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.     

Versatile modes of peptide recognition by the ClpX N domain mediate alternative adaptor‐binding specificities in different bacterial species

ClpXP, an AAA+ protease, plays key roles in protein‐quality control and many regulatory processes in bacteria. The N‐terminal domain of the ClpX component of ClpXP is involved in recognition of many protein substrates, either directly or by binding the SspB adaptor protein, which delivers specific classes of substrates for degradation. Despite very limited sequence homology between the E. coli and C. crescentus SspB orthologs, each of these adaptors can deliver substrates to the ClpXP enzyme from the other bacterial species. We show that the ClpX N domain recognizes different sequence determinants in the ClpX‐binding (XB) peptides of C. crescentus SspBα and E. coli SspB. The C. crescentus XB determinants span 10 residues and involve interactions with multiple side chains, whereas the E. coli XB determinants span half as many residues with only a few important side chain contacts. These results demonstrate that the N domain of ClpX functions as a highly versatile platform for peptide recognition, allowing the emergence during evolution of alternative adaptor‐binding specificities. Our results also reveal highly conserved residues in the XB peptides of both E. coli SspB and C. crescentus SspBα that play no detectable role in ClpX‐binding or substrate delivery.     

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.     

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.     

Flexible linkers leash the substrate binding domain of SspB to a peptide module that stabilizes delivery complexes with the AAA+ ClpXP protease

SspB dimers bind proteins bearing the ssrA-degradation tag and stimulate their degradation by the ClpXP protease. Here, E. coli SspB is shown to contain a dimeric substrate binding domain of 110-120 N-terminal residues, which binds ssrA-tagged substrates but does not stimulate their degradation. The C-terminal 40-50 residues of SspB are unstructured but are required for SspB to form substrate-delivery complexes with ClpXP. A synthetic peptide containing the 10 C-terminal residues of SspB binds ClpX, stimulates its ATPase activity, and prevents SspB-mediated delivery of GFP-ssrA for ClpXP degradation. This tripartite structure--an ssrA-tag binding and dimerization domain, a flexible linker, and a short peptide module that docks with ClpX--allows SspB to deliver tagged substrates to ClpXP without interfering with their denaturation or degradation.     

Targeted delivery of an ssrA-tagged substrate by the adaptor protein SspB to its cognate AAA+ protein ClpX

In the bacterial cytosol, degradation of ssrA-tagged proteins is primarily carried out by the proteolytic machine ClpXP in a process which is stimulated by a ClpX-specific adaptor protein, SspB. Here we elucidate the steps required for binding and transfer of ssrA-tagged substrates from SspB to ClpX. The N-terminal region of SspB is essential for its interaction with ssrA-tagged substrates, while a short conserved region at the C terminus of SspB interacts specifically with the N domain of ClpX. A single point mutation within the conserved C-terminal region of SspB is sufficient to abolish the SspB-mediated degradation of ssrA-tagged proteins by ClpXP. We propose that this region represents a common motif for the recognition of ClpX as the C-terminal region of SspB shares considerable homology with the other ClpX-specific adaptor protein, RssB. Through docking of SspB to the N-terminal domain of ClpX, the substrate is delivered to the substrate binding site in ClpX.     

The N-terminal zinc binding domain of ClpX is a dimerization domain that modulates the chaperone function

Clp ATPases are unique chaperones that promote protein unfolding and subsequent degradation by proteases. The mechanism by which this occurs is poorly understood. Here we demonstrate that the N-terminal domain of ClpX is a C4-type zinc binding domain (ZBD) involved in substrate recognition. ZBD forms a very stable dimer that is essential for promoting the degradation of some typical ClpXP substrates such as lambdaO and MuA but not GFP-SsrA. Furthermore, experiments indicate that ZBD contains a primary binding site for the lambdaO substrate and for the cofactor SspB. Removal of ZBD from the ClpX sequence renders the ATPase activity of ClpX largely insensitive to the presence of ClpP, substrates, or the SspB cofactor. All these results indicate that ZBD plays an important role in the ClpX mechanism of function and that ATP binding and/or hydrolysis drives a conformational change in ClpX involving ZBD.     

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.     

Large nucleotide-dependent movement of the N-terminal domain of the ClpX chaperone

The ClpXP ATPase-protease complex is a major component of the protein quality control machinery in the cell. A ClpX subunit consists of an N-terminal zinc binding domain (ZBD) and a C-terminal AAA+ domain. ClpX oligomerizes into a hexamer with the AAA+ domains forming the base of the hexamer and the ZBDs extending out of the base. Here, we report that ClpX switches between a capture and a feeding conformation. ZBDs in ClpX undergo large nucleotide-dependent block movement towards ClpP and into the AAA+ ring. This motion is modulated by the ClpX cofactor, SspB. Evidence for this movement was initially obtained by the surprising observation that an N-terminal extension on ClpX is clipped by bound ClpP in functional ClpXP complexes. Protease-protection, crosslinking, and light scattering experiments further support these findings.     

Communication between ClpX and ClpP during substrate processing and degradation

In the ClpXP compartmental protease, ring hexamers of the AAA(+) ClpX ATPase bind, denature and then translocate protein substrates into the degradation chamber of the double-ring ClpP(14) peptidase. A key question is the extent to which functional communication between ClpX and ClpP occurs and is regulated during substrate processing. Here, we show that ClpX-ClpP affinity varies with the protein-processing task of ClpX and with the catalytic engagement of the active sites of ClpP. Functional communication between symmetry-mismatched ClpXP rings depends on the ATPase activity of ClpX and seems to be transmitted through structural changes in its IGF loops, which contact ClpP. A conserved arginine in the sensor II helix of ClpX links the nucleotide state of ClpX to the binding of ClpP and protein substrates. A simple model explains the observed relationships between ATP binding, ATP hydrolysis and functional interactions between ClpX, protein substrates and ClpP.     

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.     

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.     

Engineering Synthetic Adaptors and Substrates for Controlled ClpXP Degradation

Facile control of targeted intracellular protein degradation has many potential uses in basic science and biotechnology. One promising approach to this goal is to redesign adaptor proteins, which can regulate proteolytic specificity by tethering substrates to energy-dependent AAA+ proteases. Using the ClpXP protease, we have probed the minimal biochemical functions required for adaptor function by designing and characterizing variant substrates, adaptors, and ClpX enzymes. We find that substrate tethering mediated by heterologous interaction domains and a small bridging molecule mimics substrate delivery by the wild-type system. These results show that simple tethering is sufficient for synthetic adaptor function. In our engineered system, tethering and proteolysis depend on the presence of the macrolide rapamycin, providing a foundation for engineering highly specific degradation of target proteins in cells. Importantly, this degradation is regulated by a small molecule without the need for new adaptor or enzyme biosynthesis.Targeted proteolytic degradation plays important roles in protein quality control and in regulating cellular circuitry in organisms ranging from bacteria to humans (1–4). In some instances, substrates are recognized directly by a protease enzyme via a degradation tag (Fig. 1A) (see Refs. 5 and 6). In other cases, adaptor proteins or multiple types of substrate sequences are also required to ensure efficient degradation (Fig. 1, B and C) (see Refs. 7 and 8).Open in a separate windowFIGURE 1.A, the ClpX component of the ClpXP protease recognizes some substrates via a degradation tag, denatures the substrate, and then translocates the unfolded protein into ClpP for degradation. B, adaptor-assisted binding of a substrate to ClpXP. C, self-tethering of a substrate to ClpXP.Experimentally induced degradation can be used as a tool to probe the role of specific proteins in cellular processes. For example, a protein that is normally stable can be modified to make its degradation conditionally dependent on the presence of an adaptor, allowing studies of the consequences of depletion after induction of adaptor synthesis (7, 9). Such systems complement methods, such as RNA interference, that rely upon repressing biosynthesis of the target protein but offer significant advantages when rapid depletion of otherwise long-lived proteins is the goal (10–12). We are interested in engineering synthetic adaptor systems to control targeted intracellular degradation.ClpXP is a AAA+ protease present in bacteria and mitochondria that consists of two components, ClpX and ClpP. Hexamers of ClpX recognize degradation tags in specific substrate proteins, unfold them in a reaction that requires ATP hydrolysis, and then use additional cycles of ATP hydrolysis to translocate the unfolded polypeptide into an interior chamber of ClpP, where proteolysis takes place (see Fig. 1A). The simplest way in which an adaptor could stimulate degradation is by tethering a specific substrate to a protease, thereby increasing its effective concentration and facilitating proteolysis (see Fig. 1B; for review, see Ref. 3). The SspB adaptor, for example, appears to function by this mechanism. SspB enhances ClpXP degradation of certain substrates, including N-RseA and proteins bearing the ssrA-degradation tag (2, 8, 13). ClpXP degrades these substrates in the absence of SspB, but K_m for degradation is substantially lower when this adaptor is present. Two features of SspB are consistent with a tethering mechanism. It has a substrate-binding domain with a groove that binds a portion of the ssrA tag or a sequence in N-RseA, and it contains a flexible C-terminal extension terminating with a peptide motif (XB) that binds to the N-terminal domain of ClpX (14–19). Mutations that prevent SspB binding to ClpX or block substrate binding to SspB eliminate stimulation of degradation (13, 16, 20).It has not been rigorously established, however, that tethering per se is sufficient for the activity of any adaptor. Based on biochemical experiments, for instance, Thibault et al. (21) proposed that the adaptor activity of SspB is mediated, in part, by its ability to direct the movement of the N-terminal domains of ClpX, and thereby to regulate the delivery of tagged substrates to ClpXP. For some adaptors, tethering of the substrate to the protease is not sufficient for degradation. For example, the ClpS adaptor tethers N-end rule substrates to the AAA+ ClpAP protease (22–24), but some ClpS mutants mediate efficient substrate tethering to ClpAP without facilitating degradation (25). In such cases, more complicated transactions between the adaptor and the protease appear to be needed to ensure that the substrate is properly delivered to the protease. Moreover, in some instances, adaptors play roles in substrate delivery but are also required for assembly of the active protease (26).The studies reported here were motivated by two major goals. First, we wished to test if a completely synthetic adaptor system could be used to regulate substrate degradation. Second, we sought to design a proteolysis system that could be controlled by the presence or absence of a small molecule. To define the minimal biochemical properties required for adaptor-protein function, we engineered and characterized synthetic variants of adaptors, substrates, and the ClpXP protease. We reasoned that if specialized interactions between SspB and the N-terminal domain of ClpX were a requisite part of substrate delivery, then replacing either component would preclude efficient degradation. By contrast, we found that rapid degradation of an otherwise poor substrate was possible in the absence of SspB and the N-domain as long as substrate·enzyme tethering was maintained by other interaction domains. These results show that tethering alone is sufficient for synthetic- adaptor function. We were also able to control degradation in vitro and in vivo using systems in which a small molecule, rapamycin, drives assembly of tethered proteolytic complexes. Thus, targeted degradation can be engineered to depend, in a conditional fashion, on the presence of a small molecule. In principle, degradation under small molecule control has many of the advantages of chemical genetics (27), but should be even simpler and more widely applicable as a method of functional inhibition. In addition, controlling degradation in this fashion is possible even when biosynthesis of new macromolecules is precluded.     

Engineering an SspB-mediated degron for novel controllable protein degradation

Elegant controllable protein degradation tools have great applications in metabolic engineering and synthetic biology designs. SspB-mediated ClpXP proteolysis system is well characterized, and SspB acts as an adaptor tethering ssrA-tagged substrates to the ClpXP protease. This degron was applied in metabolism optimization, but the efficiency was barely satisfactory. Limited high-quality tools are available for controllable protein degradation. By coupling structure-guided modeling and directed evolution, we establish state-of-the-art high-throughput screening strategies for engineering both degradation efficiency and SspB-ssrA binding specificity of this degron. The reliability of our approach is confirmed by functional validation of both SspB and ssrA mutants using fluorescence assays and metabolic engineering of itaconic acid or ferulic acid biosynthesis. Isothermal titration calorimetry analysis and molecular modeling revealed that an appropriate instead of excessively strong interaction between SspB and ssrA benefited degradation efficiency. Mutated SspB-ssrA pairs with 7–22-fold higher binding K_D than the wild-type pair led to higher degradation efficiency, revealing the advantage of directed evolution over rational design in degradation efficiency optimization. Furthermore, an artificial SspB-ssrA pair exhibiting low crosstalk of interactions with the wild-type SspB-ssrA pair was also developed. Efforts in this study have demonstrated the plasticity of SspB-ssrA binding pocket for designing high-quality controllable protein degradation tools. The obtained mutated degrons enriched the tool box of metabolic engineering designs.     

Versatile modes of peptide recognition by the AAA+ adaptor protein SspB

Energy-dependent proteases often rely on adaptor proteins to modulate substrate recognition. The SspB adaptor binds peptide sequences in the stress-response regulator RseA and in ssrA-tagged proteins and delivers these molecules to the AAA+ ClpXP protease for degradation. The structure of SspB bound to an ssrA peptide is known. Here, we report the crystal structure of a complex between SspB and its recognition peptide in RseA. Notably, the RseA sequence is positioned in the peptide-binding groove of SspB in a direction opposite to the ssrA peptide, the two peptides share only one common interaction with the adaptor, and the RseA interaction site is substantially larger than the overlapping ssrA site. This marked diversity in SspB recognition of different target proteins indicates that it is capable of highly flexible and dynamic substrate delivery.     

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.     

Distinct static and dynamic interactions control ATPase-peptidase communication in a AAA+ protease

In the ClpXP proteolytic machine, ClpX uses the energy of ATP hydrolysis to unfold protein substrates and translocate them through a central pore and into the degradation chamber of ClpP. Here, we demonstrate a bipartite system of ClpX-ClpP interactions that serves multiple functional roles. High-affinity contacts between six loops near the periphery of the hexameric ClpX ring and a ClpP ring establish correct positioning and increase degradation activity but are insensitive to nucleotide state. These static peripheral interactions maintain a stable ClpXP complex, while other parts of this machine change conformation hundreds of times per minute. By contrast, relatively weak axial contacts between loops at the bottom of the ClpX central channel and N-terminal loops of ClpP vary dynamically with the nucleotide state of individual ClpX subunits, control ATP-hydrolysis rates, and facilitate efficient protein unfolding. Thus, discrete static and dynamic interactions mediate binding and communication between ClpX and ClpP.     

Control of Substrate Gating and Translocation into ClpP by Channel Residues and ClpX Binding

ClpP is a self-compartmentalized protease, which has very limited degradation activity unless it associates with ClpX to form ClpXP or with ClpA to form ClpAP. Here, we show that ClpX binding stimulates ClpP cleavage of peptides larger than a few amino acids and enhances ClpP active-site modification. Stimulation requires ATP binding but not hydrolysis by ClpX. The magnitude of this enhancement correlates with increasing molecular weight of the molecule entering ClpP. Amino-acid substitutions in the channel loop or helix A of ClpP enhance entry of larger substrates into the free enzyme, eliminate ClpX binding in some cases, and are not further stimulated by ClpX binding in other instances. These results support a model in which the channel residues of free ClpP exclude efficient entry of all but the smallest peptides into the degradation chamber, with ClpX binding serving to relieve these inhibitory interactions. Specific ClpP channel variants also prevent ClpXP translocation of certain amino-acid sequences, suggesting that the wild-type channel plays an important role in facilitating broad translocation specificity. In combination with previous studies, our results indicate that collaboration between ClpP and its partner ATPases opens a gate that functions to exclude larger substrates from isolated ClpP.     

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.     

ClpXP, an ATP-powered unfolding and protein-degradation machine

ClpXP is a AAA+ protease that uses the energy of ATP binding and hydrolysis to perform mechanical work during targeted protein degradation within cells. ClpXP consists of hexamers of a AAA+ ATPase (ClpX) and a tetradecameric peptidase (ClpP). Asymmetric ClpX hexamers bind unstructured peptide tags in protein substrates, unfold stable tertiary structure in the substrate, and then translocate the unfolded polypeptide chain into an internal proteolytic compartment in ClpP. Here, we review our present understanding of ClpXP structure and function, as revealed by two decades of biochemical and biophysical studies.