Dynamics of substrate denaturation and translocation by the ClpXP degradation machine

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.     

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.     

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.     

Effects of protein stability and structure on substrate processing by the ClpXP unfolding and degradation machine.

ClpXP is an ATP-dependent protease that denatures native proteins and translocates the denatured polypeptide into an interior peptidase chamber for degradation. To address the mechanism of these processes, Arc repressor variants with dramatically different stabilities and unfolding half-lives varying from months to seconds were targeted to ClpXP by addition of the ssrA degradation tag. Remarkably, ClpXP degraded each variant at a very similar rate and hydrolyzed approximately 150 molecules of ATP for each molecule of substrate degraded. The hyperstable substrates did, however, slow the ClpXP ATPase cycle. These results confirm that ClpXP uses an active mechanism to denature its substrates, probably one that applies mechanical force to the native structure. Furthermore, the data suggest that denaturation is inherently inefficient or that significant levels of ATP hydrolysis are required for other reaction steps. ClpXP degraded disulfide-cross-linked dimers efficiently, even when just one subunit contained an ssrA tag. This result indicates that the pore through which denatured proteins enter the proteolytic chamber must be large enough to accommodate simultaneous passage of two or three polypeptide chains.     

Engineering controllable protein degradation

Complex biological networks are regulated via alterations in protein expression, degradation, and function. Synthetic control of these processes allows dissection of natural systems and the design of new networks. In E. coli, the adaptor SspB tethers ssrA-tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. To engineer controlled degradation, we have designed a series of modified ssrA tags that have weakened interactions with ClpXP. When SspB is present, ClpXP degrades purified substrates bearing these engineered peptide tags 100-fold more efficiently. Importantly, substrates bearing these tags are stable in the absence of SspB in vivo but are rapidly degraded upon SspB induction. Our studies supply a conceptual foundation and working components for controllable degradation, improve mechanistic understanding of adaptor-mediated proteolysis, and demonstrate that the relative importance of adaptor proteins in degradation is correlated with the strength of protease-substrate contacts.     

Altered specificity of a AAA+ protease

ClpXP, an ATP-dependent protease, degrades hundreds of different intracellular proteins. ClpX chooses substrates by binding peptide tags, typically displayed at the N or C terminus of the protein to be degraded. Here, we identify a ClpX mutant that displays a 300-fold change in substrate specificity, resulting in decreased degradation of ssrA-tagged substrates but improved degradation of proteins with other classes of degradation signals. The altered-specificity mutation occurs within "RKH" loops, which surround the entrance to the central pore of the ClpX hexamer and are highly conserved in the ClpX subfamily of AAA+ ATPases. These results support a major role for the RKH loops in substrate recognition and suggest that ClpX specificity represents an evolutionary compromise that has optimized degradation of multiple types of substrates rather than any single class.     

Linkage between ATP consumption and mechanical unfolding during the protein processing reactions of an AAA+ degradation machine

Proteolytic machines powered by ATP hydrolysis bind proteins with specific peptide tags, denature these substrates, and translocate them into a sequestered compartment for degradation. To determine how ATP is used during individual reaction steps, we assayed ClpXP degradation of ssrA-tagged titin variants with different stabilities in native and denatured forms. The rate of ATP turnover was 4-fold slower during denaturation than translocation. Importantly, this reduced turnover rate was constant during denaturation of native variants with different stabilities, but total ATP consumption increased with substrate stability, suggesting an iterative application of a uniform, mechanical unfolding force. Destabilization of substrate structure near the degradation tag accelerated degradation and dramatically reduced ATP consumption, revealing an important role for local protein stability in resisting denaturation. The ability to denature more stable proteins simply by using more ATP endows ClpX with a robust unfolding activity required for its biological roles in degradation and complex disassembly.     

Trans-targeting of protease substrates by conformationally activating a regulable ClpX-recognition motif

Conversion of bacteriophage Mu repressor to ClpXP-sensitive form correlates with induced local flexibility at the ClpX recognition motif located at the C-terminal end. Changing the C-terminal valine to an alanine (RepV196A) caused the degradation tag to be constitutively active like that of mutant repressors called Vir, which have a dominant ClpXP-sensitive conformation. However, unlike Vir, RepV196A was unable to convert wild-type repressor (Rep) to the ClpXP-sensitive form. In mixtures with Rep, only RepV196A was rapidly degraded by ClpXP. Unlike Rep, RepV196A was ClpXP sensitive without induced C-terminal flexibility. And unlike adaptor proteins that tether and deliver substrates to ClpX for trans-targeting, Vir promoted rapid degradation of Rep by ClpX deleted for the tethering site that binds adaptor proteins. Therefore, Rep's ClpX recognition motif has regulable properties, allowing an alternative trans-targeting mechanism in which an inactive degradation tag is turned on by induced conformational changes.     

Altered tethering of the SspB adaptor to the ClpXP protease causes changes in substrate delivery

SspB is a dimeric adaptor protein that increases the rate at which ssrA-tagged substrates are degraded by tethering them to the ClpXP protease. Each SspB subunit consists of a folded domain that forms the dimer interface and a flexible C-terminal tail. Ternary delivery complexes are stabilized by three sets of tethering interactions. The C-terminal XB peptide of each SspB subunit binds ClpX, the body of SspB binds one part of the ssrA-tag sequence, and ClpX binds another part of the tag. To test the functional importance of these tethering interactions, we engineered monomeric SspB variants and dimeric variants with different length linkers between the SspB body and the XB peptide and employed substrates with degradation tags that bind ClpX weakly and/or contain extensions between the binding sites for SspB and ClpX. We find that monomeric SspB variants can enhance ClpXP degradation of a subset of substrates, that doubling the number of tethering interactions stimulates degradation via changes in Km and Vmax, and that major alterations in the length of the 48-residue SspB linker cause only small changes in the efficiency of substrate delivery. These results indicate that the properties of the degradation tag and the number of SspB.ClpX tethering interactions are the major factors that determine the extent to which the substrate and ClpX are engaged in ternary delivery complexes.     

A context-dependent ClpX recognition determinant located at the C terminus of phage Mu repressor

The bacteriophage Mu immunity repressor is a conformationally sensitive sensor that can be interconverted between forms resistant to and sensitive to degradation by ClpXP protease. Protease-sensitive repressor molecules with an altered C-terminal sequence promote rapid degradation of the wild-type repressor by inducing its C-terminal end to become exposed. Here we determined that the last 5 C-terminal residues (CTD5) of the wild-type repressor contain the motif required for recognition by the ClpX molecular chaperone, a motif that is strongly dependent upon the context in which it is presented. Although attachment of the 11-residue ssrA degradation tag to the C terminus of green fluorescent protein (GFP) promoted its rapid degradation by ClpXP, attachment of 5-27 C-terminal residues of the repressor failed to promote degradation. Disordered peptides derived from 41 and 35 C-terminal residues of CcdA (CcdA41) and thioredoxin (TrxA35), respectively, activated CTD5 when placed as linkers between GFP and repressor C-terminal sequences. However, when the entire thioredoxin sequence was included as a linker to promote an ordered configuration of the TrxA35 peptide, the resulting substrate was not degraded. In addition, a hybrid tag, in which CTD5 replaced the 3-residue recognition motif of the ssrA tag, was inactive when attached directly to GFP but active when attached through the CcdA41 peptide. Thus, CTD5 is sufficient to act as a recognition motif but has requirements for its presentation not shared by the ssrA tag. We suggest that activation of CTD5 may require presentation on a disordered or flexible domain that confers ligand flexibility.     

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.     

Kinetics of protein substrate degradation by HslUV

The HslUV protease-chaperone complex degrades specific protein substrates in an ATP-dependent reaction. Current models propose that the HslU chaperone, a AAA protein of the Clp/Hsp100 family, binds and unfolds substrates and translocates the polypeptide into the catalytic cavity of the HslV protease. These processes are being characterized using substrates that are targeted to HslUV with a carboxy-terminal fusion of the natural substrate SulA or the carboxy-terminal 11 amino acid residues thereof. In a tandem fusion of green fluorescent protein with SulA, HslUV degrades the SulA moiety but not green fluorescent protein. Wild type and mutant Arc repressor variants are degraded; over a range of substrate stabilities, the specific rate of degradation and its dependence on substrate stability is similar to that of ClpXP. For a hyperstable Arc variant having an intermolecular disulfide bond, the rate of degradation by HslUV is an order of magnitude slower than by ClpXP. Similarity in degradation rates for a subset of substrates by HslUV and ClpXP suggests a similarity in mechanism of the apparent rate-limiting steps of unfolding and translocation by the chaperone components HslU and ClpX. The fall-off in degradation by HslUV for the more stable substrates that are degraded by ClpXP is consistent with the two systems acting on different spectra of biological substrates.     

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.     

Binding and degradation of heterodimeric substrates by ClpAP and ClpXP

ClpA and ClpX function both as molecular chaperones and as the regulatory components of ClpAP and ClpXP proteases, respectively. ClpA and ClpX bind substrate proteins through specific recognition signals, catalyze ATP-dependent protein unfolding of the substrate, and when in complexes with ClpP translocate the unfolded polypeptide into the cavity of the ClpP peptidase for degradation. To examine the mechanism of interaction of ClpAP with dimeric substrates, single round binding and degradation experiments were performed, revealing that ClpAP degraded both subunits of a RepA homodimer in one cycle of binding. Furthermore, ClpAP was able to degrade both protomers of a RepA heterodimer in which only one subunit contained the ClpA recognition signal. In contrast, ClpXP degraded both subunits of a dimeric substrate only when both protomers contained a recognition signal. These data suggest that ClpAP and ClpXP may recognize and bind substrates in significantly different ways.     

Structural basis of SspB-tail recognition by the zinc binding domain of ClpX

The degradation of ssrA(AANDENYALAA)-tagged proteins in the bacterial cytosol is carried out by the ClpXP protease and is markedly stimulated by the SspB adaptor protein. It has previously been reported that the amino-terminal zinc-binding domain of ClpX (ZBD) is involved in complex formation with the SspB-tail (XB: ClpX-binding motif). In an effort to better understand the recognition of SspB by ClpX and the mechanism of delivery of ssrA-tagged substrates to ClpXP, we have determined the structures of ZBD alone at 1.5, 2.0, and 2.5 A resolution in each different crystal form and also in complex with XB peptide at 1.6 A resolution. The XB peptide forms an antiparallel beta-sheet with two beta-strands of ZBD, and the structure shows a 1:1 stoichiometric complex between ZBD and XB, suggesting that there are two independent SspB-tail-binding sites in ZBD. The high-resolution ZBD:XB complex structure, in combination with biochemical analyses, can account for key determinants in the recognition of the SspB-tail by ClpX and sheds light on the mechanism of delivery of target proteins to the prokaryotic degradation machine.     

Cytoplasmic degradation of ssrA-tagged proteins

Degradation of ssrA-tagged proteins is a central feature of protein-quality control in all bacteria. In Escherichia coli, the ATP-dependent ClpXP and ClpAP proteases are thought to participate in this process, but their relative contributions to degradation of ssrA-tagged proteins in vivo have been uncertain because two adaptor proteins, ClpS and SspB, can modulate proteolysis of these substrates. Here, intracellular levels of these protease components and adaptors were determined during exponential growth and as cells entered early stationary phase. Levels of ClpA and ClpP increased about threefold during this transition, whereas ClpX, ClpS and SspB levels remained nearly constant. Using GFP-ssrA expressed from the chromosome as a degradation reporter, the effects of altered concentrations of different protease components or adaptor proteins were explored. Both ClpXP and ClpAP degraded GFP-ssrA in the cell, demonstrating that wild-type levels of SspB and ClpS do not inhibit ClpAP completely. Upon entry into stationary phase, increased levels of ClpAP resulted in increased degradation of ssrA-tagged substrates. As measured by maximum turnover rates, ClpXP degradation of GFP-ssrA in vivo was significantly more efficient than in vitro. Surprisingly, ClpX-dependent ClpP-independent degradation of GFP-ssrA was also observed. Thus, unfolding of this substrate by ClpX appears to enhance intracellular degradation by other proteases.     

Asymmetric interactions of ATP with the AAA+ ClpX6 unfoldase: allosteric control of a protein machine

ATP hydrolysis by AAA+ ClpX hexamers powers protein unfolding and translocation during ClpXP degradation. Although ClpX is a homohexamer, positive and negative allosteric interactions partition six potential nucleotide binding sites into three classes with asymmetric properties. Some sites release ATP rapidly, others release ATP slowly, and at least two sites remain nucleotide free. Recognition of the degradation tag of protein substrates requires ATP binding to one set of sites and ATP or ADP binding to a second set of sites, suggesting a mechanism that allows repeated unfolding attempts without substrate release over multiple ATPase cycles. Our results rule out concerted hydrolysis models involving ClpX(6)*ATP(6) or ClpX(6)*ADP(6) and highlight structures of hexameric AAA+ machines with three or four nucleotides as likely functional states. These studies further emphasize commonalities between distant AAA+ family members, including protein and DNA translocases, helicases, motor proteins, clamp loaders, and other ATP-dependent enzymes.     

Protein inactivation in mycobacteria by controlled proteolysis and its application to deplete the beta subunit of RNA polymerase

Using a component of the Escherichia coli protein degradation machinery, we have established a system to regulate protein stability in mycobacteria. A protein tag derived from the E. coli SsrA degradation signal did not affect several reporter proteins in wild-type Mycobacterium smegmatis or Mycobacterium tuberculosis. Expression of the adaptor protein SspB, which recognizes this modified tag and helps deliver tagged proteins to the protease ClpXP, strongly decreased the activities and protein levels of different reporters. This inactivation did not occur when the function of ClpX was inhibited. Using this system, we constructed a conditional M. smegmatis knockdown mutant in which addition of anhydrotetracycline (atc) caused depletion of the beta subunit of RNA polymerase, RpoB. The impact of atc on this mutant was dose-dependent. Very low amounts of atc did not prevent growth but increased sensitivity to an antibiotic that inactivates RpoB. Intermediate amounts of RpoB knockdown resulted in bacteriostasis and a more substantial depletion led to a decrease in viability by up to 99%. These studies identify SspB-mediated proteolysis as an efficient approach to conditionally inactivate essential proteins in mycobacteria. They further demonstrate that depletion of RpoB by ～ 93% is sufficient to cause death of M. smegmatis.