The membrane-cytoplasmic linker defines activity of FtsH proteases in Pseudomonas aeruginosa clone C.

Pandemic Pseudomonas aeruginosa clone C strains encode two inner-membrane associated ATP-dependent FtsH proteases. PaftsH1 is located on the core genome and supports cell growth and intrinsic antibiotic resistance, whereas PaftsH2, a xenolog acquired through horizontal gene transfer from a distantly related species, is unable to functionally replace PaftsH1. We show that purified PaFtsH2 degrades fewer substrates than PaFtsH1. Replacing the 31-amino acid-extended linker region of PaFtsH2 spanning from the C-terminal end of the transmembrane helix-2 to the first seven highly divergent residues of the cytosolic AAA+ ATPase module with the corresponding region of PaFtsH1 improves hybrid-enzyme substrate processing in vitro and enables PaFtsH2 to substitute for PaFtsH1 in vivo. Electron microscopy indicates that the identity of this linker sequence influences FtsH flexibility. We find membrane-cytoplasmic (MC) linker regions of PaFtsH1 characteristically glycine-rich compared to those from FtsH2. Consequently, introducing three glycines into the membrane-proximal end of PaFtsH2's MC linker is sufficient to elevate its activity in vitro and in vivo. Our findings establish that the efficiency of substrate processing by the two PaFtsH isoforms depends on MC linker identity and suggest that greater linker flexibility and/or length allows FtsH to degrade a wider spectrum of substrates. As PaFtsH2 homologs occur across bacterial phyla, we hypothesize that FtsH2 is a latent enzyme but may recognize specific substrates or is activated in specific contexts or biological niches. The identity of such linkers might thus play a more determinative role in the functionality of and physiological impact by FtsH proteases than previously thought.

A closed translocation channel in the substrate-free AAA+ ClpXP protease diminishes rogue degradation.

AAA+ proteases degrade intracellular proteins in a highly specific manner. E. coli ClpXP, for example, relies on a C-terminal ssrA tag or other terminal degron sequences to recognize proteins, which are then unfolded by ClpX and subsequently translocated through its axial channel and into the degradation chamber of ClpP for proteolysis. Prior cryo-EM structures reveal that the ssrA tag initially binds to a ClpX conformation in which the axial channel is closed by a pore-2 loop. Here, we show that substrate-free ClpXP has a nearly identical closed-channel conformation. We destabilize this closed-channel conformation by deleting residues from the ClpX pore-2 loop. Strikingly, open-channel ClpXP variants degrade non-native proteins lacking degrons faster than the parental enzymes in vitro but degraded GFP-ssrA more slowly. When expressed in E. coli, these open channel variants behave similarly to the wild-type enzyme in assays of filamentation and phage-Mu plating but resulted in reduced growth phenotypes at elevated temperatures or when cells were exposed to sub-lethal antibiotic concentrations. Thus, channel closure is an important determinant of ClpXP degradation specificity.

Lon degrades stable substrates slowly but with enhanced processivity, redefining the attributes of a successful AAA+ protease.

Lon is a widely distributed AAA+ (ATPases associated with diverse cellular activities) protease known for degrading poorly folded and damaged proteins and is often classified as a weak protein unfoldase. Here, using a Lon-degron pair from Mesoplasma florum (MfLon and MfssrA, respectively), we perform ensemble and single-molecule experiments to elucidate the molecular mechanisms underpinning MfLon function. Notably, we find that MfLon unfolds and degrades stably folded substrates and that translocation of these unfolded polypeptides occurs with a ∼6-amino-acid step size. Moreover, the time required to hydrolyze one ATP corresponds to the dwell time between steps, indicating that one step occurs per ATP-hydrolysis-fueled "power stroke." Comparison of MfLon to related AAA+ enzymes now provides strong evidence that HCLR-clade enzymes function using a shared power-stroke mechanism and, surprisingly, that MfLon is more processive than ClpXP and ClpAP. We propose that ample unfoldase strength and substantial processivity are features that contribute to the Lon family's evolutionary success.

The SspB adaptor drives structural changes in the AAA+ ClpXP protease during ssrA-tagged substrate delivery.

Energy-dependent protein degradation by the AAA+ ClpXP protease helps maintain protein homeostasis in bacteria and eukaryotic organelles of bacterial origin. In Escherichia coli and many other proteobacteria, the SspB adaptor assists ClpXP in degrading ssrA-tagged polypeptides produced as a consequence of tmRNA-mediated ribosome rescue. By tethering these incomplete ssrA-tagged proteins to ClpXP, SspB facilitates their efficient degradation at low substrate concentrations. How this process occurs structurally is unknown. Here, we present a cryo-EM structure of the SspB adaptor bound to a GFP-ssrA substrate and to ClpXP. This structure provides evidence for simultaneous contacts of SspB and ClpX with the ssrA tag within the tethering complex, allowing direct substrate handoff concomitant with the initiation of substrate translocation. Furthermore, our structure reveals that binding of the substrate·adaptor complex induces unexpected conformational changes within the spiral structure of the AAA+ ClpX hexamer and its interaction with the ClpP tetradecamer.

FtsH degrades kinetically stable dimers of cyclopropane fatty acid synthase via an internal degron.

Targeted protein degradation plays important roles in stress responses in all cells. In E. coli, the membrane-bound AAA+ FtsH protease degrades cytoplasmic and membrane proteins. Here, we demonstrate that FtsH degrades cyclopropane fatty acid (CFA) synthase, whose synthesis is induced upon nutrient deprivation and entry into stationary phase. We find that neither the disordered N-terminal residues nor the structured C-terminal residues of the kinetically stable CFA-synthase dimer are required for FtsH recognition and degradation. Experiments with fusion proteins support a model in which an internal degron mediates FtsH recognition as a prelude to unfolding and proteolysis. These findings elucidate the terminal step in the life cycle of CFA synthase and provide new insight into FtsH function.

AAA+ protease-adaptor structures reveal altered conformations and ring specialization.

ClpAP, a two-ring AAA+ protease, degrades N-end-rule proteins bound by the ClpS adaptor. Here we present high-resolution cryo-EM structures of Escherichia coli ClpAPS complexes, showing how ClpA pore loops interact with the ClpS N-terminal extension (NTE), which is normally intrinsically disordered. In two classes, the NTE is bound by a spiral of pore-1 and pore-2 loops in a manner similar to substrate-polypeptide binding by many AAA+ unfoldases. Kinetic studies reveal that pore-2 loops of the ClpA D1 ring catalyze the protein remodeling required for substrate delivery by ClpS. In a third class, D2 pore-1 loops are rotated, tucked away from the channel and do not bind the NTE, demonstrating asymmetry in engagement by the D1 and D2 rings. These studies show additional structures and functions for key AAA+ elements. Pore-loop tucking may be used broadly by AAA+ unfoldases, for example, during enzyme pausing/unloading.

Structure and function of ClpXP, a AAA+ proteolytic machine powered by probabilistic ATP hydrolysis.

ClpXP is an archetypical AAA+ protease, consisting of ClpX and ClpP. ClpX is an ATP-dependent protein unfoldase and polypeptide translocase, whereas ClpP is a self-compartmentalized peptidase. ClpXP is currently the only AAA+ protease for which high-resolution structures exist, the molecular basis of recognition for a protein substrate is understood, extensive biochemical and genetic analysis have been performed, and single-molecule optical trapping has allowed direct visualization of the kinetics of substrate unfolding and translocation. In this review, we discuss our current understanding of ClpXP structure and function, evaluate competing sequential and probabilistic mechanisms of ATP hydrolysis, and highlight open questions for future exploration.

FtsH degrades dihydrofolate reductase by recognizing a partially folded species.

AAA+ proteolytic machines play essential roles in maintaining and rebalancing the cellular proteome in response to stress, developmental cues, and environmental changes. Of the five AAA+ proteases in Escherichia coli, FtsH is unique in its attachment to the inner membrane and its function in degrading both membrane and cytosolic proteins. E. coli dihydrofolate reductase (DHFR) is a stable and biophysically well-characterized protein, which a previous study found resisted FtsH degradation despite the presence of an ssrA degron. By contrast, we find that FtsH degrades DHFR fused to a long peptide linker and ssrA tag. Surprisingly, we also find that FtsH degrades DHFR with shorter linkers and ssrA tag, and without any linker or tag. Thus, FtsH must be able to recognize a sequence element or elements within DHFR. We find that FtsH degradation of DHFR is noncanonical in the sense that it does not rely upon recognition of an unstructured polypeptide at or near the N-terminus or C-terminus of the substrate. Results using peptide-array experiments, mutant DHFR proteins, and fusion proteins suggest that FtsH recognizes an internal sequence in a species of DHFR that is partially unfolded. Overall, our findings provide insight into substrate recognition by FtsH and indicate that its degradation capacity is broader than previously reported.

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.

Heat activates the AAA+ HslUV protease by melting an axial autoinhibitory plug.

At low temperatures, protein degradation by the AAA+ HslUV protease is very slow. New crystal structures reveal that residues in the intermediate domain of the HslU6 unfoldase can plug its axial channel, blocking productive substrate binding and subsequent unfolding, translocation, and degradation by the HslV12 peptidase. Biochemical experiments with wild-type and mutant enzymes support a model in which heat-induced melting of this autoinhibitory plug activates HslUV proteolysis.

ClpP1P2 peptidase activity promotes biofilm formation in Pseudomonas aeruginosa.

Caseinolytic proteases (Clp) are central to bacterial proteolysis and control cellular physiology and stress responses. They are composed of a double-ring compartmentalized peptidase (ClpP) and a AAA+ unfoldase (ClpX or ClpA/ClpC). Unlike many bacteria, the opportunistic pathogen Pseudomonas aeruginosa contains two ClpP homologs: ClpP1 and ClpP2. The specific functions of these homologs, however, are largely elusive. Here, we report that the active form of PaClpP2 is a part of a heteromeric PaClpP17 P27 tetradecamer that is required for proper biofilm development. PaClpP114 and PaClpP17 P27 complexes exhibit distinct peptide cleavage specificities and interact differentially with P. aeruginosa ClpX and ClpA. Crystal structures reveal that PaClpP2 has non-canonical features in its N- and C-terminal regions that explain its poor interaction with unfoldases. However, experiments in vivo indicate that the PaClpP2 peptidase active site uniquely contributes to biofilm development. These data strongly suggest that the specificity of different classes of ClpP peptidase subunits contributes to the biological outcome of proteolysis. This specialized role of PaClpP2 highlights it as an attractive target for developing antimicrobial agents that interfere specifically with late-stage P. aeruginosa development.

ClpAP proteolysis does not require rotation of the ClpA unfoldase relative to ClpP.

AAA+ proteases perform regulated protein degradation in all kingdoms of life and consist of a hexameric AAA+ unfoldase/translocase in complex with a self-compartmentalized peptidase. Based on asymmetric features of cryo-EM structures and a sequential hand-over-hand model of substrate translocation, recent publications have proposed that the AAA+ unfoldases ClpA and ClpX rotate with respect to their partner peptidase ClpP to allow function. Here, we test this model by covalently crosslinking ClpA to ClpP to prevent rotation. We find that crosslinked ClpAP complexes unfold, translocate, and degrade protein substrates in vitro, albeit modestly slower than uncrosslinked enzyme controls. Rotation of ClpA with respect to ClpP is therefore not required for ClpAP protease activity, although some flexibility in how the AAA+ ring docks with ClpP may be necessary for optimal function.

Modular and coordinated activity of AAA+ active sites in the double-ring ClpA unfoldase of the ClpAP protease.

ClpA is a hexameric double-ring AAA+ unfoldase/translocase that functions with the ClpP peptidase to degrade proteins that are damaged or unneeded. How the 12 ATPase active sites of ClpA, 6 in the D1 ring and 6 in the D2 ring, work together to fuel ATP-dependent degradation is not understood. We use site-specific cross-linking to engineer ClpA hexamers with alternating ATPase-active and ATPase-inactive modules in the D1 ring, the D2 ring, or both rings to determine if these active sites function together. Our results demonstrate that D2 modules coordinate with D1 modules and ClpP during mechanical work. However, there is no requirement for adjacent modules in either ring to be active for efficient enzyme function. Notably, ClpAP variants with just three alternating active D2 modules are robust protein translocases and function with double the energetic efficiency of ClpAP variants with completely active D2 rings. Although D2 is the more powerful motor, three or six active D1 modules are important for high enzyme processivity, which depends on D1 and D2 acting coordinately. These results challenge sequential models of ATP hydrolysis and coupled mechanical work by ClpAP and provide an engineering strategy that will be useful in testing other aspects of ClpAP mechanism.

Multistep substrate binding and engagement by the AAA+ ClpXP protease.

Escherichia coli ClpXP is one of the most thoroughly studied AAA+ proteases, but relatively little is known about the reactions that allow it to bind and then engage specific protein substrates before the adenosine triphosphate (ATP)-fueled mechanical unfolding and translocation steps that lead to processive degradation. Here, we employ a fluorescence-quenching assay to study the binding of ssrA-tagged substrates to ClpXP. Polyphasic stopped-flow association and dissociation kinetics support the existence of at least three distinct substrate-bound complexes. These kinetic data fit well to a model in which ClpXP and substrate form an initial recognition complex followed by an intermediate complex and then, an engaged complex that is competent for substrate unfolding. The initial association and dissociation steps do not require ATP hydrolysis, but subsequent forward and reverse kinetic steps are accelerated by faster ATP hydrolysis. Our results, together with recent cryo-EM structures of ClpXP bound to substrates, support a model in which the ssrA degron initially binds in the top portion of the axial channel of the ClpX hexamer and then is translocated deeper into the channel in steps that eventually pull the native portion of the substrate against the channel opening. Reversible initial substrate binding allows ClpXP to check potential substrates for degrons, potentially increasing specificity. Subsequent substrate engagement steps allow ClpXP to grip a wide variety of sequences to ensure efficient unfolding and translocation of almost any native substrate.

Structural basis of ClpXP recognition and unfolding of ssrA-tagged substrates.

When ribosomes fail to complete normal translation, all cells have mechanisms to ensure degradation of the resulting partial proteins to safeguard proteome integrity. In Escherichia coli and other eubacteria, the tmRNA system rescues stalled ribosomes and adds an ssrA tag or degron to the C-terminus of the incomplete protein, which directs degradation by the AAA+ ClpXP protease. Here, we present cryo-EM structures of ClpXP bound to the ssrA degron. C-terminal residues of the ssrA degron initially bind in the top of an otherwise closed ClpX axial channel and subsequently move deeper into an open channel. For short-degron protein substrates, we show that unfolding can occur directly from the initial closed-channel complex. For longer degron substrates, our studies illuminate how ClpXP transitions from specific recognition into a nonspecific unfolding and translocation machine. Many AAA+ proteases and protein-remodeling motors are likely to employ similar multistep recognition and engagement strategies.

The Intrinsically Disordered N-terminal Extension of the ClpS Adaptor Reprograms Its Partner AAA+ ClpAP Protease.

Adaptor proteins modulate substrate selection by AAA+ proteases. The ClpS adaptor delivers N-degron substrates to ClpAP but inhibits degradation of substrates bearing ssrA tags or other related degrons. How ClpS inhibits degradation of such substrates is poorly understood. Here, we demonstrate that ClpS impedes recognition of ssrA-tagged substrates by a non-competitive mechanism and also slows subsequent unfolding/translocation of these substrates as well as of N-degron substrates. This suppression of mechanical activity is largely a consequence of the ability of ClpS to repress ATP hydrolysis by ClpA, but several lines of evidence show that ClpS's inhibition of substrate binding and its ATPase repression are separable activities. Using ClpS mutants and ClpS-ClpA chimeras, we establish that engagement of the intrinsically disordered N-terminal extension of ClpS by ClpA is both necessary and sufficient to inhibit multiple steps of ClpAP-catalyzed degradation. These observations reveal how an adaptor can simultaneously modulate the catalytic activity of a AAA+ enzyme, efficiently promote recognition of some substrates, suppress recognition of other substrates, and thereby affect degradation of its menu of substrates in a specific manner. We propose that similar mechanisms are likely to be used by other adaptors to regulate substrate choice and the catalytic activity of molecular machines.

Regulation of Antimycin Biosynthesis Is Controlled by the ClpXP Protease.

The survival of any microbe relies on its ability to respond to environmental change. Use of extracytoplasmic function (ECF) RNA polymerase sigma (σ) factors is a major strategy enabling dynamic responses to extracellular signals. Streptomyces species harbor a large number of ECF σ factors, nearly all of which are uncharacterized, but those that have been characterized generally regulate genes required for morphological differentiation and/or response to environmental stress, except for σAntA, which regulates starter-unit biosynthesis in the production of antimycin, an anticancer compound. Unlike a canonical ECF σ factor, whose activity is regulated by a cognate anti-σ factor, σAntA is an orphan, raising intriguing questions about how its activity may be controlled. Here, we reconstituted in vitro ClpXP proteolysis of σAntA but not of a variant lacking a C-terminal di-alanine motif. Furthermore, we show that the abundance of σAntAin vivo was enhanced by removal of the ClpXP recognition sequence and that levels of the protein rose when cellular ClpXP protease activity was abolished. These data establish direct proteolysis as an alternative and, thus far, unique control strategy for an ECF RNA polymerase σ factor and expands the paradigmatic understanding of microbial signal transduction regulation.IMPORTANCE Natural products produced by Streptomyces species underpin many industrially and medically important compounds. However, the majority of the ∼30 biosynthetic pathways harbored by an average species are not expressed in the laboratory. This unrevealed biochemical diversity is believed to comprise an untapped resource for natural product drug discovery. Major roadblocks preventing the exploitation of unexpressed biosynthetic pathways are a lack of insight into their regulation and limited technology for activating their expression. Our findings reveal that the abundance of σAntA, which is the cluster-situated regulator of antimycin biosynthesis, is controlled by the ClpXP protease. These data link proteolysis to the regulation of natural product biosynthesis for the first time to our knowledge, and we anticipate that this will emerge as a major strategy by which actinobacteria regulate production of their natural products. Further study of this process will advance understanding of how expression of secondary metabolism is controlled and will aid pursuit of activating unexpressed biosynthetic pathways.

Mitochondrial ClpX activates an essential biosynthetic enzyme through partial unfolding.

Mitochondria control the activity, quality, and lifetime of their proteins with an autonomous system of chaperones, but the signals that direct substrate-chaperone interactions and outcomes are poorly understood. We previously discovered that the mitochondrial AAA+ protein unfoldase ClpX (mtClpX) activates the initiating enzyme for heme biosynthesis, 5-aminolevulinic acid synthase (ALAS), by promoting cofactor incorporation. Here, we ask how mtClpX accomplishes this activation. Using S. cerevisiae proteins, we identified sequence and structural features within ALAS that position mtClpX and provide it with a grip for acting on ALAS. Observation of ALAS undergoing remodeling by mtClpX revealed that unfolding is limited to a region extending from the mtClpX-binding site to the active site. Unfolding along this path is required for mtClpX to gate cofactor binding to ALAS. This targeted unfolding contrasts with the global unfolding canonically executed by ClpX homologs and provides insight into how substrate-chaperone interactions direct the outcome of remodeling.

Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate.

ClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies of the E. coli enzyme that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel, as observed for other AAA+ proteases and protein-remodeling machines. Strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been inferred from these structural features for other AAA+ unfoldases, but biochemical and single-molecule biophysical studies indicate that ClpXP operates by a probabilistic mechanism in which five to eight residues are translocated for each ATP hydrolyzed. We propose structure-based models that could account for the functional results.

The Non-dominant AAA+ Ring in the ClpAP Protease Functions as an Anti-stalling Motor to Accelerate Protein Unfolding and Translocation.

ATP-powered unfoldases containing D1 and D2 AAA+ rings play important roles in protein homeostasis, but uncertainty about the function of each ring remains. Here we use single-molecule optical tweezers to assay mechanical unfolding and translocation by a variant of the ClpAP protease containing an ATPase-inactive D1 ring. This variant displays substantial mechanical defects in both unfolding and translocation of protein substrates. Notably, when D1 is hydrolytically inactive, ClpAP often stalls for times as long as minutes, and the substrate can back-slip through the enzyme when ATP concentrations are low. The inactive D1 variant also has more difficulty traveling in the N-to-C direction on a polypeptide track than it does moving in a C-to-N direction. These results indicate that D1 normally functions as an auxiliary/regulatory motor to promote uninterrupted enzyme advancement that is fueled largely by the D2 ring.