (Dougan et al. 2002;Neuwald et al. 1999), which form large hexameric ring structures in an ATP-dependent manner (Lee et al. 2003;Wang et al. 2001;Zolkiewski et al. 1999). Hsp100/Clp superfamily proteins contain central components of the protein quality-control system that degrade or disaggregate unfolded and aggregated proteins (Maurizi and Xia 2004;Mogk et al. 2008;Sauer et al. 2004). ...
... In any case, a direct physical interaction between the ClpT proteins and the Hsp100 chaperones is necessary. Hsp100 chaperones have protein remodeling activities; i.e., the ability to change the biological activity of a protein complex by modifying its structure [39,40]. We speculate that chloroplastic Hsp100 chaperones may exert this ability in order to remodel the ClpT proteins. ...
Background
The caseinolytic protease (Clp) is crucial for chloroplast biogenesis and proteostasis. The Arabidopsis Clp consists of two heptameric rings (P and R rings) assembled from nine distinct subunits. Hsp100 chaperones (ClpC1/2 and ClpD) are believed to dock to the axial pores of Clp and then transfer unfolded polypeptides destined to degradation. The adaptor proteins ClpT1 and 2 attach to the protease, apparently blocking the chaperone binding sites. This competition was suggested to regulate Clp activity. Also, monomerization of ClpT1 from dimers in the stroma triggers P and R rings association. So, oligomerization status of ClpT1 seems to control the assembly of the Clp protease.ResultsIn this work, ClpT1 was obtained in a recombinant form and purified. In solution, it mostly consists of monomers while dimers represent a small fraction of the population. Enrichment of the dimer fraction could only be achieved by stabilization with a crosslinker reagent. We demonstrate that ClpT1 specifically interacts with the Hsp100 chaperones ClpC2 and ClpD. In addition, ClpT1 stimulates the ATPase activity of ClpD by more than 50% when both are present in a 1:1 molar ratio. Outside this optimal proportion, the stimulatory effect of ClpT1 on the ATPase activity of ClpD declines.Conclusions
The accessory protein ClpT1 behaves as a monomer in solution. It interacts with the chloroplastic Hsp100 chaperones ClpC2 and ClpD and tightly modulates the ATPase activity of the latter. Our results provide new experimental evidence that may contribute to revise and expand the existing models that were proposed to explain the roles of this poorly understood regulatory protein.
... Hsp104/ClpB belongs to the Hsp100/Clp superfamily of ATPase associated with various cellular activities (AAA+) (Dougan et al. 2002;Neuwald et al. 1999), which form large hexameric ring structures in an ATP-dependent manner (Lee et al. 2003;Wang et al. 2001;Zolkiewski et al. 1999). Hsp100/Clp superfamily proteins contain central components of the protein quality-control system that degrade or disaggregate unfolded and aggregated proteins (Maurizi and Xia 2004;Mogk et al. 2008;Sauer et al. 2004). ...
... Hsp104/ClpB belongs to the Hsp100/Clp superfamily of ATPase associated with various cellular activities (AAA+) (Dougan et al. 2002;Neuwald et al. 1999), which form large hexameric ring structures in an ATP-dependent manner (Lee et al. 2003;Wang et al. 2001;Zolkiewski et al. 1999). Hsp100/Clp superfamily proteins contain central components of the protein quality-control system that degrade or disaggregate unfolded and aggregated proteins (Maurizi and Xia 2004;Mogk et al. 2008;Sauer et al. 2004). ...
Several molecular chaperones are known to be involved in bacteria stress response. To investigate the role of chaperone ClpB in rhizobia stress tolerance as well as in the rhizobia-plant symbiosis process, the clpB gene from a chickpea microsymbiont, strain Mesorhizobium ciceri LMS-1, was identified and a knockout mutant was obtained. The ClpB knockout mutant was tested to several abiotic stresses, showing that it was unable to grow after a heat shock and it was more sensitive to acid shock than the wild-type strain. A plant-growth assay performed to evaluate the symbiotic performance of the clpB mutant showed a higher proportion of ineffective root nodules obtained with the mutant than with the wild-type strain. Nodulation kinetics analysis showed a 6- to 8-day delay in nodule appearance in plants inoculated with the ΔclpB mutant. Analysis of nodC gene expression showed lower levels of transcript in the ΔclpB mutant strain. Analysis of histological sections of nodules formed by the clpB mutant showed that most of the nodules presented a low number of bacteroids. No differences in the root infection abilities of green fluorescent protein-tagged clpB mutant and wild-type strains were detected. To our knowledge, this is the first study that presents evidence of the involvement of the chaperone ClpB from rhizobia in the symbiotic nodulation process.
... They possess a wide range of functional roles, including protein folding assistance, protein degradation, disaggregation of denatured polypeptides and assembly of large molecular complexes123. Most (if not all) Clp/ Hsp100 proteins act as independent chaperones but some associate with oligomeric barrel-shaped proteases to degrade irreversibly damaged proteins [4,5]. Notable examples are ClpA and ClpX from Escherichia coli and ClpC from Bacillus subtilis and Synechococcus elongatus, which couple to their respective ClpP protease678. ...
Clp/Hsp100 chaperones are involved in protein quality control. They act as independent units or in conjunction with a proteolytic core to degrade irreversibly damaged proteins. Clp chaperones from plant chloroplasts have been also implicated in the process of precursor import, along with Hsp70 chaperones. They are thought to pull the precursors in as the transit peptides enter the organelle. How Clp chaperones identify their substrates and engage in their processing is not known. This information may lie in the position, sequence or structure of the Clp recognition motifs.
We tested the influence of the position of the transit peptide on the interaction with two chloroplastic Clp chaperones, ClpC2 and ClpD from Arabidopsis thaliana (AtClpC2 and AtClpD). The transit peptide of ferredoxin-NADP+ reductase was fused to either the N- or C-terminal end of glutathione S-transferase. Another fusion with the transit peptide interleaved between two folded proteins was used to probe if AtClpC2 and AtClpD could recognize tags located in the interior of a polypeptide. We also used a mutated transit peptide that is not targeted by Hsp70 chaperones (TP1234), yet it is imported at a normal rate. The fusions were immobilized on resins and the purified recombinant chaperones were added. After a washing protocol, the amount of bound chaperone was assessed. Both AtClpC2 and AtClpD interacted with the transit peptides when they were located at the N-terminal position of a protein, but not when they were allocated to the C-terminal end or at the interior of a polypeptide.
AtClpC2 and AtClpD have a positional preference for interacting with a transit peptide. In particular, the localization of the signal sequence at the N-terminal end of a protein seems mandatory for interaction to take place. Our results have implications for the understanding of protein quality control and precursor import in chloroplasts.
... The ADEPs were later chemically optimized to address issues related to potency and aqueous solubility (Hinzen et al., 2006). The protein target of the ADEPs, ClpP protease, is a tetradecameric serine protease comprised of two stacked heptameric rings, which, in Escherichia coli, can form complexes with the AAA+ ATPase chaperones ClpX or ClpA (Katayama et al., 1988;Maurizi and Xia, 2004). ClpX and ClpA are hexameric chaperones that bind on one or both ends of the ClpP protease. ...
ClpP is a cylindrical serine protease whose ability to degrade proteins is regulated by the unfoldase ATP-dependent chaperones. ClpP on its own can only degrade small peptides. Here, we used ClpP as a target in a high-throughput screen for compounds, which activate the protease and allow it to degrade larger proteins, hence, abolishing the specificity arising from the ATP-dependent chaperones. Our screen resulted in five distinct compounds, which we designate as Activators of Self-Compartmentalizing Proteases 1 to 5 (ACP1 to 5). The compounds are found to stabilize the ClpP double-ring structure. The ACP1 chemical structure was considered to have drug-like characteristics and was further optimized to give analogs with bactericidal activity. Hence, the ACPs represent classes of compounds that can activate ClpP and that can be developed as potential novel antibiotics.
... DE-TA dominant-negative function AAA+ ATPases form typical homohexameric ring structures, which are essential for their ATPase activity [46,63]. ...
DYT1 dystonia is an autosomal-dominant movement disorder, characterised by early onset of involuntary sustained muscle contractions. It is caused by a 3-bp deletion in the DYT1 gene, which results in the deletion of a single glutamate residue in the C-terminus of the protein torsinA. TorsinA is a member of the AAA-ATPase family of; chaperones with multiple functions in the cell. There is no evidence of neurodegeneration in DYT1 dystonia, which suggests that mutant torsinA leads to functional neuronal abnormalities leading to dystonic movements. In the recent years, different functional roles have been attributed to torsinA, including being a component of the cytoskeleton and the nuclear envelope, and involvement in the secretory pathway and synaptic vesicle machinery. The aim of this review is to summarise these findings and the different models proposed, which have contributed to our current understanding of the function of torsinA.
... To date an HSP100 mitochondrial homologue has not been found in humans (Tatsuta 2009). This family possesses ATPase activity and represses the formation of aggregates prompted by denatured proteins (Maurizi and Xia 2004). Additionally, if aggregation occurs due to the inability of the heat shock proteins to handle an overwhelming amount of denatured proteins, HSP100 family proteins have the ability to facilitate the resolubilization of aggregates (Glover and Tkach 2001). ...
Heat shock protein 90 (HSP90) is an abundant molecular chaperone that regulates the functional stability of client oncoproteins, such as STAT3, Raf-1 and Akt, which play a role in the survival of malignant cells. The chaperone function of HSP90 is driven by the binding and hydrolysis of ATP. The geldanamycin analog, 17-AAG, binds to the ATP pocket of HSP90 leading to the degradation of client proteins. However, treatment with 17-AAG results in the elevation of the levels of antiapoptotic proteins HSP70 and HSP27, which may lead to cell death resistance. The increase in HSP70 and HSP27 protein levels is due to the activation of the transcription factor HSF-1 binding to the promoter region of HSP70 and HSP27 genes. HSF-1 binding subsequently promotes HSP70 and HSP27 gene expression. Based on this, I hypothesized that inhibition of transcription/translation of HSP or client proteins would enhance 17-AAG-mediated cytotoxicity. Multiple myeloma (MM) cell lines MM.1S, RPMI-8226, and U266 were used as a model. To test this hypothesis, two different strategies were used.
For the first approach, a transcription inhibitor was combined with 17-AAG. The established transcription inhibitor Actinomycin D (Act D), used in the clinic, intercalates into DNA and blocks RNA elongation. Stress inducible (HSP90á, HSP70 and HSP27) and constitutive (HSP90â and HSC70) mRNA and protein levels were measured using real time RT-PCR and immunoblot assays. Treatment with 0.5 µM 17-AAG for 8 hours resulted in the induction of all HSP transcript and protein levels in the MM cell lines. This induction of HSP mRNA levels was diminished by 0.05 µg/mL Act D for 12 hours in the combination treatment, except for HSP70. At the protein level, Act D abrogated the 17-AAG-mediated induction of all HSP expression levels, including HSP70. Cytotoxic evaluation (Annexin V/7-AAD assay) of Act D in combination with 17-AAG suggested additive or more than additive interactions.
For the second strategy, an agent that affected bioenergy production in addition to targeting transcription and translation was used. Since ATP is necessary for the proper folding and maturation of client proteins by HSP90, ATP depletion should lead to a decrease in client protein levels. The transcription and translation inhibitor 8-Chloro-Adenosine (8-Cl-Ado), currently in clinical trials, is metabolized into its cytotoxic form 8-Cl-ATP causing a parallel decrease of the cellular ATP pool. Treatment with 0.5 µM 17-AAG for 8 hours resulted in the induction of all HSP transcript and protein levels in the three MM cell lines evaluated. In the combination treatment, 10 µM 8-Cl-Ado for 20 hours did not abrogate the induction of HSP mRNA or protein levels. Since cellular bioenergy is necessary for the stabilization of oncoproteins by HSP90, immunoblot assays analyzing for expression levels of client proteins such as STAT3, Raf-1, and Akt were performed. Immunoblot assays detecting for the phosphorylation status of the translation repressor 4E-BP1, whose activity is modulated by upstream kinases sensitive to changes in ATP levels, were also performed. The hypophosphorylated state of 4E-BP1 leads to translation repression. Data indicated that treatment with 17-AAG alone resulted in a minor (
... Bacterial ClpB is an AAA+ molecular machine that uses the energy of ATP binding and hydrolysis to extract unfolded protein monomers from aggregates, and to remodel and translocate them through its axial channel for productive reactivation [1][2][3]. Disaggregation and reactivation of protein aggregates in the cell requires the combined action of ClpB and the DnaK system, that alone are unable to reactivate large insoluble aggregates [4][5][6][7]. As a member of class I of the Hsp100 chaperone family, ClpB contains two nucleotide binding sites (NBD) per protomer [8]. ...
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.
Antimicrobial resistance (AMR) to currently available antibiotics/drugs is a global threat. It is desirable to develop new drugs that work through a novel target(s) to avoid drug resistance. This review discusses the potential of the caseinolytic protease P (ClpP) peptidase complex as a novel target for finding novel antibiotics, emphasising the ClpP’s structure and function. ClpP contributes to the survival of bacteria via its ability to destroy misfolded or aggregated proteins. In consequence, its inhibition may lead to microbial death. Drugs inhibiting ClpP activity are currently being tested, but no drug against this target has been approved yet. It was demonstrated that Nblocked dipeptides are essential for activating ClpP’s proteolytic activity. Hence, compounds mimicking these dipeptides could act as inhibitors of the formation of an active ClpP complex. Drugs, including Bortezomib, Cisplatin, Cefmetazole, and Ixazomib, inhibit ClpP activation. However, they were not approved as drugs against the target because of their high toxicity, likely due to the presence of strong electrophiles in their warheads. The modifications of these warheads could be a good strategy to reduce the toxicity of these molecules. For instance, a boronate warhead was replaced by a chloromethyl ketone, and this new molecule was shown to exhibit selectivity for prokaryotic ClpP. A better understanding of the structure and function of the ClpP complex would benefit the search for compounds mimicking N-blocked dipeptides that would inhibit ClpP complex activity and cause bacterial death.
Multi-drug-resistant bacteria present an urgent threat to modern medicine, creating a desperate need for antibiotics with new modes of action. As natural products remain an unsurpassed source for clinically viable antibiotic compounds, we investigate the mechanism of action of armeniaspirol. The armeniaspirols are a structurally unique class of Gram-positive antibiotic discovered from Streptomyces armeniacus for which resistance cannot be readily obtained. We show that armeniaspirol inhibits the ATP-dependent proteases ClpXP and ClpYQ in vitro and in the model Gram-positive Bacillus subtilis. This inhibition dysregulates the divisome and elongasome supported by an upregulation of key proteins FtsZ, DivIVA, and MreB inducing cell division arrest. The inhibition of ClpXP and ClpYQ to dysregulate cell division represents a unique antibiotic mechanism of action and armeniaspirol is the only known natural product inhibitor of the coveted anti-virulence target ClpP. Thus, armeniaspirol possesses a promising lead scaffold for antibiotic development with unique pharmacology.
Obligate intracellular malaria parasites reside within a vacuolar compartment generated during invasion which is the principal interface between pathogen and host. To subvert their host cell and support their metabolism, these parasites coordinate a range of transport activities at this membrane interface that are critically important to parasite survival and virulence, including nutrient import, waste efflux, effector protein export, and uptake of host cell cytosol. Here, we review our current understanding of the transport mechanisms acting at the malaria parasite vacuole during the blood and liver-stages of development with a particular focus on recent advances in our understanding of effector protein translocation into the host cell by the Plasmodium Translocon of EXported proteins (PTEX) and small molecule transport by the PTEX membrane-spanning pore EXP2. Comparison to Toxoplasma gondii and other related apicomplexans is provided to highlight how similar and divergent mechanisms are employed to fulfill analogous transport activities.
Heat shock proteins (HSP) are class of conserved and ubiquitous molecular chaperones present in all living organisms from primitive bacteria to humans. Numerous evidences accumulated last decades have proven multiple functions of HSP in aquatic organisms. Besides fundamentally respond to thermal stress, HSP are also stimulated by other extracellular stresses including salinity, pH, hypoxia, pollutants and pathogens. The induction of HSP towards multiple environmental stressors is to protect aquatic organisms. The mechanism and pathway involved in HSP induction are relatively complicated but are systematic to most cellular stressors. Given the vital functions of HSP in cellular protein protection and immunity defences, HSP-based vaccines and HSP-induced compounds have been developed and applied for the health management of aquatic organisms. Aquaculture species treated with these products have shown increased HSP productions, which have effectively, protect them against various stresses. Application of HSP based therapy in aquaculture practices proves as a promising approach in boosting aquaculture production and sustaining food security.
Proteins with genetic or acquired defects tend to misfold, aggregate, and precipitate, impoverishing the pool of potentially useful protein molecules available for folding. In addition, defective peptides and their precipitates can be toxic. Therefore, faulty peptides must be salvaged, i.e., folded, or eliminated by proteases. This article presents proteases that occur in prokaryotes, archaea and bacteria, and the eukaryotic cell organelles, including those derived from prokaryotes.
Cells respond to an increased extracellular tonicity initially with a net uptake of inorganic electrolytes to restore ionic balance with the environment and to counteract cell shrinkage. However, high intracellular concentrations of inorganic ions may lead to enhanced accumulation of misfolded or aggregated proteins. To prevent excess protein denaturation, cells induce various osmoprotective mechanisms. One of the most conserved mechanisms is the expression of protein-stabilizing heat shock proteins (HSPs). Elevated cellular levels of HSPs (or their homologues) have been found in numerous organisms during osmotic stress, such as the enterobacterium Escherichia coli, the yeast strain Saccharomyces cerevisiae, the plant Arabidopsis thaliana (Thale cress), the nematode Caenorhabditis elegans, the fish Sparus sarba (silver sea bream) as well as in mammals like mice or rats, illustrating the important osmoprotective function of HSPs in the whole alive nature. The present article provides an overview of HSP function during osmotic stress in various pro-and eukaryotic organisms. The main focus lies on the role of HSPs in mammalian cells and especially in cells of the mammalian kidney, where cells are routinely exposed to high osmolarities in the renal medulla. The abundance of these HSPs in the kidney often correlates with the corticomedullary osmotic gradient, with low amounts in the isoosmotic cortex and highest amounts in the inner medulla, contributing to the remarkable abillity of these cells to deal with the high osmolarities observed in these tissues.
Proteasomes are large ATP-dependent proteases implicated in degradation of key regulatory cellular proteins and abnormal cellular proteins. The current work is the first characterization of two AAA-ATPase proteasomal regulatory proteins PAN A and PAN B in halophilic archaea. The first part of the manuscript characterizes the mode of regulation of the two PANs in the extremely halophilic archaoen Halobacterium salinarium. Our experiments show the existence of two isoforms of each PAN proteins in the cells. The two isoforms differ in their N-terminal regions and result probably from the use of two alternative transcription start sites. Primer extension methods demonstrate the existence of heterogeneity in PAN's mRNA as revealed by mapping of the 5' non coding regions. The second part of the manuscript shed lights on oligomerisation and association of the PAN proteins in cellulo. Fractionation of cell free extracts on sucrose gradients indicates a weak degree of oligomerisation of the PANs and the absence of stable complexes with the proteasome 20 S. The last part of this work focuses on the regulation of PAN A and PAN B gene expression in response to stress including heat shock and salt shock. Our results clearly show that the two proteasome activating proteins are not regulated in a similar way in response to environmental stress
Extreme halophiles are microorganisms adapted to low water activity living at the upper salt concentration that life can tolerate. We review here recent data that specify the main factors, which determine their peculiar salt-dependent biochemistry. The data suggested that evolution proceeds by stage to modify the molecular dynamics properties of the entire proteome. Extreme halophiles therefore represent tractable models to understand how fast and to what extent microorganisms adapt to environmental changes. Halophiles are also robust organisms, capable to resist multiple stressors. Preliminary studies indicated that they have developed a cellular response specifically aimed to survive when the salt condition fluctuates. Because of these properties halophilic organisms deserve special attention in the search for traces of life on other planets.
In this study, we investigated the antimicrobial effect of tea polyphenols (TP) against Serratia marcescens and examined the related mechanism. Morphology changes of S. marcescens were first observed by transmission electron microscopy after treatment with TP, which indicated that the primary inhibition action of TP was to damage the bacterial cell membranes. The permeability of the outer and inner membrane of S. marcescens dramatically increased after TP treatment, which caused severe disruption of cell membrane, followed by the release of small cellular molecules. Furthermore, a proteomics approach based on two-dimensional gel electrophoresis and MALDI-TOF/TOF MS analysis was used to study the difference of membrane protein expression in the control and TP treatment S. marcescens. The results showed that the expression of some metabolism enzymes and chaperones in TP-treated S. marcescens significantly increased compared to the untreated group, which might result in the metabolic disorder of this bacteria. Taken together, our results first demonstrated that TP had a significant growth inhibition effect on S. marcescens through cell membrane damage.
ClpB is a hexameric molecular chaperone that, together with the DnaK system, has the ability to disaggregate stress-denatured proteins. The hexamer is a highly dynamic complex, able to reshuffle subunits. To further characterize the biological implications of the ClpB oligomerization state, the association equilibrium of the wild-type (wt) protein and of two deletion mutants, which lack part or the whole M domain, was quantitatively analyzed under different experimental conditions, using several biophysical [analytical ultracentrifugation, composition-gradient (CG) static light scattering, and circular dichroism] and biochemical (ATPase and chaperone activity) methods. We have found that (i) ClpB self-associates from monomers to form hexamers and higher-order oligomers that have been tentatively assigned to dodecamers, (ii) oligomer dissociation is not accompanied by modifications of the protein secondary structure, (iii) the M domain is engaged in intersubunit interactions that stabilize the protein hexamer, and (iv) the nucleotide-induced rearrangement of ClpB affects the protein oligomeric core, in addition to the proposed radial extension of the M domain. The difference in the stability of the ATP- and ADP-bound states [ΔΔG(ATP-ADP) = -10 kJ/mol] might explain how nucleotide exchange promotes the conformational change of the protein particle that drives its functional cycle.
The use of food products containing probiotic microorganisms is of increasing economic importance. The health promoting effects of selected probiotic strains has been substantiated in controlled clinical studies. The probiotic nature of the health promoting bacteria is not well studied compared to the virulence of pathogenic bacteria. Microorganisms used in food technology and probiotics are exposed to technological and digestive stresses, such as temperature changes and low pH. Virulence and stress responses are closely related in several Gram-positive bacteria while extremely little is known about the possible overlap of stress responses and the probiotic nature of the bacteria. The available data concerning stress responses of lactobacilli and bifidobacteria mainly cover physiological changes in these bacteria when subject to stress, such as high temperature and low pH, and their ability to survive in different challenges. ClpATPases are a family of stress proteins that are known as virulence factors in a number of pathogenic bacteria, such as Staphylococcus aureus and Streptococcus pneumoniae, and regulators of several vital biological processes in Gram-positive bacteria with a low G+C content. In the first part of this thesis, clpL ATPase encoding genes and their protein products were characterized in two potentially probiotic lactobacilli, Lactobacillus rhamnosus E-97800 and L. gasseri ATCC 33323. Southern blot analysis revealed that among four L. rhamnosus strains only L. rhamnosus E-97800 carried two clpL genes, assigned as clpL1 and clpL2. Expression of both genes were induced after heat stress >20- and 3-fold, respectively. The clpL2 region was found to be mobilized after prolonged cultivation of E-97800 at a high temperature. The sequence analyses revealed that clpL2 shared 98 % identity to L. plantarum clpL gene, clpL2 is flanked by inverted repeat highly identical to the repeats of a functional insertion element in a L. plantarum strain. The data indicate that L. rhamnosus E-97800 has acquired clpL2 region via horizontal gene transfer, propably the donor being a lactobacillar strain. Moreover, the low G+C content (40 %) of clpL2 compared to clpL1 (49 %) and to the average L. rhamnosus entries at GenBank (48 %) together with high identity of clpL2 to the L. plantarum clpL gene indicate that the clpL2 containing element has been transposed relatively recently. Homology searches using clpL genes as query sequences revealed that the number of paralogous clpL genes varies among lactic acid bacteria (LAB). However, the putative selective advantage of this extra clpL paralog to host bacteria remains to be studied, since the stress tolerance of the clpL2-deficient strain was not altered compared to its parental strain. We demonstrated that a CIRCE element, which is known to mediate HrcA-dependent regulation, is located upstream of the clpL1 in L. rhamnosus and clpL in L. gasseri which together with the strong induction fold of clpL1 during heat stress suggest HrcA-mediated regulation of clpL genes. Moreover, we showed that purified HrcA protein is able to specifically bind to the promoter region of clpL in L. gasseri. Thus, the expression of clpL is most likely regulated by the HrcA/CIRCE system in these lactobacilli representing a novel regulon. In the second part of this work, two-dimensional electrophoresis-based (2-DE) tools were applied to investigate stress responses in selected probiotic bacteria. Since probiotic bacteria are adapted to grow in an environment rich in nutrients, the optimization of growth conditions for efficient metabolic labelling to examine protein synthesis kinetics in defined media is needed. A semi-defined medium for metabolic labelling with [35S]methionine for Bifidobacterium longum was developed. This medium was shown to support efficient protein radiolabelling. In addition, chemically defined media and experimental conditions supporting efficient protein radiolabelling with [35S]methionine were developed, and proved to be applicable for a number of lactobacillar strains to investigate their stress responses. Fluorescence 2-D difference gel electrophoresis (DIGE) was applied to study the heat shock response of L. gasseri ATCC 33323. In addition to classical chaperons DnaK and GroEL, four Clp AAA+ (ATPases associated with a variety of cellular activities) ATPases were detected and found to be increased in abundance after a heat shock. One of these, clpL, was deleted by using a thermosensitive vector. It was shown that the functional clpL gene is essential for the development of constitutive and induced thermotolerance in L. gasseri. The expression of several HSPs (heat shock proteins) was at the same level in both clpL deficient and its parental strain indicating that ClpL is not involved in modulation of the heat shock response in L. gasseri. Instead, ClpL probably prevents aggregation of non-native proteins generated by stress and thus the clpL gene might have industrial potential. Probioottisten, terveyttä edistävien, bakteerien käyttö elintarvikkeissa on lisääntymässä. Probioottisten bakteerien tehokkuus ja turvallisuus on osoitettu kliinisissä tutkimuksissa, mutta mekanismeja probioottisten vaikutusten taustalla ei tunneta. Probioottisten bakteereiden biologisista prosesseista tiedetään paljon vähemmän kuin tautia aiheuttavien bakteereiden taudinaiheuttamismekanismeista. Myöskään ei tiedetä miksi vain osa tietyn bakteerisuvun kannoista omaa probioottisia piirteitä. Probioottibakteeri altistuu vaikeille ja muuttuville olosuhteille koko elinkaarensa ajan. Probioottibakteeri joutuu sietämään tuotannon aikana lämmönvaihteluja, varastoinnin aikana suurta happopitoisuutta ja viimein ihmisen ruuansulatuskanavassa vatsa- ja sappihappoja. Tieto biologisista mekanismeista, joiden avulla probioottibakteeri selviytyy stressistä tai sopeutuu siihen, on hyvin rajallista. ClpATPaasit muodostavat proteiiniperheen, joka on säilynyt evoluutiossa kaikissa eliöissä, ja ne ovat tärkeitä säätelijöitä monissa elintärkeissä biologisissa prosesseissa, joissa taudinaiheuttajabakteerit elävät. ClpATPaasien mahdollista roolia probioottisten bakteereiden terveydelle edullisten ominaisuuksien taustalla ei tiedetä. Väitöskirjani ensimmäisessä osassa karakterisoitiin ClpL ATPaasia koodittavia geenejä eräissä laktobasilleissa, joiden suvussa on probioottisia kantoja. Osoittautui, että vain yhdessä neljästä tutkitusta Lactobacillus rhamnosus bakteerikannasta oli kaksi ClpL ATPaasia koodittavaa geeniä. Molemmat clpL-geenit, clpL1 ja clpL2, aktivoituivat korkean lämpötilan aiheuttamassa strressitilassa. clpL2-geeni osoittautui ns. hyppivä geeniksi. Tämä ylimääräinen clpL2-geeni on mitä ilmeisimmin siirtynyt hiljattain jostakin toisesta lactobasillista tähän L. rhamnosus bakteerikantaan. L. rhamnosus bakteerikannalla, josta puuttui ClpL2-proteiinia tuottava geeni, ei havaittu stressisiedoltaan muuttuneita fenotyyppejä. Lactobacillus gasseri bakteerilla, jolla ei ollut toimivaa clpL-geeniä, oli lämmönsiedoltaan ja korkeaan lämpöön sopeutumiseltaan alentunut fenotyyppi, Lisäksi osoitettiin, että clpL-geenin ilmentymistä säätelee L. gasseri -bakteerissa ns. HrcA-proteiini. HrcA-proteiini on tunnettu lämpöstressivasteen säätelijä, mutta sen ei ole osoitettu säätelevän clpL-geenejä muissa bakteerilajeissa. Väitöskirjani toisessa osassa optimoitiin menetelmiä tutkia lukuisten proteiinien ilmentymistä samaan aikaan erilaisissa stressiolosuhteissa proteomiikan keinoin. Proteomiikan menetelmin saadaan tietoa, mitkä proteiinit ovat tärkeitä ko. olosuhteessa ja siitä, mitkä proteiinit mahdollisesti muodostavat toiminnallisia kokonaisuuksia. Proteomiikan menetelmillä voi olla mahdollista tulevaisuudessa tunnistaa probioottisten ominaisuuksien kannalta tärkeitä proteiineja. Tässä työssä tutkittu clpL-geeni omaa huomattavaa kaupallista ja teknologista mielenkiintoa, sillä se on lupaava ehdokas kehitettäväksi mittariksi mittaamaan bakteerin sopeutumista tuotannon stressiolosuhteisiin.
Intra- and extracellular protein misfolding and aggregation is likely to contribute to a number of age-related central nervous system diseases ("proteinopathies"). Therefore, molecular chaperones, such as heat shock proteins (HSPs), that regulate protein folding, misfolding and adaption to cellular stress are emerging as therapeutic targets. Here we review the current knowledge of HSP-modulating drugs and discuss the opportunities and difficulties of their therapeutic use to treat proteinopathies such as Alzheimer's- and Parkinson's disease, the polyglutamine- and prion disorders and Amyotrophic Lateral Sclerosis.
Das hochkonservierte Dank-DnaJ-GrpE Chaperon System schützt Zellen gegen stark erhöhte Umgebungstemperaturen. Unter extremen oxidativen Stressbedingungen verliert DnaK jedoch reversibel seine Aktivität. Dies beruht auf der, durch oxidativen Stress hervorgerufenen Abnahme der intrazellulären ATP Konzentration. Nun werden aggregationssensitive Proteine von dem redox regulierten, ATP-unabhängigen Chaperon Hsp33 gebunden bis Nicht-Stressbedingungen wiederhergestellt sind. DnaK reaktiviert und kann nun die Rückfaltung der an Hsp33 gebundenen Proteine unterstützen. Studien an dem Co-Chaperon DnaJ zeigten eine unabhängige Funktion für die beiden hochkonservierten Zink Zentren. Zn-Zentrum I ist für die autonome Chaperonaktivität von DnaJ verantwortlich, während Zn-Zentrum II unabdingbar für die katalytische Aktivität der DnaK/DnaJ/GrpE Maschinerie ist. Zusätzlich zur J-Domäne von DnaJ muss DnaK mit Zn-Zentrum II in Kontakt treten, um seine hohe Substrataffinität zu erreichen.
Point mutations in either of the two nucleotide-binding domains
(NBD) of Hsp104 (NBD1 and NBD2) eliminate its thermotolerance function
in vivo. In vitro, NBD1 mutations
virtually eliminate ATP hydrolysis with little effect on
hexamerization; analogous NBD2 mutations reduce ATPase activity and
severely impair hexamerization. We report that high protein
concentrations overcome the assembly defects of NBD2 mutants and
increase ATP hydrolysis severalfold, changing
Vmax with little effect on
Km. In a complementary fashion, the
detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
inhibits hexamerization of wild-type (WT) Hsp104, lowering
Vmax with little effect on
Km. ATP hydrolysis exhibits a Hill
coefficient between 1.5 and 2, indicating that it is influenced by
cooperative subunit interactions. To further analyze the effects of
subunit interactions on Hsp104, we assessed the effects of mutant
Hsp104 proteins on WT Hsp104 activities. An NBD1 mutant that
hexamerizes but does not hydrolyze ATP reduces the ATPase activity of
WT Hsp104 in vitro. In vivo, this mutant
is not toxic but specifically inhibits the thermotolerance function of
WT Hsp104. Thus, interactions between subunits influence the ATPase
activity of Hsp104, play a vital role in its biological functions, and
provide a mechanism for conditionally inactivating Hsp104 function
in vivo.
ClpX and ClpA are molecular chaperones that interact with specific proteins and, together with ClpP, activate their ATP-dependent
degradation. The chaperone activity is thought to convert proteins into an extended conformation that can access the sequestered
active sites of ClpP. We now show that ClpX can catalyze unfolding of a green fluorescent protein fused to a ClpX recognition
motif (GFP-SsrA). Unfolding of GFP-SsrA depends on ATP hydrolysis. GFP-SsrA unfolded either by ClpX or by treatment with denaturants
binds to ClpX in the presence of adenosine 5′-O-(3-thiotriphosphate) and is released slowly (t1/2 ≈ 15 min). Unlike ClpA, ClpX cannot trap unfolded proteins in stable complexes unless they also have a high-affinity binding
motif. Addition of ATP or ADP accelerates release (t1/2 ≈ 1 min), consistent with a model in which ATP hydrolysis induces a conformation of ClpX with low affinity for unfolded substrates.
Proteolytically inactive complexes of ClpXP and ClpAP unfold GFP-SsrA and translocate the protein to ClpP, where it remains
unfolded. Complexes of ClpXP with translocated substrate within the ClpP chamber retain the ability to unfold GFP-SsrA. Our
results suggest a bipartite mode of interaction between ClpX and substrates. ClpX preferentially targets motifs exposed in
specific proteins. As the protein is unfolded by ClpX, additional motifs are exposed that facilitate its retention and favor
its translocation to ClpP for degradation.
Escherichia coli ClpX, a member of the Clp family of ATPases, has ATP-dependent chaperone activity and is required for specific ATP-dependent proteolytic activities expressed by ClpP. Gel filtration and electron microscopy showed that ClpX subunits (Mr 46, 000) associate to form a six-membered ring (Mr approximately 280, 000) that is stabilized by binding of ATP or nonhydrolyzable analogs of ATP. ClpP, which is composed of two seven-membered rings stacked face-to-face, interacts with the nucleotide-stabilized hexamer of ClpX to form a complex that could be isolated by gel filtration. Electron micrographs of negatively stained ClpXP preparations showed side views of 1:1 and 2:1 ClpXP complexes in which ClpP was flanked on either one or both sides by a ring of ClpX. Thus, as was seen for ClpAP, a symmetry mismatch exists in the bonding interactions between the seven-membered rings of ClpP and the six-membered rings of ClpX. Competition studies showed that ClpA may have a slightly higher affinity (approximately 2-fold) for binding to ClpP. Mixed complexes of ClpA, ClpX, and ClpP with the two ATPases bound simultaneously to opposite faces of a single ClpP molecule were seen by electron microscopy. In the presence of ATP or nonhydrolyzable analogs of ATP, ClpXP had nearly the same activity as ClpAP against oligopeptide substrates (>10,000 min-1/tetradecamer of ClpP). Thus, ClpX and ClpA interactions with ClpP result in structurally analogous complexes and induce similar conformational changes that affect the accessibility and the catalytic efficiency of ClpP active sites.
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.
ClpA, the regulatory subunit of Clp protease from Escherichia coli, has two ATP-binding sites in non-homologous regions of the protein, referred to as domain I and domain II. We have mutated the invariant lysine in the ATP-binding sites of domain I and domain II and studied the enzymatic properties of the purified mutant ClpA proteins. The domain I mutant, ClpA-K220Q, was unable to form a hexamer in the presence of nucleotide, but the comparable domain II mutant, ClpA-K501Q, associated into a hexamer in the presence of ATP, indicating that nucleotide binding to domain I favors a conformation required to stabilize the quaternary structure of ClpA. ClpA-K220Q was defective in ATPase activity and in the ability to activate protein and peptide degradation by ClpP, but the defects could be partially overcome by formation of hybrid hexamers with wild-type ClpA. Another domain I mutant, ClpA-K220R, readily formed hexamers in the presence of ATP and retained > or = 60% of the wild-type ATPase activity and ability to activate ClpP. These results indicate that hexamer formation is a prerequisite for expression of enzymatic activity. Domain II mutants ClpA-K501Q and ClpA-K501R had very low ATPase activity (< 10% of wild-type) and a severe defect in activation of protein degradation, which requires ATP hydrolysis. Domain II mutants were able to activate ClpP to degrade a peptide whose degradation required nucleotide binding but not hydrolysis. Nucleotide binding to domain II of ClpA is important to form a productive complex with ClpP, and domain II appears to be primarily responsible for an energy-requiring step in the catalytic cycle unique to the degradation of large proteins.
The heat shock protein ClpB in Escherichia coli is a protein-activated ATPase and consists of two proteins with sizes of 93 and 79 kDa. By polymerase chain reaction-aided site-directed mutagenesis, both the proteins have been shown to be encoded by the same reading frame of the clpB gene, the 93-kDa protein (ClpB93) from the 5'-end AUG translational initiation site and the 79-kDa protein (ClpB79) from the 149th codon (an internal GUG start site). Both the purified ClpB93 and ClpB79 proteins behave as tetrameric complexes with a very similar size of about 350 kDa upon gel filtration on a Superose-6 column. Both appear to be exclusively localized to the cytosol of E. coli. Both show inherent ATPase activities and have an identical Km of 1.1 mM for ATP. The ATPase activity of ClpB93 is as markedly stimulated by proteins, including casein and insulin, as that of wild-type ClpB, but the same proteins show little or no effect on ClpB79. Because ClpB79 lacks the 148 N-terminal sequence of ClpB93 but retains the two consensus sequences for adenine nucleotide binding, the N-terminal portion appears to contain a site(s) or domain(s) responsible for protein binding. Furthermore, ClpB79 is capable of inhibiting the casein-activated ATPase activity of ClpB93 in a dose-dependent manner but without any effect on its inherent ATPase activity. In addition, ClpB93 mixed with differing amounts of ClpB79 behave as tetrameric molecules, although its protein-activated ATPase activity is gradually reduced. These results suggest that tetramer formation between ClpB93 and ClpB79 may be responsible for the inhibition of the activity.
CcdA, the antidote protein of the ccd post-segregational killing system carried by the F plasmid, was degraded in vitro by purified Lon protease from Escherichia coli. CcdA had a low affinity for Lon (Km ≥200 µM), and the peptide bond turnover number was ∼10 min−1. CcdA formed tight complexes with purified CcdB, the killer protein encoded in the ccd operon, and fluorescence and hydrodynamic measurements suggested that interaction with CcdB converted CcdA to a more compact
conformation. CcdB prevented CcdA degradation by Lon and blocked the ability of CcdA to activate the ATPase activity of Lon,
suggesting that Lon may recognize bonding domains of proteins exposed when their partners are absent. Degradation of CcdA
required ATP hydrolysis; however, CcdA41, consisting of the carboxyl-terminal 41 amino acids of CcdA and lacking the α-helical
secondary structure present in CcdA, was degraded without ATP hydrolysis. Lon cleaved CcdA primarily between aliphatic and
hydrophilic residues, and CcdA41 was cleaved at the same peptide bonds, indicating that ATP hydrolysis does not affect cleavage
specificity. CcdA lost α-helical structure at elevated temperatures (Tm ∼50°C), and its degradation became independent of ATP hydrolysis at this temperature. ATP hydrolysis may be needed to disrupt
interactions that stabilize the secondary structure of proteins allowing the disordered protein greater access to the proteolytic
active sites.
N-ethylmaleimide-sensitive fusion protein (NSF) is a cytosolic ATPase required for many intracellular vesicle fusion reactions. NSF consists of an amino-terminal region that interacts with other components of the vesicle trafficking machinery, followed by two homologous ATP-binding cassettes, designated D1 and D2, that possess essential ATPase and hexamerization activities, respectively. The crystal structure of D2 bound to Mg2+-AMPPNP has been determined at 1.75 A resolution. The structure consists of a nucleotide-binding and a helical domain, and it is unexpectedly similar to the first two domains of the clamp-loading subunit delta' of E. coli DNA polymerase III. The structure suggests several regions responsible for coupling of ATP hydrolysis to structural changes in full-length NSF.
Heat shock protein 101 (HSP101) cDNA and genomic clones from maize have been isolated. The structure of maize HSP101 reveals the presence of six exons interrupted by five introns. Maize HSP101 contains a predicted open reading frame that translates into a 912-aa sequence with a mass of 101kDa. Initiation of transcription was mapped 146 bases upstream of the AUG codon. Five heat shock element (HSE) boxes were found within the proximal 289 bases of the promoter region. Southern blot analysis of genomic DNA indicates that the maize genome contains only one copy of HSP101. A protein sequence comparison showed that maize Hsp101 belongs to the heat shock 100kDa and caseino-lytic protease B protein family (Hsp100/ClpB) that plays important roles in bacteria and yeast in the survival to extremely high temperatures and the control of proteolysis. Accumulation of HSP101 mRNA was strong under heat shock conditions, but not detectable after cold or osmotic stress treatments or by exogenous application of ABA. The analysis of the predicted supersecondary structure of maize Hsp101 showed that a coiled-coil located in the middle region of the protein is evolutionarily conserved in all members of the Clp A, B and C subfamilies. It is proposed that these supersecondary structures may have important roles in Clp function.
It has previously been established that sequences at the C termini of polypeptide substrates are critical for efficient hydrolysis
by the ClpP/ClpX ATP-dependent protease. We report for the bacteriophage λ O replication protein, however, that N-terminal
sequences play the most critical role in facilitating proteolysis by ClpP/ClpX. The N-terminal portion of λ O is degraded
at a rate comparable with that of wild type O protein, whereas the C-terminal domain of O is hydrolyzed at least 10-fold more
slowly. Consistent with these results, deletion of the first 18 amino acids of λ O blocks degradation of the N-terminal domain,
whereas proteolysis of the O C-terminal domain is only slightly diminished as a result of deletion of the C-terminal 15 amino
acids. We demonstrate that ClpX retains its capacity to bind to the N-terminal domain following removal of the first 18 amino
acids of O. However, ClpX cannot efficiently promote the ATP-dependent binding of this truncated O polypeptide to ClpP, the
catalytic subunit of the ClpP/ClpX protease. Based on our results with λ O protein, we suggest that two distinct structural
elements may be required in substrate polypeptides to enable efficient hydrolysis by the ClpP/ClpX protease: (i) a ClpX-binding
site, which may be located remotely from substrate termini, and (ii) a proper N- or C-terminal sequence, whose exposure on
the substrate surface may be induced by the binding of ClpX.
ClpA, a member of the Clp/Hsp100 family of ATPases, is both an ATP-dependent molecular chaperone and the regulatory component
of ClpAP protease. We demonstrate that chaperone and protease activities occur concurrently in ClpAP complexes during a single
round of RepA binding to ClpAP and ATP-dependent release. This result was substantiated with a ClpA mutant, ClpA(K220V), carrying
an amino acid substitution in the N-terminal ATP binding site. ClpA(K220V) is unable to activate RepA, but the presence of
ClpP or chemically inactivated ClpP restores its ability to activate RepA. The presence of ClpP simultaneously facilitates
degradation of RepA. ClpP must remain bound to ClpA(K220V) for these effects, indicating that both chaperone and proteolytic
activities of the mutant complex occur concurrently. ClpA(K220V) itself is able to form stable complexes with RepA in the
presence of a poorly hydrolyzed ATP analog, adenosine 5′-O-(thiotriphosphate), and to release RepA upon exchange of adenosine 5′-O-(thiotriphosphate) with ATP. However, the released RepA is inactive in DNA binding, indicating that the N-terminal ATP binding
site is essential for the chaperone activity of ClpA. Taken together, these results suggest that substrates bound to the complex
of the proteolytic and ATPase components can be partitioned between release/reactivation and translocation/degradation.
ClpB is a heat-shock protein fromEscherichia coli with an unknown function. We studied a possible molecular chaperone activity of ClpB in vitro. Firefly luciferase was denatured in urea and then diluted into the refolding buffer (in the presence of 5 mm ATP and 0.1 mg/ml bovine serum albumin). Spontaneous reactivation of luciferase was very weak (less than 0.02% of the native
activity) because of extensive aggregation. Conventional chaperone systems (GroEL/GroES and DnaK/DnaJ/GrpE) or ClpB alone
did not reactivate luciferase under those conditions. However, ClpB together with DnaK/DnaJ/GrpE greatly enhanced the luciferase
activity regain (up to 57% of native activity) by suppressing luciferase aggregation. This coordinated function of ClpB and
DnaK/DnaJ/GrpE required ATP hydrolysis, although the ClpB ATPase was not activated by native or denatured luciferase. When
the chaperones were added to the luciferase refolding solutions after 5–25 min of refolding, ClpB and DnaK/DnaJ/GrpE recovered
the luciferase activity from preformed aggregates. Thus, we have identified a novel multi-chaperone system from E. coli, which is analogous to the Hsp104/Ssa1/Ydj1 system from yeast. ClpB is the only known bacterial Hsp100 protein capable of
cooperating with other heat-shock proteins in suppressing and reversing protein aggregation.
We systematically analyzed the capability of the major cytosolic chaperones of Escherichia coli to cope with protein misfolding and aggregation during heat stress in vivo and in cell extracts. Under physiological heat stress conditions, only the DnaK system efficiently prevented the aggregation of thermolabile proteins, a surprisingly high number of 150-200 species, corresponding to 15-25% of detected proteins. Identification of thermolabile DnaK substrates by mass spectrometry revealed that they comprise 80% of the large (>/=90 kDa) but only 18% of the small (</=30 kDa) cytosolic proteins and include essential proteins. The DnaK system in addition acts with ClpB to form a bi-chaperone system that quantitatively solubilizes aggregates of most of these proteins. Efficient solubilization also occurred in an in vivo order-of-addition experiment in which aggregates were formed prior to induction of synthesis of the bi-chaperone system. Our data indicate that large-sized proteins are most vulnerable to thermal unfolding and aggregation, and that the DnaK system has central, dual protective roles for these proteins by preventing their aggregation and, cooperatively with ClpB, mediating their disaggregation. Keywords: chaperones/heat-shock response/Hsp70/protein denaturation/thermotolerance
Bacteria contain a remarkable RNA molecule - known alternatively as SsrA RNA, tmRNA, or 10Sa RNA - that acts both as a tRNA and as an mRNA to direct the modification of proteins whose biosynthesis has stalled or has been interrupted. These incomplete proteins are marked for degradation by cotranslational addition of peptide tags to their C-termini in a reaction that is mediated by ribosome-bound SsrA RNA and an associated protein factor, SmpB. This system plays a key role in intracellular protein quality control and also provides a mechanism to clear jammed or obstructed ribosomes. Here the structural, functional and phylogenetic properties of this unique RNA and its associated factors are reviewed, and the intracellular proteases that act to degrade the proteins tagged by this system are also discussed.
ClpX and ClpA are molecular chaperones that interact with specific proteins and, together with ClpP, activate their ATP-dependent degradation. The chaperone activity is thought to convert proteins into an extended conformation that can access the sequestered active sites of ClpP. We now show that ClpX can catalyze unfolding of a green fluorescent protein fused to a ClpX recognition motif (GFP-SsrA). Unfolding of GFP-SsrA depends on ATP hydrolysis. GFP-SsrA unfolded either by ClpX or by treatment with denaturants binds to ClpX in the presence of adenosine 5'-O-(3-thiotriphosphate) and is released slowly (t(1/2) approximately 15 min). Unlike ClpA, ClpX cannot trap unfolded proteins in stable complexes unless they also have a high-affinity binding motif. Addition of ATP or ADP accelerates release (t(1/2) approximately 1 min), consistent with a model in which ATP hydrolysis induces a conformation of ClpX with low affinity for unfolded substrates. Proteolytically inactive complexes of ClpXP and ClpAP unfold GFP-SsrA and translocate the protein to ClpP, where it remains unfolded. Complexes of ClpXP with translocated substrate within the ClpP chamber retain the ability to unfold GFP-SsrA. Our results suggest a bipartite mode of interaction between ClpX and substrates. ClpX preferentially targets motifs exposed in specific proteins. As the protein is unfolded by ClpX, additional motifs are exposed that facilitate its retention and favor its translocation to ClpP for degradation.
ClpA, a member of the Clp/Hsp100 ATPase family, is a molecular chaperone and regulatory component of ClpAP protease. We explored the mechanism of protein recognition by ClpA using a high affinity substrate, RepA, which is activated for DNA binding by ClpA and degraded by ClpAP. By characterizing RepA derivatives with N- or C-terminal deletions, we found that the N-terminal portion of RepA is required for recognition. More precisely, RepA derivatives lacking the N-terminal 5 or 10 amino acids are degraded by ClpAP at a rate similar to full-length RepA, whereas RepA derivatives lacking 15 or 20 amino acids are degraded much more slowly. Thus, ClpA recognizes an N-terminal signal in RepA beginning in the vicinity of amino acids 10-15. Moreover, peptides corresponding to RepA amino acids 4-13 and 1-15 inhibit interactions between ClpA and RepA. We constructed fusions of RepA and green fluorescent protein, a protein not recognized by ClpA, and found that the N-terminal 15 amino acids of RepA are sufficient to target the fusion protein for degradation by ClpAP. However, fusion proteins containing 46 or 70 N-terminal amino acids of RepA are degraded more efficiently in vitro and are noticeably stabilized in vivo in clpADelta and clpPDelta strains compared with wild type.
Point mutations in either of the two nucleotide-binding domains (NBD) of Hsp104 (NBD1 and NBD2) eliminate its thermotolerance function in vivo. In vitro, NBD1 mutations virtually eliminate ATP hydrolysis with little effect on hexamerization; analogous NBD2 mutations reduce ATPase activity and severely impair hexamerization. We report that high protein concentrations overcome the assembly defects of NBD2 mutants and increase ATP hydrolysis severalfold, changing V(max) with little effect on K(m). In a complementary fashion, the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate inhibits hexamerization of wild-type (WT) Hsp104, lowering V(max) with little effect on K(m). ATP hydrolysis exhibits a Hill coefficient between 1.5 and 2, indicating that it is influenced by cooperative subunit interactions. To further analyze the effects of subunit interactions on Hsp104, we assessed the effects of mutant Hsp104 proteins on WT Hsp104 activities. An NBD1 mutant that hexamerizes but does not hydrolyze ATP reduces the ATPase activity of WT Hsp104 in vitro. In vivo, this mutant is not toxic but specifically inhibits the thermotolerance function of WT Hsp104. Thus, interactions between subunits influence the ATPase activity of Hsp104, play a vital role in its biological functions, and provide a mechanism for conditionally inactivating Hsp104 function in vivo.
p97, an abundant hexameric ATPase of the AAA family, is involved in homotypic membrane fusion. It is thought to disassemble SNARE complexes formed during the process of membrane fusion. Here, we report two structures: a crystal structure of the N-terminal and D1 ATPase domains of murine p97 at 2.9 A resolution, and a cryoelectron microscopy structure of full-length rat p97 at 18 A resolution. Together, these structures show that the D1 and D2 hexamers pack in a tail-to-tail arrangement, and that the N domain is flexible. A comparison with NSF D2 (ATP complex) reveals possible conformational changes induced by ATP hydrolysis. Given the D1 and D2 packing arrangement, we propose a ratchet mechanism for p97 during its ATP hydrolysis cycle.
Escherichia coli ClpA and ClpX are ATP-dependent protein unfoldases that each interact with the protease, ClpP, to promote specific protein degradation. We have used limited proteolysis and deletion analysis to probe the conformations of ClpA and ClpX and their interactions with ClpP and substrates. ATP gamma S binding stabilized ClpA and ClpX such that that cleavage by lysylendopeptidase C occurred at only two sites. Both proteins were cleaved within in a loop preceding an alpha-helix-rich C-terminal domain. Although the loop varies in size and composition in Clp ATPases, cleavage occurred within and around a conserved triad, IG(F/L). Binding of ClpP blocked this cleavage, and prior cleavage at this site rendered both ClpA and ClpX defective in binding and activating ClpP, suggesting that this site is involved in interactions with ClpP. ClpA was also cut at a site near the junction of the two ATPase domains, whereas the second cleavage site in ClpX lay between its N-terminal and ATPase domains. ClpP did not block cleavage at these other sites. The N-terminal domain of ClpX dissociated upon cleavage, and the remaining ClpXDeltaN remained as a hexamer, associated with ClpP, and expressed ATPase, chaperone, and proteolytic activity. A truncated mutant of ClpA lacking its N-terminal 153 amino acids also formed a hexamer, associated with ClpP, and expressed these activities. We propose that the N-terminal domains of ClpX and ClpA lie on the outside ring surface of the holoenzyme complexes where they contribute to substrate binding or perform a gating function affecting substrate access to other binding sites and that a loop on the opposite face of the ATPase rings stabilizes interactions with ClpP and is involved in promoting ClpP proteolytic activity.
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.
Molecular chaperones assist protein folding by facilitating their “forward” folding and preventing aggregation. However, once aggregates have formed, these chaperones cannot facilitate protein disaggregation. Bacterial ClpB and its eukaryotic homolog Hsp104 are essential proteins of the heat-shock response, which have the remarkable capacity to rescue stress-damaged proteins from an aggregated state. We have determined the structure of Thermus thermophilus ClpB (TClpB) using a combination of X-ray crystallography and cryo-electron microscopy (cryo-EM). Our single-particle reconstruction shows that TClpB forms a two-tiered hexameric ring. The ClpB/Hsp104-linker consists of an 85 Å long and mobile coiled coil that is located on the outside of the hexamer. Our mutagenesis and biochemical data show that both the relative position and motion of this coiled coil are critical for chaperone function. Taken together, we propose a mechanism by which an ATP-driven conformational change is coupled to a large coiled-coil motion, which is indispensable for protein disaggregation.
SspB, a specificity factor for the ATP-dependent ClpXP protease, stimulates proteolysis of protein substrates bearing the ssrA degradation tag. The SspB protein is shown here to form a stable homodimer with two independent binding sites for ssrA-tagged proteins or peptides. SspB by itself binds to ClpX and stimulates the ATPase activity of this enzyme. In the presence of ATPγS, a ternary complex of SspB, GFP-ssrA, and the ClpX ATPase was sufficiently stable to isolate by gel-filtration or ion-exchange chromatography. This complex consists of one SspB dimer, two molecules of GFP-ssrA, and one ClpX hexamer. SspB dimers do not commit bound substrates to ClpXP degradation but increase the affinity and cooperativity of binding of ssrA-tagged substrates to ClpX, facilitating enhanced degradation at low substrate concentrations.
The σS (RpoS) subunit of RNA polymerase is the master regulator of the general stress response in Escherichia coli and related bacteria. While rapidly growing cells contain very little σS, exposure to many different stress conditions results in rapid and strong σS induction. Consequently, transcription of numerous σS-dependent genes is activated, many of which encode gene products with stress-protective functions. Multiple signal integration in the control of the cellular σS level is achieved by rpoS transcriptional and translational control as well as by regulated σS proteolysis, with various stress conditions differentially affecting these levels of σS control. Thus, a reduced growth rate results in increased rpoS transcription whereas high osmolarity, low temperature, acidic pH, and some late-log-phase signals stimulate the translation of already present rpoS mRNA. In addition, carbon starvation, high osmolarity, acidic pH, and high temperature result in stabilization of σS, which, under nonstress conditions, is degraded with a half-life of one to several minutes. Important cis-regulatory determinants as well as trans-acting regulatory factors involved at all levels of σS regulation have been identified. rpoS translation is controlled by several proteins (Hfq and HU) and small regulatory RNAs that probably affect the secondary structure of rpoS mRNA. For σS proteolysis, the response regulator RssB is essential. RssB is a specific direct σS recognition factor, whose affinity for σS is modulated by phosphorylation of its receiver domain. RssB delivers σS to the ClpXP protease, where σS is unfolded and completely degraded. This review summarizes our current knowledge about the molecular functions and interactions of these components and tries to establish a framework for further research on the mode of multiple signal input into this complex regulatory system.
Proteolysis by cytoplasmic, energy-dependent proteases plays a critical role in many regulatory circuits, keeping basal levels of regulatory proteins low and rapidly removing proteins when they are no longer needed. In bacteria, four families of energy-dependent proteases carry out degradation. In all of them, substrates are first recognized and bound by ATPase domains and then unfolded and translocated to a sequestered proteolytic chamber. Substrate selection depends not on ubiquitin but on intrinsic recognition signals within the proteins and, in some cases, on adaptor or effector proteins that participate in delivering the substrate to the protease. For some, the activity of these adaptors can be regulated, which results in regulated proteolysis. Recognition motifs for proteolysis are frequently found at the N and C termini of substrates. Proteolytic switches appear to be critical for cell cycle development in Caulobacter crescentus, for proper sporulation in Bacillus subtilis, and for the transition in and out of stationary phase in Escherichia coli. In eukaryotes, the same proteases are found in organelles, where they also play important roles.
Molecular chaperones assist protein folding by facilitating their "forward" folding and preventing aggregation. However, once aggregates have formed, these chaperones cannot facilitate protein disaggregation. Bacterial ClpB and its eukaryotic homolog Hsp104 are essential proteins of the heat-shock response, which have the remarkable capacity to rescue stress-damaged proteins from an aggregated state. We have determined the structure of Thermus thermophilus ClpB (TClpB) using a combination of X-ray crystallography and cryo-electron microscopy (cryo-EM). Our single-particle reconstruction shows that TClpB forms a two-tiered hexameric ring. The ClpB/Hsp104-linker consists of an 85 A long and mobile coiled coil that is located on the outside of the hexamer. Our mutagenesis and biochemical data show that both the relative position and motion of this coiled coil are critical for chaperone function. Taken together, we propose a mechanism by which an ATP-driven conformational change is coupled to a large coiled-coil motion, which is indispensable for protein disaggregation.
Proteolysis functions as a precise regulatory mechanism for a broad spectrum of cellular processes. Such control impacts not only on the stability of key metabolic enzymes but also on the effective removal of terminally damaged polypeptides. Much of this directed protein turnover is performed by proteases that require ATP and, of those in bacteria, the Clp protease from Escherichia coli is one of the best characterized to date. The Clp holoenzyme consists of two adjacent heptameric rings of the proteolytic subunit known as ClpP, which are flanked by a hexameric ring of a regulatory subunit from the Clp/Hsp100 chaperone family at one or both ends. The recently resolved three-dimensional structure of the E. coli ClpP protein has provided new insights into its interaction with the regulatory/chaperone subunits. In addition, an increasing number of studies over the last few years have recognized the added complexity and functional importance of ClpP proteins in other eubacteria and, in particular, in photosynthetic organisms ranging from cyanobacteria to higher plants. The goal of this review is to summarize these recent findings and to highlight those areas that remain unresolved.
Self-association of ClpB (a mixture of 95- and 80-kDa subunits) has been studied with gel filtration chromatography, analytical ultracentrifugation, and electron microscopy. Monomeric ClpB predominates at low protein concentration (0.07 mg0mL), while an oligomeric form is highly populated at >4 mg/mL. The oligomer formation is enhanced in the presence of 2 mM ATP or adenosine 5′-O-thiotriphosphate (ATPS). In contrast, 2 mMADP inhibits full oligomerization of ClpB. The apparent size of the ATP- or ATPS-induced oligomer, as determined by gel filtration, sedimentation velocity and electron microscopy image averaging, and the molecular weight, as determined by sedimentation equilibrium, are consistent with those of a ClpB hexamer. These results indicate that the oligomerization reactions of ClpB are similar to those of other Hsp100 proteins.
Proteolytic cleavage of lexA repressor is an early step in derepression of the SOS regulatory system of Escherichia coli. In vivo and in vitro data have indicated a role for recA protein in this specific proteolytic reaction. I show here that, under certain conditions, specific in vitro cleavage of highly-purified lexA protein can take place in the absence of recA protein. This autodigestion reaction cleaved the same alanine-glycine bond as did the recA-dependent cleavage reaction. Several lines of evidence argued that it was not due to a contaminating protease activity. Autodigestion was stimulated by alkaline pH. It occurred in the presence of EDTA but was stimulated several fold by the presence of Ca2+, Co2+, or Mg2+. The reaction appeared to be first-order, and its rate was independent of protein concentration over a wide range, strongly suggesting that it is intramolecular. Purified phage lambda repressor also broke down under similar conditions to yield products like those resulting from recA protein action. Phage lambda repressor broke down at a far slower rate than did lexA, as previously observed in the recA-catalyzed in vitro reaction and in vivo. This correlation between the two types of cleavage also extended to the reactions with mutant repressor proteins; taken together with the site specificity, it suggests that autodigestion and recA-dependent cleavage follow, at least in part, a similar reaction pathway. These findings indicate that specific cleavage of lexA protein can be catalyzed by the protein itself and suggest that recA protein plays an indirect stimulatory role, perhaps as an allosteric effector, in the recA-dependent reaction, rather than acting directly as a protease. The protease active site and the recA-recognition site lie in the central or COOH-terminal portion of the lexA protein, since a tryptic fragment containing these portions of lexA protein could take part in both reactions.
In Escherichia coli, the sigma factor, RpoS, is a central regulator in stationary-phase cells. We have identified a gene, sprE (stationary-phase regulator), as essential for the negative regulation of rpoS expression. SprE negatively regulates the rpoS gene product at the level of protein stability, perhaps in response to nutrient availability. The ability of SprE to destabilize RpoS is dependent on the ClpX/ClpP protease. Based on homology, SprE is a member of the response regulator family of proteins. SprE is the first response regulator identified that is implicated in the control of protein stability. Moreover, SprE is the first reported protein that appears to regulate rpoS in response to a specific environmental parameter.
The HSP100/Clp proteins are a newly discovered family with a great diversity of functions, such as increased tolerance to high temperatures, promotion of proteolysis of specific cellular substrates and regulation of transcription. HSP100/Clp proteins are also synthesized in a variety of specific patterns and, in eukaryotes, are localized to different subcellular compartments. Recent data suggest that a common ability to disassemble higher-order protein structures and aggregates unifies the molecular functions of this diverse family.
ClpX, a molecular chaperone and the regulatory subunit of the ClpXP protease, is shown to contain tandem modular domains that bind to the C-terminal sequences of target proteins in a manner that parallels functional specificity. Nuclear magnetic resonance studies show that these C-terminal sequences are displayed as disordered peptides on the surface of otherwise folded proteins. The ClpX substrate-binding domains are homologous to sequences in other Clp/Hsp100 proteins and are related more distantly to PDZ domains, which also mediate C-terminal specific protein-protein interactions. Conservation of these binding domains indicates that the mode of substrate recognition characterized here for ClpX will be a conserved feature among Clp/Hsp100 family members and a distinguishing characteristic between this chaperone family and the Hsp70 chaperones.
Hsp104 is a stress tolerance factor that promotes the reactivation of heat-damaged proteins in yeast by an unknown mechanism. Herein, we demonstrate that Hsp104 functions in this process directly. Unlike other chaperones, Hsp104 does not prevent the aggregation of denatured proteins. However, in concert with Hsp40 and Hsp70, Hsp104 can reactivate proteins that have been denatured and allowed to aggregate, substrates refractory to the action of other chaperones. Hsp104 cooperates with the chaperones present in reticulocyte lysates but not with DnaK of E. coli. We conclude that Hsp104 has a protein remodeling activity that acts on trapped, aggregated proteins and requires specific interactions with conventional chaperones to promote refolding of the intermediates it produces.
N-ethylmaleimide-sensitive factor (NSF) is a hexameric ATPase which primes and/or dissociates SNARE complexes involved in intracellular fusion events. Each NSF protomer contains three domains: an N-terminal domain required for SNARE binding and two ATPase domains, termed D1 and D2, with D2 being required for oligomerization. We have determined the 1.9 A crystal structure of the D2 domain of NSF complexed with ATP using multi-wavelength anomalous dispersion phasing. D2 consists of a nucleotide binding subdomain with a Rossmann fold and a C-terminal subdomain, which is structurally unique among nucleotide binding proteins. There are interactions between the ATP moiety and both the neighboring D2 protomer and the C-terminal subdomain that may be important for ATP-dependent oligomerization. Of particular importance are three well-ordered and conserved lysine residues that form ionic interactions with the beta- and gamma-phosphates, one of which likely contributes to the low hydrolytic activity of D2.
ClpAP, a typical energy-dependent protease, consists of a proteolytic component (ClpP) and a chaperone-like ATPase (ClpA). ClpP is composed of two apposed heptameric rings, whereas in the presence of ATP or ATPgammaS, ClpA is a single hexameric ring. Formation of ClpAP complexes involves a symmetry mismatch as sixfold ClpA stacks axially on one or both faces of sevenfold ClpP. We have analyzed these structures by cryo-electron microscopy. Our three-dimensional reconstruction of ClpA at 29-A resolution shows the monomer to be composed of two domains of similar size that, in the hexamer, form two tiers enclosing a large cavity. Cylindrical reconstruction of ClpAP reveals three compartments: the digestion chamber inside ClpP; a compartment between ClpP and ClpA; and the cavity inside ClpA. They are connected axially via narrow apertures, implying that substrate proteins should be unfolded to allow translocation into the digestion chamber. The cavity inside ClpA is structurally comparable to the "Anfinsen cage" of other chaperones and may play a role in the unfolding of substrates. A geometrical description of the symmetry mismatch was obtained by using our model of ClpA and the crystal structure of ClpP (Wang et al., 1997, Cell 91, 447-456) to identify the particular side views presented by both molecules in individual complexes. The interaction is characterized by a key pair of subunits, one of each protein. A small turn (8.6(o) = 2pi/42; equivalent to a 4-A shift) would transfer the key interaction to another pair of subunits. We propose that nucleotide hydrolysis results in rotation, facilitating the processive digestion of substrate proteins.
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.
Proteolysis functions as a precise regulatory mechanism for a broad spectrum of cellular processes. Such control impacts not only on the stability of key metabolic enzymes but also on the effective removal of terminally damaged polypeptides. Much of this directed protein turnover is performed by proteases that require ATP and, of those in bacteria, the Clp protease from Escherichia coli is one of the best characterized to date. The Clp holoenzyme consists of two adjacent heptameric rings of the proteolytic subunit known as ClpP, which are flanked by a hexameric ring of a regulatory subunit from the Clp/Hsp100 chaperone family at one or both ends. The recently resolved three-dimensional structure of the E. coli ClpP protein has provided new insights into its interaction with the regulatory/chaperone subunits. In addition, an increasing number of studies over the last few years have recognized the added complexity and functional importance of ClpP proteins in other eubacteria and, in particular, in photosynthetic organisms ranging from cyanobacteria to higher plants. The goal of this review is to summarize these recent findings and to highlight those areas that remain unresolved.
Chaperone rings play a vital role in the opposing ATP-mediated
processes of folding and degradation of many cellular proteins, but the
mechanisms by which they assist these life and death actions are only
beginning to be understood. Ring structures present an advantage to
both processes, providing for compartmentalization of the substrate
protein inside a central cavity in which multivalent, potentially
cooperative interactions can take place between the substrate and a
high local concentration of binding sites, while access of other
proteins to the cavity is restricted sterically. Such restriction
prevents outside interference that could lead to nonproductive fates of
the substrate protein while it is present in non-native form, such as
aggregation. At the step of recognition, chaperone rings recognize
different motifs in their substrates, exposed hydrophobicity in the
case of protein-folding chaperonins, and specific “tag” sequences
in at least some cases of the proteolytic chaperones. For both folding
and proteolytic complexes, ATP directs conformational changes in the
chaperone rings that govern release of the bound polypeptide. In the
case of chaperonins, ATP enables a released protein to pursue the
native state in a sequestered hydrophilic folding chamber, and, in the
case of the proteases, the released polypeptide is translocated into a
degradation chamber. These divergent fates are at least partly governed
by very different cooperating components that associate with the
chaperone rings: that is, cochaperonin rings on one hand and
proteolytic ring assemblies on the other. Here we review the structures
and mechanisms of the two types of chaperone ring system.
Recent work suggests that two unrelated phenotypes, [PSI+] and [URE3], in the yeast Saccharomyces cerevisiae are transmitted by non-covalent changes in the physical states of their protein determinants, Sup35p and Ure2p, rather than by changes in the genes that encode these proteins. The mechanism by which alternative protein states are self-propagating is the key to understanding how proteins function as elements of epigenetic inheritance. Here, we focus on recent molecular-genetic analysis of the inheritance of the [PSI+] factor of S. cerevisiae. Insights into this process might be extendable to a group of mammalian diseases (the amyloidoses), which are also believed to be a manifestation of self-perpetuating changes in protein conformation.
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.
The SulA protein is a cell division inhibitor in Escherichia coli, and is specifically degraded by Lon protease. To study the recognition site of SulA for Lon, we prepared a mutant SulA protein lacking the C-terminal 8 amino acid residues (SA8). This deletion protein was accumulated and stabilized more than native SulA in lon(+) cells in vivo. Moreover, the deletion SulA fused to maltose binding protein was not degraded by Lon protease, and did not stimulate the ATPase or peptidase activity of Lon in vitro, probably due to the much reduced interaction with Lon. A BIAcore study showed that SA8 directly interacts with Lon. These results suggest that SA8 of SulA was recognized by Lon protease. The SA8 peptide, KIHSNLYH, specifically inhibited the degradation of native SulA by Lon protease in vitro, but not that of casein. A mutant SA8, KAHSNLYH, KIASNLYH, or KIHSNAYH, also inhibited the degradation of SulA, while such peptides as KIHSNLYA did not. These results show that SulA has the specified rows of C-terminal 8 residues recognized by Lon, leading to facilitated binding and subsequent cleavage by Lon protease both in vivo and in vitro.
ClpXP is a protein machine composed of the ClpX ATPase, a member of the Clp/Hsp100 family of remodeling enzymes, and the ClpP peptidase. Here, ClpX and ClpXP are shown to catalyze denaturation of GFP modified with an ssrA degradation tag. ClpX translocates this denatured protein into the proteolytic chamber of ClpP and, when proteolysis is blocked, also catalyzes release of denatured GFP-ssrA from ClpP in a reaction that requires ATP and additional substrate. Kinetic experiments reveal that multiple reaction steps require collaboration between ClpX and ClpP and that denaturation is the rate-determining step in degradation. These insights into the mechanism of ClpXP explain how it executes efficient degradation in a manner that is highly specific for tagged proteins, irrespective of their intrinsic stabilities.
The heat-shock protein ClpB is a protein-activated ATPase that is essential for survival of Escherichia coli at high temperatures. ClpB has also recently been suggested to function as a chaperone in reactivation of aggregated proteins. In addition, the clpB gene has been shown to contain two translational initiation sites and therefore encode two polypeptides of different size. To determine the structural organization of ClpB, the ClpB proteins were subjected to chemical cross-linking analysis and electron microscopy. The average images of the ClpB proteins with end-on orientation revealed a seven-membered, ring-shaped structure with a central cavity. Their side-on view showed a two-layered structure with an equal distribution of mass across the equatorial plane of the complex. Since the ClpB subunit has two large regions containing consensus sequences for nucleotide binding, each layer of the ClpB heptamer appears to represent the side projection of one of the major domains arranged on a ring. In the absence of salt and ATP, the ClpB proteins showed a high tendency to form a heptamer. However, they dissociated into various species of oligomers with smaller sizes, depending on salt concentration. Above 0.2 M NaCl, the ClpB proteins behaved most likely as a monomer in the absence of ATP, but assembled into a heptamer in its presence. Furthermore, mutations of the first ATP-binding site, but not the second site, prevented the ATP-dependent oligomerization of the ClpB proteins in the presence of 0.3 M NaCl. These results indicate that ClpB has a heptameric ring-shaped structure with a central cavity and this structural organization requires ATP binding to the first nucleotide-binding site localized to the N-terminal half of the ATPase.
HslUV is a "prokaryotic proteasome" composed of the HslV protease and the HslU ATPase, a chaperone of the Clp/Hsp100 family. The 3.4 A crystal structure of an HslUV complex is presented here. Two hexameric ATP binding rings of HslU bind intimately to opposite sides of the HslV protease; the HslU "intermediate domains" extend outward from the complex. The solution structure of HslUV, derived from small angle X-ray scattering data under conditions where the complex is assembled and active, agrees with this crystallographic structure. When the complex forms, the carboxy-terminal helices of HslU distend and bind between subunits of HslV, and the apical helices of HslV shift substantially, transmitting a conformational change to the active site region of the protease.
Binding and internalization of a protein substrate by E. coli ClpXP was investigated by electron microscopy. In sideviews of ATP gamma S-stabilized ClpXP complexes, a narrow axial channel was visible in ClpX, surrounded by protrusions on its distal surface. When substrate lambda O protein was added, extra density attached to this surface. Upon addition of ATP, this density disappeared as lambda O was degraded. When ATP was added to proteolytically inactive ClpXP-lambda O complexes, the extra density transferred to the center of ClpP and remained inside ClpP after separation from ClpX. We propose that substrates of ATP-dependent proteases bind to specific sites on the distal surface of the ATPase, and are subsequently unfolded and translocated into the internal chamber of the protease.
The molecular chaperone protein Hsp78, a member of the Clp/Hsp100 family localized in the mitochondria of Saccharomyces cerevisiae, is required for maintenance of mitochondrial functions under heat stress. To characterize the biochemical mechanisms of Hsp78 function, Hsp78 was purified to homogeneity and its role in the reactivation of chemically and heat-denatured substrate protein was analyzed in vitro. Hsp78 alone was not able to mediate reactivation of firefly luciferase. Rather, efficient refolding was dependent on the simultaneous presence of Hsp78 and the mitochondrial Hsp70 machinery, composed of Ssc1p/Mdj1p/Mge1p. Bacterial DnaK/DnaJ/GrpE, which cooperates with the Hsp78 homolog, ClpB in Escherichia coli, could not substitute for the mitochondrial Hsp70 system. However, efficient Hsp78-dependent refolding of luciferase was observed if DnaK was replaced by Ssc1p in these experiments, suggesting a specific functional interaction of both chaperone proteins. These findings establish the cooperation of Hsp78 with the Hsp70 machinery in the refolding of heat-inactivated proteins and demonstrate a conserved mode of action of ClpB homologs.
The sigma(S) subunit of Escherichia coli RNA polymerase regulates the expression of stationary phase and stress response genes. Control over sigma(S) activity is exercised in part by regulated degradation of sigma(S). In vivo, degradation requires the ClpXP protease together with RssB, a protein homologous to response regulator proteins. Using purified components, we reconstructed the degradation of sigma(S) in vitro and demonstrate a direct role for RssB in delivering sigma(S) to ClpXP. RssB greatly stimulates sigma(S) degradation by ClpXP. Acetyl phosphate, which phosphorylates RssB, is required. RssB participates in multiple rounds of sigma(S) degradation, demonstrating its catalytic role. RssB promotes sigma(S) degradation specifically; it does not affect degradation of other ClpXP substrates or other proteins not normally degraded by ClpXP. sigma(S) and RssB form a stable complex in the presence of acetyl phosphate, and together they form a ternary complex with ClpX that is stabilized by ATP[gamma-S]. Alone, neither sigma(S) nor RssB binds ClpX with high affinity. When ClpP is present, a larger sigma(S)--RssB--ClpXP complex forms. The complex degrades sigma(S) and releases RssB from ClpXP in an ATP-dependent reaction. Our results illuminate an important mechanism for regulated protein turnover in which a unique targeting protein, whose own activity is regulated through specific signaling pathways, catalyzes the delivery of a specific substrate to a specific 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.
Detection of similarity is particularly difficult for small proteins and thus connections between many of them remain unnoticed. Structure and sequence analysis of several metal-binding proteins reveals unexpected similarities in structural domains classified as different protein folds in SCOP and suggests unification of seven folds that belong to two protein classes. The common motif, termed treble clef finger in this study, forms the protein structural core and is 25-45 residues long. The treble clef motif is assembled around the central zinc ion and consists of a zinc knuckle, loop, beta-hairpin and an alpha-helix. The knuckle and the first turn of the helix each incorporate two zinc ligands. Treble clef domains constitute the core of many structures such as ribosomal proteins L24E and S14, RING fingers, protein kinase cysteine-rich domains, nuclear receptor-like fingers, LIM domains, phosphatidylinositol-3-phosphate-binding domains and His-Me finger endonucleases. The treble clef finger is a uniquely versatile motif adaptable for various functions. This small domain with a 25 residue structural core can accommodate eight different metal-binding sites and can have many types of functions from binding of nucleic acids, proteins and small molecules, to catalysis of phosphodiester bond hydrolysis. Treble clef motifs are frequently incorporated in larger structures or occur in doublets. Present analysis suggests that the treble clef motif defines a distinct structural fold found in proteins with diverse functional properties and forms one of the major zinc finger groups.
The AAA+ superfamily of ATPases, which contain a homologous ATPase module, are found in all kingdoms of living organisms where they participate in diverse cellular processes including membrane fusion, proteolysis and DNA replication. Recent structural studies have revealed that they usually form ring-shaped oligomers, which are crucial for their ATPase activities and mechanisms of action. These ring-shaped oligomeric complexes are versatile in their mode of action, which collectively seem to involve some form of disruption of molecular or macromolecular structure; unfolding of proteins, disassembly of protein complexes, unwinding of DNA, or alteration of the state of DNA-protein complexes. Thus, the AAA+ proteins represent a novel type of molecular chaperone. Comparative analyses have also revealed significant similarities and differences in structure and molecular mechanism between AAA+ ATPases and other ring-shaped ATPases.