Diverse pore loops of the AAA+ ClpX machine mediate unassisted and adaptor-dependent recognition of ssrA-tagged substrates

ClpX, an archetypal proteolytic AAA+ unfoldase, must engage the ssrA tags of appropriate substrates prior to ATP-dependent unfolding and translocation of the denatured polypeptide into ClpP for degradation. Here, specificity-transplant and disulfide-crosslinking experiments reveal that the ssrA tag interacts with different loops that form the top, middle, and lower portions of the central channel of the ClpX hexamer. Our results support a two-step binding mechanism, in which the top loop serves as a specificity filter and the remaining loops form a binding site for the peptide tag relatively deep within the pore. Crosslinking experiments suggest a staggered arrangement of pore loops in the hexamer and nucleotide-dependent changes in pore-loop conformations. This mechanism of initial tag binding would allow ATP-dependent conformational changes in one or more pore loops to drive peptide translocation, force unfolding, and mediate threading of the denatured protein through the ClpX pore.     

Roles of the N-domains of the ClpA unfoldase in binding substrate proteins and in stable complex formation with the ClpP protease

The hexameric cylindrical Hsp100 chaperone ClpA mediates ATP-dependent unfolding and translocation of recognized substrate proteins into the coaxially associated serine protease ClpP. Each subunit of ClpA is composed of an N-terminal domain of approximately 150 amino acids at the top of the cylinder followed by two AAA+ domains. In earlier studies, deletion of the N-domain was shown to have no effect on the rate of unfolding of substrate proteins bearing a C-terminal ssrA tag, but it did reduce the rate of degradation of these proteins (Lo, J. H., Baker, T. A., and Sauer, R. T. (2001) Protein Sci. 10, 551-559; Singh, S. K., Rozycki, J., Ortega, J., Ishikawa, T., Lo, J., Steven, A. C., and Maurizi, M. R. (2001) J. Biol. Chem. 276, 29420-29429). Here we demonstrate, using both fluorescence resonance energy transfer to measure the arrival of substrate at ClpP and competition between wild-type and an inactive mutant form of ClpP, that this effect on degradation is caused by diminished stability of the ClpA-ClpP complex during translocation and proteolysis, effectively disrupting the targeting of unfolded substrates to the protease. We have also examined two larger ssrA-tagged substrates, CFP-GFP-ssrA and luciferase-ssrA, and observed different behaviors. CFP-GFP-ssrA is not efficiently unfolded by the truncated chaperone whereas luciferase-ssrA is, suggesting that the former requires interaction with the N-domains, likely via the body of the protein, to stabilize its binding. Thus, the N-domains play a key allosteric role in complex formation with ClpP and may also have a critical role in recognizing certain tag elements and binding some substrate proteins.     

Division of labor between the pore-1 loops of the D1 and D2 AAA+ rings coordinates substrate selectivity of the ClpAP protease

ClpAP, an ATP-dependent protease consisting of ClpA, a double-ring hexameric unfoldase of the ATPases associated with diverse cellular activities superfamily, and the ClpP peptidase, degrades damaged and unneeded proteins to support cellular proteostasis. ClpA recognizes many protein substrates directly, but it can also be regulated by an adapter, ClpS, that modifies ClpA’s substrate profile toward N-degron substrates. Conserved tyrosines in the 12 pore-1 loops lining the central channel of the stacked D1 and D2 rings of ClpA are critical for degradation, but the roles of these residues in individual steps during direct or adapter-mediated degradation are poorly understood. Using engineered ClpA hexamers with zero, three, or six pore-1 loop mutations in each ATPases associated with diverse cellular activities superfamily ring, we found that active D1 pore loops initiate productive engagement of substrates, whereas active D2 pore loops are most important for mediating the robust unfolding of stable native substrates. In complex with ClpS, active D1 pore loops are required to form a high affinity ClpA•ClpS•substrate complex, but D2 pore loops are needed to “tug on” and remodel ClpS to transfer the N-degron substrate to ClpA. Overall, we find that the pore-1 loop tyrosines in D1 are critical for direct substrate engagement, whereas ClpS-mediated substrate delivery requires unique contributions from both the D1 and D2 pore loops. In conclusion, our study illustrates how pore loop engagement, substrate capture, and powering of the unfolding/translocation steps are distributed between the two rings of ClpA, illuminating new mechanistic features that may be common to double-ring protein unfolding machines.     

Both ATPase Domains of ClpA Are Critical for Processing of Stable Protein Structures

ClpA is a ring-shaped hexameric chaperone that binds to both ends of the protease ClpP and catalyzes the ATP-dependent unfolding and translocation of substrate proteins through its central pore into the ClpP cylinder. Here we study the relevance of ATP hydrolysis in the two ATPase domains of ClpA. We designed ClpA Walker B variants lacking ATPase activity in the first (D1) or the second ATPase domain (D2) without impairing ATP binding. We found that the two ATPase domains of ClpA operate independently even in the presence of the protease ClpP or the adaptor protein ClpS. Notably, ATP hydrolysis in the first ATPase module is sufficient to process a small, single domain protein of low stability. Substrate proteins of moderate local stability were efficiently processed when D1 was inactivated. However, ATP hydrolysis in both domains was required for efficiently processing substrates of high local stability. Furthermore, we provide evidence for the ClpS-dependent directional translocation of N-end rule substrates from the N to C terminus and propose a mechanistic model for substrate handover from the adaptor protein to the chaperone.     

Nucleotide-dependent substrate handoff from the SspB adaptor to the AAA+ ClpXP protease

The SspB adaptor enhances ClpXP degradation by binding the ssrA degradation tag of substrates and the AAA+ ClpX unfoldase. To probe the mechanism of substrate delivery, we engineered a disulfide bond between the ssrA tag and SspB and demonstrated otherwise normal interactions by solving the crystal structure. Although the covalent link prevents adaptor.substrate dissociation, ClpXP degraded GFP-ssrA that was disulfide bonded to the adaptor. Thus, crosslinked substrate must be handed directly from SspB to ClpX. The ssrA tag in the covalent adaptor complex interacted with ClpX.ATPgammaS but not ClpX.ADP, suggesting that handoff occurs in the ATP bound enzyme. By contrast, SspB alone bound ClpX in both nucleotide states. Similar handoff mechanisms will undoubtedly be used by many AAA+ adaptors and enzymes, allowing assembly of delivery complexes in either nucleotide state, engagement of the recognition tag in the ATP state, and application of an unfolding force to the attached protein following hydrolysis.     

Crystallographic investigation of peptide binding sites in the N-domain of the ClpA chaperone

Escherichia coli ClpA, an Hsp100/Clp chaperone and an integral component of the ATP-dependent ClpAP protease, participates in the dissolution and degradation of regulatory proteins and protein aggregates. ClpA consists of three functional domains: an N-terminal domain and two ATPase domains, D1 and D2. The N-domain is attached to D1 by a mobile linker and is made up of two tightly bound, identically folded alpha-helical bundles related by a pseudo 2-fold symmetry. Between the halves of the pseudo-dimer is a large flexible acidic loop that becomes better ordered upon binding of the small adaptor protein, ClpS. We have identified a number of structural features in the N-domain, including a Zn(++) binding motif, several interfaces for binding to ClpS, and a prominent hydrophobic surface area that binds peptides in different configurations. These structural motifs may contribute to binding of protein or peptide substrates with weak affinity and broad specificity. Kinetic studies comparing wild-type ClpA to a mutant ClpA with its N-domain deleted show that the N-domains contribute to the binding of a non-specific protein substrate but not of a folded substrate with the specific SsrA recognition tag. A functional model is proposed in which the N-domains in ClpA function as tentacles to weakly hold on to proteins thereby enhancing local substrate concentration.     

Examination of the nucleotide‐linked assembly mechanism of E. coli ClpA

Escherichia coli ClpA is a AAA+ (ATPase Associated with diverse cellular Activities) chaperone that catalyzes the ATP‐dependent unfolding and translocation of substrate proteins targeted for degradation by a protease, ClpP. ClpA hexamers associate with one or both ends of ClpP tetradecamers to form ClpAP complexes. Each ClpA protomer contains two nucleotide‐binding sites, NBD1 and NBD2, and self‐assembly into hexamers is thermodynamically linked to nucleotide binding. Despite a number of studies aimed at characterizing ClpA and ClpAP‐catalyzed substrate unfolding and degradation, respectively, to date the field is unable to quantify the concentration of ClpA hexamers available to interact with ClpP for any given nucleotide and total ClpA concentration. In this work, sedimentation velocity studies are used to quantitatively examine the self‐assembly of a ClpA Walker B variant in the presence of ATP. In addition to the hexamerization, we observe the formation of a previously unreported ClpA dodecamer in the presence of ATP. Further, we report apparent equilibrium constants for the formation of each ClpA oligomer obtained from direct boundary modeling of the sedimentation velocity data. The energetics of nucleotide binding to NBD1 and NBD2 are revealed by examining the dependence of the apparent association equilibrium constants on free nucleotide concentration.     

Synchrotron protein footprinting supports substrate translocation by ClpA via ATP-induced movements of the D2 loop

Synchrotron X-ray protein footprinting is used to study structural changes upon formation of the ClpA hexamer. Comparative solvent accessibilities between ClpA monomer and ClpA hexamer samples are in agreement throughout most of the sequence, with calculations based on two previously proposed hexameric models. The data differ substantially from the proposed models in two parts of the structure: the D1 sensor 1 domain and the D2 loop region. The results suggest that these two regions can access alternate conformations in which their solvent protection is greater than that in the structural models based on crystallographic data. In combination with previously reported structural data, the footprinting data provide support for a revised model in which the D2 loop contacts the D1 sensor 1 domain in the ATP-bound form of the complex. These data provide the first direct experimental support for the nucleotide-dependent D2 loop conformational change previously proposed to mediate substrate translocation.     

A Single ClpS Monomer Is Sufficient to Direct the Activity of the ClpA Hexamer

ClpS is an adaptor protein that interacts with ClpA and promotes degradation of proteins with N-end rule degradation motifs (N-degrons) by ClpAP while blocking degradation of substrates with other motifs. Although monomeric ClpS forms a 1:1 complex with an isolated N-domain of ClpA, only one molecule of ClpS binds with high affinity to ClpA hexamers (ClpA₆). One or two additional molecules per hexamer bind with lower affinity. Tightly bound ClpS dissociates slowly from ClpA₆ with a t_½ of ∼3 min at 37 °C. Maximum activation of degradation of the N-end rule substrate, LR-GFP_Venus, occurs with a single ClpS bound per ClpA₆; one ClpS is also sufficient to inhibit degradation of proteins without N-degrons. ClpS competitively inhibits degradation of unfolded substrates that interact with ClpA N-domains and is a non-competitive inhibitor with substrates that depend on internal binding sites in ClpA. ClpS inhibition of substrate binding is dependent on the order of addition. When added first, ClpS blocks binding of both high and low affinity substrates; however, when substrates first form committed complexes with ClpA₆, ClpS cannot displace them or block their degradation by ClpP. We propose that the first molecule of ClpS binds to the N-domain and to an additional functional binding site, sterically blocking binding of non-N-end rule substrates as well as additional ClpS molecules to ClpA₆. Limiting ClpS-mediated substrate delivery to one per ClpA₆ avoids congestion at the axial channel and allows facile transfer of proteins to the unfolding and translocation apparatus.     

Effects of local protein stability and the geometric position of the substrate degradation tag on the efficiency of ClpXP denaturation and degradation

ClpX and related AAA+ ATPases of the Clp/Hsp100 family are able to denature native proteins. Here, we explore the role of protein stability in ClpX denaturation and subsequent ClpP degradation of model substrates bearing ssrA degradation tags at different positions. ClpXP degraded T. thermophilus RNase-H* with a C-terminal ssrA tag very efficiently, despite the very high global stability of this thermophilic protein. In fact, global thermodynamic stability appears to play little role in susceptibility to degradation, as a far less stable RNase-H*-ssrA mutant was degraded more slowly than wild type by ClpXP and a completely unfolded mutant variant was degraded less than twice as fast as the wild-type parent. When ssrA peptide tags were covalently linked to surface cysteines at positions 114 or 140 of RNase-H*, the conjugates were proteolyzed very slowly. This resistance to degradation was not caused by inaccessibility of the ssrA tag or an inability of ClpXP to degrade proteins with side-chain linked ssrA tags. Our results support a model in which ClpX denatures proteins by initially unfolding structural elements attached to the degradation tag, suggest an important role for the position of the degradation tag and direction of force application, and correlate well with the mapping of local protein stability within RNase-H* by native-state hydrogen exchange.     

Characterization of the N-terminal repeat domain of Escherichia coli ClpA-A class I Clp/HSP100 ATPase

The ClpA, ClpB, and ClpC subfamilies of the Clp/HSP100 ATPases contain a conserved N-terminal region of approximately 150 residues that consists of two approximate sequence repeats. This sequence from the Escherichia coli ClpA enzyme is shown to encode an independent structural domain (the R domain) that is monomeric and approximately 40% alpha-helical. A ClpA fragment lacking the R domain showed ATP-dependent oligomerization, protein-stimulated ATPase activity, and the ability to complex with the ClpP peptidase and mediate degradation of peptide and protein substrates, including casein and ssrA-tagged proteins. Compared with the activities of the wild-type ClpA, however, those of the ClpA fragment missing the R domain were reduced. These results indicate that the R domain is not required for the basic recognition, unfolding, and translocation functions that allow ClpA-ClpP to degrade some protein substrates, but they suggest that it may play a role in modulating these activities.     

Structure of a delivery protein for an AAA+ protease in complex with a peptide degradation tag

Substrate selection by AAA+ ATPases that function to unfold proteins or alter protein conformation is often regulated by delivery or adaptor proteins. SspB is a protein dimer that binds to the ssrA degradation tag and delivers proteins bearing this tag to ClpXP, an AAA+ protease, for degradation. Here, we describe the structure of the peptide binding domain of H. influenzae SspB in complex with an ssrA peptide at 1.6 A resolution. The ssrA peptides are bound in well-defined clefts located at the extreme ends of the SspB homodimer. SspB contacts residues within the N-terminal and central regions of the 11 residue ssrA tag but leaves the C-terminal residues exposed and positioned to dock with ClpX. This structure, taken together with biochemical analysis of SspB, suggests mechanisms by which proteins like SspB escort substrates to AAA+ ATPases and enhance the specificity and affinity of target recognition.     

ATP hydrolysis by the proteasome regulatory complex PAN serves multiple functions in protein degradation

To clarify the role of ATP in proteolysis, we studied archaeal 20S proteasomes and the PAN (proteasome-activating nucleotidase) regulatory complex, a homolog of the eukaryotic 19S ATPases. PAN's ATPase activity was stimulated similarly by globular (GFPssrA) and unfolded (casein) substrates, and by the ssrA recognition peptide. Denaturation of GFPssrA did not accelerate its degradation or eliminate the requirement for PAN and ATP. During degradation of one molecule of globular or unfolded substrates, 300-400 ATP molecules were hydrolyzed. An N-terminal deletion in the 20S alpha subunits caused opening of the substrate-entry channel and rapid degradation of unfolded proteins without PAN; however, degradation of globular GFPssrA still required PAN's ATPase activity, even after PAN-catalyzed unfolding. Thus, substrate binding activates ATP hydrolysis, which promotes three processes: substrate unfolding, gate opening in the 20S, and protein translocation.     

Crystal structure of ClpA,an Hsp100 chaperone and regulator of ClpAP protease

Escherichia coli ClpA, an Hsp100/Clp chaperone and an integral component of the ATP-dependent ClpAP protease, participates in regulatory protein degradation and the dissolution and degradation of protein aggregates. The crystal structure of the ClpA subunit reveals an N-terminal domain with pseudo-twofold symmetry and two AAA(+) modules (D1 and D2) each consisting of a large and a small sub-domain with ADP bound in the sub-domain junction. The N-terminal domain interacts with the D1 domain in a manner similar to adaptor-binding domains of other AAA(+) proteins. D1 and D2 are connected head-to-tail consistent with a cooperative and vectorial translocation of protein substrates. In a planar hexamer model of ClpA, built by assembling ClpA D1 and D2 into homohexameric rings of known structures of AAA(+) modules, the differences in D1-D1 and D2-D2 interfaces correlate with their respective contributions to hexamer stability and ATPase activity.     

Conserved residues in the N-domain of the AAA+ chaperone ClpA regulate substrate recognition and unfolding

Protein degradation in the cytosol of Escherichia coli is carried out by a variety of different proteolytic machines, including ClpAP. The ClpA component is a hexameric AAA+ (ATPase associated with various cellular activities) chaperone that utilizes the energy of ATP to control substrate recognition and unfolding. The precise role of the N-domains of ClpA in this process, however, remains elusive. Here, we have analysed the role of five highly conserved basic residues in the N-domain of ClpA by monitoring the binding, unfolding and degradation of several different substrates, including short unstructured peptides, tagged and untagged proteins. Interestingly, mutation of three of these basic residues within the N-domain of ClpA (H94, R86 and R100) did not alter substrate degradation. In contrast mutation of two conserved arginine residues (R90 and R131), flanking a putative peptide-binding groove within the N-domain of ClpA, specifically compromised the ability of ClpA to unfold and degrade selected substrates but did not prevent substrate recognition, ClpS-mediated substrate delivery or ClpP binding. In contrast, a highly conserved tyrosine residue lining the central pore of the ClpA hexamer was essential for the degradation of all substrate types analysed, including both folded and unstructured proteins. Taken together, these data suggest that ClpA utilizes two structural elements, one in the N-domain and the other in the pore of the hexamer, both of which are required for efficient unfolding of some protein substrates.     

Stepwise unfolding of a β barrel protein by the AAA+ ClpXP protease

In the AAA+ ClpXP protease, ClpX uses the energy of ATP binding and hydrolysis to unfold proteins before translocating them into ClpP for degradation. For proteins with C-terminal ssrA tags, ClpXP pulls on the tag to initiate unfolding and subsequent degradation. Here, we demonstrate that an initial step in ClpXP unfolding of the 11-stranded β barrel of superfolder GFP-ssrA involves extraction of the C-terminal β strand. The resulting 10-stranded intermediate is populated at low ATP concentrations, which stall ClpXP unfolding, and at high ATP concentrations, which support robust degradation. To determine if stable unfolding intermediates cause low-ATP stalling, we designed and characterized circularly permuted GFP variants. Notably, stalling was observed for a variant that formed a stable 10-stranded intermediate but not for one in which this intermediate was unstable. A stepwise degradation model in which the rates of terminal-strand extraction, strand refolding or recapture, and unfolding of the 10-stranded intermediate all depend on the rate of ATP hydrolysis by ClpXP accounts for the observed changes in degradation kinetics over a broad range of ATP concentrations. Our results suggest that the presence or absence of unfolding intermediates will play important roles in determining whether forced enzymatic unfolding requires a minimum rate of ATP hydrolysis.     

Evidence for an unfolding/threading mechanism for protein disaggregation by Saccharomyces cerevisiae Hsp104

Saccharomyces cerevisiae Hsp104, a hexameric member of the Hsp100/Clp subfamily of AAA+ ATPases with two nucleotide binding domains (NBD1 and 2), refolds aggregated proteins in conjunction with Hsp70 molecular chaperones. Hsp104 may act as a "molecular crowbar" to pry aggregates apart and/or may extract proteins from aggregates by unfolding and threading them through the axial channel of the Hsp104 hexamer. Targeting Tyr-662, located in a Gly-Tyr-Val-Gly motif that forms part of the axial channel loop in NBD2, we created conservative (Phe and Trp) and non-conservative (Ala and Lys) amino acid substitutions. Each of these Hsp104 derivatives was comparable to the wild type protein in their ability to hydrolyze ATP, assemble into hexamers, and associate with heat-shock-induced aggregates in living cells. However, only those with conservative substitutions complemented the thermotolerance defect of a Deltahsp104 yeast strain and promoted refolding of aggregated protein in vitro. Monitoring fluorescence from Trp-662 showed that titration of fully assembled molecules with either ATP or ADP progressively quenches fluorescence, suggesting that nucleotide binding determines the position of the loop within the axial channel. A Glu to Lys substitution at residue 645 in the NBD2 axial channel strongly alters the nucleotide-induced change in fluorescence of Trp-662 and specifically impairs in protein refolding. These data establish that the structural integrity of the axial channel through NBD2 is required for Hsp104 function and support the proposal that Hsp104 and ClpB use analogous unfolding/threading mechanisms to promote disaggregation and refolding that other Hsp100s use to promote protein degradation.     

The ClpS adaptor mediates staged delivery of N-end rule substrates to the AAA+ ClpAP protease

The ClpS adaptor delivers N-end rule substrates to ClpAP, an energy-dependent AAA+ protease, for degradation. How ClpS binds specific N-end residues is known in atomic detail and clarified here, but the delivery mechanism is poorly understood. We show that substrate binding is enhanced when ClpS binds hexameric ClpA. Reciprocally, N-end rule substrates increase ClpS affinity for ClpA(6). Enhanced binding requires the N-end residue and a peptide bond of the substrate, as well as multiple aspects of ClpS, including a side chain that contacts the substrate α-amino group and the flexible N-terminal extension (NTE). Finally, enhancement also needs the N domain and AAA+ rings of ClpA, connected by a long linker. The NTE can be engaged by the ClpA translocation pore, but ClpS resists unfolding/degradation. We propose a staged-delivery model that illustrates how intimate contacts between the substrate, adaptor, and protease reprogram specificity and coordinate handoff from the adaptor to the protease.     

Peptide and protein binding in the axial channel of Hsp104. Insights into the mechanism of protein unfolding

The AAA+ molecular chaperone Hsp104 mediates the extraction of proteins from aggregates by unfolding and threading them through its axial channel in an ATP-driven process. An Hsp104-binding peptide selected from solid phase arrays enhanced the refolding of a firefly luciferase-peptide fusion protein. Analysis of peptide binding using tryptophan fluorescence revealed two distinct binding sites, one in each AAA+ module of Hsp104. As a further indication of the relevance of peptide binding to the Hsp104 mechanism, we found that it competes with the binding of a model unfolded protein, reduced carboxymethylated alpha-lactalbumin. Inactivation of the pore loops in either AAA+ module prevented stable peptide and protein binding. However, when the loop in the first AAA+ was inactivated, stimulation of ATPase turnover in the second AAA+ module of this mutant was abolished. Drawing on these data, we propose a detailed mechanistic model of protein unfolding by Hsp104 in which an initial unstable interaction involving the loop in the first AAA+ module simultaneously promotes penetration of the substrate into the second axial channel binding site and activates ATP turnover in the second AAA+ module.     

The ClpP double ring tetradecameric protease exhibits plastic ring-ring interactions, and the N termini of its subunits form flexible loops that are essential for ClpXP and ClpAP complex formation

ClpP is a conserved serine-protease with two heptameric rings that enclose a large chamber containing the protease active sites. Each ClpP subunit can be divided into a handle region, which mediates ring-ring interactions, and a head domain. ClpP associates with the hexameric ATPases ClpX and ClpA, which can unfold and translocate substrate proteins through the ClpP axial pores into the protease lumen for degradation. We have determined the x-ray structure of Streptococcus pneumoniae ClpP(A153P) at 2.5 A resolution. The structure revealed two novel features of ClpP which are essential for ClpXP and ClpAP functional activities. First, the Ala --> Pro mutation disrupts the handle region, resulting in an altered ring-ring dimerization interface, which, in conjunction with biochemical data, demonstrates the unusual plasticity of this region. Second, the structure shows the existence of a flexible N-terminal loop in each ClpP subunit. The loops line the axial pores in the ClpP tetradecamer and then protrude from the protease apical surface. The sequence of the N-terminal loop is highly conserved in ClpP across all kingdoms of life. These loops are essential determinants for complex formation between ClpP and ClpX/ClpA. Mutation of several amino acid residues in this loop or the truncation of the loop impairs ClpXP and ClpAP complex formation and prevents the coupling between ClpX/ClpA and ClpP activities.