Cryo-EM structures of human ClpXP reveal mechanisms of assembly and proteolytic activation

Domain: neuroscience
Published: 2026-01-27
Editorial outcome: major_revision

Abstract
The human ClpXP complex (hClpXP) orchestrates mitochondrial protein quality control through targeted degradation of misfolded and unnecessary proteins. While bacterial ClpXP systems are well characterized, the assembly and regulation of human ClpXP remain poorly understood. In this study, we elucidate the complete assembly pathway of hClpXP through high-resolution cryo-electron microscopy (cryo-EM) structures. Our findings confirm that hClpP exists as a single-ring heptamer in isolation and reveal a previously undocumented initial assembly complex in which hexameric hClpX first engages with heptameric hClpP. We further demonstrate how this interaction drives substantial conformational rearrangements that facilitate the formation of tetradecameric hClpP within the fully assembled complex. Notably, we characterize a unique eukaryotic sequence in hClpX, termed the E-loop, which plays a critical role in stabilizing hexamer assembly and maintaining ATPase activity. Additionally, we show that peptide binding at the hClpP active site triggers further structural changes essential for achieving full proteolytic competence. Together, these structures provide unprecedented mechanistic insights into the stepwise assembly and activation of hClpXP, significantly advancing our understanding of this essential mitochondrial protein degradation machinery.

Introduction
Mitochondria are busy signaling hubs where a multitude of cellular processes converge, including oxidative phosphorylation, calcium homeostasis, redox signaling, and apoptosis1–3. Maintaining internal protein homeostasis (proteostasis) involves a sophisticated and tightly controlled protein quality control network that ensures proper import, folding, and turnover of proteins4. In humans, disruptions in this network are associated with aging and can lead to a wide range of diseases, including cancer, neurological disorders, diabetes, and skin disorders5. The ClpXP complex is one of the key ATP-dependent unfoldase-proteases involved in this protein quality control network, specifically targeting and degrading misfolded, damaged, or surplus proteins in the matrix of mitochondria. While all other mitochondrial proteases are encoded by a single gene, ClpXP is an assembly of two distinct proteins: ClpX, an ATPase associated with diverse cellular activities (AAA+) with unfoldase activity, and ClpP, a conserved serine protease. Together, these components work in concert to effectively degrade damaged proteins, as well as regulating essential cellular functions, including heme biosynthesis6,7, mitochondrial DNA nucleoid distribution8, and reactive oxygen species (ROS) production9.

Despite extensive biochemical and structural characterization of unicellular ClpXP systems10–14, many of the molecular details underlying human ClpXP (hClpXP) assembly and proteolytic activation are uncharacterized. ClpXP systems are broadly conserved across all forms of life, but the specific requirements of eukaryotic proteostasis have driven ClpX to evolve distinct regulatory components. For example, hClpX features a unique eukaryotic-specific insertion within its ATPase domain, previously termed the E-domain15 (Supplementary Figs. 1a, 2a). The function of this insertion was unknown, but its positioning at the distal end of hClpX suggests a potential role in substrate recognition16 (Supplementary Fig. 1d). Additionally, hClpP possesses an additional 28 amino acids at its C-terminus, termed the C-terminal extension (Supplementary Fig. 2b). Deleting this extension promotes tighter interaction between hClpP and hClpX17, but the precise underlying mechanism remains unclear.

The hClpP protease is also evolutionarily divergent. Bacterial ClpP assembles as a tetradecamer composed of two stacked heptameric rings that enclose 14 proteolytic active sites within the chamber18, but this is not necessarily the case for hClpP. Some studies suggest that hClpP exists as a tetradecamer19–21, whereas others indicate that ClpP assembles as a stable heptamer and only forms a tetradecamer upon binding with hClpX17,22. Studies employing small molecule activators of hClpP have provided some insights into its mechanism of activation21,23,24, but in the absence of a direct structural comparison between apo hClpP and hClpX-bound hClpP, the precise mechanism of hClpP activation remains elusive.

In this study, we use cryo-electron microscopy (cryo-EM) to investigate the assembly pathway and activation mechanisms of the hClpXP complex, revealing previously unobserved assemblies that distinguish hClpXP from its bacterial homologs. Our studies indicate that unbound hClpP natively adopts a single-ring asymmetric heptamer, which interacts with hClpX to form an initial assembly complex. Interaction with substrate drives formation of the fully assembled hClpXP complex, which we show transitions from a proteolytically inactive conformation to a proteolytically competent state when the peptide interacts with the protease site. Our hClpXP structures also reveal the architecture of the E-domain (hereafter referred to as the E-loop), which we show to be involved in nucleotide sensing and hClpX hexamer stabilization. Together, these findings highlight the distinct allosteric mechanisms that have evolved for hClpXP to meet the complex demands of the mitochondrial environment.

Results
Purification of the hClpXP sample for cryo-EM structural studies
We first aimed to compare the structure of the hClpXP complex to the numerous prior structures of the unicellular homologs to identify evolutionarily conserved or divergent features. However, generating a stable hClpXP complex for cryo-EM studies required substantial optimization. To structurally stabilize the hClpX construct for high-resolution structure determination, we removed the N-terminal zinc-binding domain16,25 and the C-terminal extension, both predicted to be disordered by AlphaFold (version 2) (Supplementary Fig. 1c,d). We also introduced a Walker B motif mutation (E359A), previously shown in bacterial ClpX and other AAA+ translocases, to slow ATP hydrolysis and promote hexamer stability11–13. This optimized construct is hereafter referred to as hClpXI146-ΔCTE-E359A.

While the zinc-binding domain has been characterized in bacterial ClpX and shown not to affect the structural conformation of the assembly14,26, the functional relevance of the C-terminal extension in hClpX remains unclear. To investigate its potential role in ATPase activity, we performed an NADH-coupled ATPase assay comparing hClpXI146 variants with or without the C-terminal extension. Our data indicate that the C-terminal extension does not influence ATP binding affinity but decreases ATPase activity and the degradation of unfolded substrates in the presence of hClpP (Supplementary Fig. 3a,b). While removal of the hClpX C-terminal extension reduces its enzymatic activity and helps stabilize the oligomer for cryo-EM characterization, the underlying mechanisms by which this modification alters activity remain to be elucidated through future studies.

Although our construct optimization improved the stability and yield of hClpX, we did not observe formation of stable co-assembly with wild-type hClpP (Supplementary Fig. 4a). Thus, we next sought to enhance complex formation through hClpP construct optimization. Previous studies showed that removing the C-terminal extension (CTE)17,19 and introducing a proteolytically inactivating S153A mutation13 stabilize ClpXP complex assembly. Accordingly, we generated an hClpPΔCTE-S153A construct, which exhibited improved binding to hClpX, as observed by negative stain EM (Supplementary Figs. 4a,b, 5c). Titration experiments comparing wild-type hClpP and hClpPΔCTE suggested that both proteins bind and activate hClpX similarly, but hClpPΔCTE exhibits a modestly higher substrate turnover rate, indicating that the C-terminal extension negatively regulates hClpP activity (Supplementary Fig. 3c). To further stabilize the complex for high-resolution single-particle cryo-EM analysis, we applied disuccinimidyl suberate (DSS) crosslinking27, which substantially increased the number of intact hClpXP particles (Supplementary Fig. 4c). Notably, DSS does not crosslink hClpP alone (Supplementary Fig. 4e). For simplicity, we refer to this optimized, crosslinked complex (hClpXI146-ΔCTE-E359A/hClpPΔCTE-S153A/DSS-X-linked) as hClpXPX-linked hereafter.

Cryo-EM structure of hClpXP reveals a conserved substrate translocation mechanism by hClpX
We determined the structure of the hClpXPX-linked complex at an overall resolution of ~3 Å (Fig. 1a, Supplementary Fig. 6 and Supplementary Table 1). The reconstruction is generally reminiscent of previously determined bacterial ClpXP structures, including a co-purified peptide bound within the central pore of the hClpX unfoldase, a common feature of recombinantly expressed AAA+ proteins containing a Walker B mutation12,28,29. The hexameric assembly of hClpX adopts a shallow spiral conformation consistent with bacterial ClpX and other AAA+ unfoldases11,12,14, and we labeled the subunits of hClpX in alphabetical order (A, B, C, D, E, and F) with chain A at the uppermost position of the spiral and continuing downwards, and chain F identified as the displaced “seam” subunit disengaged from the peptide substrate (Fig. 1b). Also consistent with other AAA+ unfoldases was density corresponding to an ATP nucleotide with a coordinated magnesium cofactor observed in the binding pockets of chains A through D, where the ATP β- and γ-phosphates are stabilized by interactions with K299 (Walker A motif) and R562 (sensor-II) of the same subunit, as well as R499 (arginine finger) from the adjacent protomer (Supplementary Fig. 7). The nucleotide density in lowest subunit of the spiral, chain E, was representative of ADP and lacked observable magnesium density (Supplementary Fig. 7b). Further, the arginine finger from the neighboring F subunit is shifted away to produce a more open nucleotide-binding pocket. No nucleotide density was resolved in the seam subunit F.

As previously observed in other AAA+ proteins, the nucleotide state of each protomer correlates with the extent of the subunit’s interaction with the incoming substrate within the central ATPase channel30–32. The pore-1 loop of each nucleotide-bound hClpX subunit in the right-handed spiral engages the translocating peptide via a conserved GYVG sequence, so that the aromatic-hydrophobic motif intercalates into the substrate backbone (Fig. 1d). This organization maintains grip on the incoming polypeptide to facilitate translocation towards the peptidase chamber using a putative hand-over-hand mechanism11,12,31–33.

ClpX attaches to ClpP via loops containing a conserved Ile/Leu-Gly-Phe (IGF or LGF) motif, which bind to the hydrophobic pockets formed at the inter-protomer ClpP interface11. Our hClpXP structure shows how F441 of the hClpX LGF loop forms conserved hydrophobic interactions with residues Y118, W146, Y138, and L104 in the hydrophobic pocket of hClpP (Supplementary Fig. 8). Prior cryo-EM studies of bacterial ClpXP systems have shown that one of the hydrophobic binding pockets in ClpP is empty due to the six-to-seven protomer mismatch between the unfoldase and protease. However, this empty pocket is sometimes situated between the clefts occupied by the IGF loops of ClpX subunits E and F11,12,14 while in others it is located between subunits D and E34,35. It was proposed that the ATP hydrolysis-driven motions of the ATPase subunits coordinate repositioning of the ClpX I/LGF loops as well as the stability of the N-terminal β-hairpins comprising the ClpP axial channel to facilitate substrate translocation11. However, recent studies show that the crosslinked ClpAP complex remain capable of degrading unstructured substrates, suggesting that a rotational motion may not be required for proteolysis36. Our crosslinked hClpXP abolished FITC-casein degradation activity, which may result from impaired substrate unfolding and translocation caused by the promiscuity of the DSS crosslinker (Supplementary Fig. 9a). The pore-2 loops of ClpX are also thought to coordinate substrate translocation through interactions with the axial ClpP β-hairpins that vary based on ClpX nucleotide state11–13,37. In our hClpXPX-linked reconstruction, we observe an empty pocket between hClpX chains D and E (Fig. 1c), and although our pore-2 loops are not resolved, we observe that the pore-2-adjacent residue D374 from hClpX subunit E interacts with R68 at the top of a hClpP β-hairpin (Supplementary Fig. 10). This observation is similar to the Neisseria meningitides ClpXP structure, where only a single ClpX protomer (subunit E) interacts with the arginine residue from ClpP β-hairpin at the same position11, suggesting a conserved ClpXP communication and substrate translocation mechanism.

E-domain of hClpX senses nucleotide states and promotes hexamer formation
A notable distinguishing feature of our hClpXPX-linked structure is the presence of the E-loop, a eukaryotic-specific insertion of unknown function within the AAA+ module (Fig. 2a). Within the hClpX hexamer, the E-loop extends from the large AAA+ domain of one subunit over the adjacent nucleotide binding pocket and interacts with the neighboring subunit at the interface of the large and small AAA+ domains. Despite the numerous interactions, the E-loop is generally flexible and is only partially resolved. The interface between the large and small AAA+ domains is distinct for each subunit within translocating AAA+ unfoldases due to the staircase organization of the hexamer30–32, thereby impacting E-loop interactions. Accordingly, the resolvability of the E-loop density varies among different subunits, indicating that the register of the subunit within the hexamer impacts the E-loop stability (Supplementary Fig. 11b). The least-resolved E-loop is that of the seam subunit, chain F, which extends over the ADP-bound pocket of chain E. Proceeding from the top of the ATPase spiral to the bottom, the E-loop arching across each of the preceding nucleotide binding pockets becomes increasingly ordered, with the E-loop extending over the lower-most ATP pocket being the best-resolved of the loops. Given the location of the E-loop, and that E285 within the loop is within hydrogen bonding distance of both the nucleotide ribose and R565 in the binding pocket (Fig. 2a and Supplementary Fig. 11a,b), we surmised that the E-loop may play a role in the ATPase mechanochemical cycle.

To test this hypothesis, we removed the E-loop (residues 230-304) from hClpX (hClpXdE) and evaluated its impact on ATPase activity and substrate degradation. Deletion of the E-loop decreased ATP affinity by 4-fold and decreased ATPase activity by more than half (Fig. 2b, Supplementary Fig. 3a,b). Removing the E-loop also impaired the ability of hClpXP to degrade substrates, as indicated by a 11-fold reduction in FITC-casein degradation (Fig. 2c). Negative stain EM analysis of this truncation mutant revealed an inability of the hClpXdE to form hexamers, instead assembling into higher-order oligomers, including heptameric, octameric, and nonameric assemblies in the absence of exogenous ATP (Supplementary Fig. 11c,d). Although the addition of ATP and MgCl2 facilitated the formation of some loosely organized hexamers, we also observed heptamers and unclosed ring assemblies. Given that the ClpXdE construct shows impaired substrate degradation (Supplementary Fig. 3b), these data collectively suggest that the E-loop is critical for organizing hClpX into a hexamer that is competent to engage and translocate substrate. Notably, site-specifically mutating the nucleotide-interacting residue E285 within the E-loop to alanine was as deleterious to hClpXP enzymatic activities as removal of the E-loop (Fig. 2b,c), but the mutation did not negatively affect hClpX hexamer formation (Supplementary Fig. 11e). Given our observed four-fold decrease in ATP binding affinity of the E285A mutation (equivalent to the hClpXdE construct, Supplementary Fig. 3a,b) we posit that the E285 residue plays a key role in hClpX ATPase and substrate translocation activities by sensing and communicating changes in nucleotide states between neighboring subunits.

Similar to unicellular ClpX systems, ATP binding is important for hClpX to form hexamers that are competent for substrate translocation16 (Supplementary Fig. 11c). However, our data indicate that the E-loop serves as a critical structural feature required for proper hexameric assembly of hClpX and effective ATP-driven substrate translocation. Given that the E-loop is specific to eukaryotes, these findings suggest an evolutionary adaptation of hClpX to incorporate a more complex set of structural requirements for stabilizing hexamer formation and sensing nucleotide states during substrate translocation in mitochondria.

hClpP assembles as an asymmetric heptamer whose C-terminal extension negatively regulates its activity
hClpXP complex, we next aimed to investigate the mechanism of hClpXP assembly. It is well-established that bacterial ClpP proteins form stable tetradecamers composed of two stacked heptamers, but there are conflicting results regarding whether hClpP assembles as a heptamer or tetradecamer in the absence of hClpX in solution17,20–22. When we expressed and purified our wild-type hClpP construct, we observed that it eluted from size exclusion chromatography at a mass corresponding to a heptameric assembly (Supplementary Fig. 5a). We further confirmed that hClpP predominantly exists as a heptamer in solution using mass photometry and negative-stain EM (Supplementary Fig. 5b,c) and next aimed to determine a high-resolution cryo-EM structure. We observed that the apo hClpP heptamer exhibits a strong preferred axial orientation when vitrified on cryo-EM grids (Supplementary Fig. 12a,b), which was alleviated by the addition of fluorinated fos-choline-8 detergent (Supplementary Fig. 12c). Although the majority of imaged hClpP oligomers were heptameric according to 2D classification, there was a minor population of tetradecameric hClpP, possibly induced by the high concentration of hClpP applied to the cryo-EM grid (15 mg/ml). Our 2D averages also showed that the tetradecamers were not well-resolved (circled in red in Supplementary Fig. 12d), indicative of the flexibility of transient inter-heptamer interactions. These data indicate that the hClpP heptamers may not readily form stable tetradecamers in cellular conditions and that docking of the hClpX LGF loop in the ClpP hydrophobic pocket plays a role in promoting assembly of the hClpP tetradecamer.

Despite challenges from reduced apparent particle concentration and ice thickness, further processing of our fluorinated fos-choline-8 dataset allowed us to obtain a heptameric hClpP reconstruction with a reported overall resolution of ~3.5 Å (Supplementary Fig. 12 and Supplementary Table 1), which was sufficient to generate an atomic model (Fig. 3a, b). Surprisingly, we found that the hClpP heptamer is not symmetric as expected, but rather assembles into an asymmetric oligomer in the absence of hClpX (Supplementary Fig. 13a,c). A salient feature of the asymmetric heptamer is the ordering of three of the N-terminal loops into β-hairpins, forming a partial wall around the axial hClpP pore, while these loops from the other four subunits are disordered (Fig. 3b, Supplementary Fig. 14). We surmise that this distinct asymmetric organization of the N-terminal loops forming a partial wall at the ClpP pore impacts hClpX recruitment, although further biophysical studies will be required to elucidate the possible regulatory mechanism of this unexpected structural feature.

hClpP contains an unstructured 28-residue C-terminal extension (Supplementary Fig. 2b) that is unresolved in our hClpP reconstructions. We wondered how the removal of this disordered region from ClpP increases affinity for ClpX and increases substrate degradation17 (Supplementary Fig. 3c, 4b, 15c). Prior studies of the C-terminal extension in ClpP homologs from Mycobacterium smegmatis38 and Listeria monocyotenes13 suggest that this segment may be positioned to block the interaction between the ClpX loops and their respective hydrophobic binding pockets in ClpP. In our apo hClpP structure, we noted that the two proline residues preceding the C-terminal extension are well-resolved, stably positioned atop the LGF loop binding pocket (Supplementary Fig. 15a). The location of these proline residues confirms that the C-terminal extensions are located in the vicinity of the hClpP LGF loop binding pockets, where they could potentially shield the pockets from hClpX interaction. Further, these prolines residues are positioned so that they would sterically hinder the binding of LGF loops (Supplementary Fig. 15a). We noted that these residues are shifted away from the LGF binding pocket in our hClpXP structure to accommodate the interactions necessary to form the heterocomplex (Supplementary Fig. 15b). These structural and biochemical observations suggest that the C-terminal extension negatively regulates the enzymatic activity of hClpXP.

hClpP switches from an asymmetric heptamer to asymmetric tetradecamer upon interaction with hClpX
In addition to displacing the C-terminal extension of hClpP, hClpX binding is associated with a large-scale reorganization of the hClpP heptamer. Superimposing our structures of the apo hClpP heptamer and one heptameric ring of the hClpX-bound hClpP reveals several notable conformational changes (Fig. 3c). Upon hClpX binding, the four N-terminal loops that were disordered in our asymmetric heptameric hClpP structure adopt the canonical β-hairpin fold, concurrent with expansion of the axial pore to allow unfolded substrates to access the proteolytic chamber.

Notably, we also observe a ~15° axial rotation of the subunits away from the hClpX hexamer as the hClpP heptamer transitions from an asymmetric heptamer to asymmetric assembly (Fig. 3c). The rotation of the hClpP subunits upon hClpX binding results in an inward movement of the handle regions to facilitate critical interactions at the hClpP-hClpP interface, promoting a stable tetradecamer conformation (Supplementary Fig. 16a). In the hClpX-bound hClpP tetradecameric structure, the conserved oligomerization sensors E225 and R226 from one ClpP heptamer are repositioned in proximity to form salt bridges with R226 and E225 from the opposing ClpP heptamer (Supplementary Fig. 16a,b). When we introduced the E225A and R226A mutations to hClpP, these mutations destabilize, but do not entirely abolish, hClpP tetradecamer formation in the presence of hClpX, as assessed by negative stain EM (Supplementary Fig. 4d). However, these mutations completely abolished hClpXP’s ability to degrade FITC-casein substrates, indicating that the oligomeric sensors are essential for the enzymatic activity of hClpP (Supplementary Fig. 9b). We also observe several other residues in the handle region, including E196, K202, and Y206, that interact with residues from the opposing subunit to maintain the tetradacameric state of hClpP (Supplementary Fig. 16b). Additionally, the catalytic residue D227 contributes to stabilizing the tetradecamer by forming a salt bridge with Q194. These interactions were also recently observed in a tetradecameric cryoEM structure of hClpP determined while this manuscript was under preparation39.

hClpXP forms a substrate-free initial assembly complex
To confirm that our substrate-bound ClpXP structure was not impacted by chemical crosslinking and construct engineering, we set out to determine a structure of the uncrosslinked hClpXP complex using the wild-type proteins (Supplementary Fig. 1b, 17, Supplementary Table 1). Although our negative stain images did not show assembled complexes, our observed activity of the purified components indicated that complex formation was nonetheless occurring. We posited that the low (nanomolar) concentrations of protein used for our negative staining may be promoting complex dissociation. Thus, we incubated hClpX and hClpP together at high concentration of 3 mg/ml in the presence of ATPγS and froze grids of this sample for cryoEM imaging. Although hClpX and hClpP were mostly dissociated, a large dataset of cryoEM micrographs yielded sufficient hClpXP complexes to obtain moderate-resolution maps of the hClpXP complex, with hClpX and hClpP locally refined to resolutions of ~4.5 Å and ~3.5 Å, respectively (Supplementary Fig. 17, Supplementary Table 1). We generated a model of uncrosslinked hClpXP by rigid-body fitting the crosslinked hClpX and hClpP models into the composite map of uncrosslinked hClpX and hClpP, followed by Phenix real-space refinement (Supplementary Fig. 18a). An overlay of the uncrosslinked and crosslinked models revealed an RMSD of 0.5 Å, indicating that the two structures are nearly identical within the limits of the map resolution. While the hClpP tetradecamer shares a consistent conformation, we observe subtle shifts in hClpX subunits (Supplementary Fig. 18b). Overall, this analysis suggests that the cross-linked hClpXP structures closely represent the uncross-linked full-length hClpXP structure.

During our analysis of this dataset, we were surprised to identify a population of particles corresponding to a single hClpX hexamer bound to a single hClpP heptamer ring (Fig. 4a, Supplementary Fig. 17). This organization is distinct from all previously characterized ClpXP systems, where ClpX exclusively associates with tetradecameric ClpP. This organization was absent in our crosslinked hClpXP dataset, suggesting that it represents an intermediate in the hClpXP-substrate assembly pathway. Notably, the ATPase domains of hClpX in this stacked hClpX-hClpP complex adopt a more open configuration compared to substrate-bound structure, and the central pore is devoid of substrate (Fig. 4d). Further, the arrangement of individual hClpX subunits is relatively flat, a notable departure from the conventional staircase conformation used by hClpX and other AAA+ ATPases to facilitate substrate peptide unfolding and translocation (Fig. 4d). The heptameric hClpP in this identified hClpXP complex exhibits slight asymmetry, representing an intermediate state between asymmetric hClpP heptamer and the symmetric hClpX bound hClpP tetradecamer (Supplementary Fig. 13b). These characteristics indicate this hClpXP complex represents an early stage in the assembly process, poised for subsequent substrate engagement and nucleotide exchange. Based on these observations, we term this structure the “initial assembly” complex (hClpXPIA).

Comparison of the substrate-free hClpXPIA complex with the substrate-bound hClpXP structure enables us to define the structural rearrangements associated with substrate engagement. One of the most striking rearrangements is a ~10 degree rotation of the hClpX relative to hClpP upon substrate binding (Fig. 4c), which prompted us to examine the interactions between the hClpX LGF loops and the hydrophobic pockets of the hClpP heptamer. During this inter-oligomeric rotation, the hClpX LGF loops remain positioned in their respective hClpP pockets with the exception of the LGF loop from the “E” subunit, which shifts from the binding pocket formed by hClpP chains I and J to the neighboring pocket (Fig. 4a,b). It is proposed that the ClpX I/LGF loops dynamically engage and disengage from one ClpP binding pocket to the next during substrate processing, and the clockwise movement of this LGF loop is consistent with the directionality associated with hClpX enzymatic activity11. This repositioning of the LGF loop during the transition from initial assembly complex to fully assembled complex suggests a mechanistic link between substrate engagement, ATPase activation, and formation of the hClpP tetradecamer.

Our hClpXPX-linked structure showed that the “E” subunit of hClpX interacts closely with an N-terminal β-hairpin of hClpP (Supplementary Fig. 10), and our isolated hClpP heptamer structure also presented an asymmetric arrangement of N-terminal β-hairpins, with some more stably directed upward (Fig. 3b, Supplementary Fig. 14). This asymmetric arrangement of hClpP β-hairpins may play a role in the initial recruitment of hClpX by providing recognition elements that establish a preferred asymmetric distribution of LGF loops in the hydrophobic hClpP binding pockets. Such a directed assembly mechanism could establish an energetically favorable substrate recruitment complex, poised to reposition the first LGF loop to the next binding pocket upon substrate engagement as the first step of substrate processing.

Substrate binding is required for hClpP activation
Prior structural studies of bacterial ClpP revealed three distinct states of the tetradecameric assembly, defined as extended, compact, and compressed18. The extended state is considered to be the proteolytically active conformation, whereas the compact and compressed states are regarded as transition states posited to facilitate peptide exit10,18. However, in seeming contradiction to these results from unicellular systems, the crystal structure of apo hClpP revealed a tetradecamer in an extended state19, while crystal structures of hClpP bound to small molecule activators showed tetradecamers in a compact state21,23,24,40. While it is possible that hClpP has evolved a fundamentally distinct proteolytic mechanism, it is also conceivable that these structural conformations were not representative of their corresponding proteolytic states due to crystallization conditions. We thus aimed to clarify these structure-activity relationships with cryo-EM analyses.

Prior single particle cryo-EM studies of bacterial ClpXP complexes indicate that ClpX binding promotes ClpP to adopt the proteolytically competent extended conformation10. Given the level of structural conservation observed between bacterial ClpXP and our hClpXP complexes, we expected this feature to be preserved. Intriguingly, our hClpXP complex, despite being poised for degradation with a substrate peptide in the hClpX central pore, contains hClpP in a compact conformation that is presumed to be proteolytically inactive (Fig. 5a). In this inactive conformation, the catalytic histidine is orientated away from the nucleophilic serine such that the necessary hydrogen bond network required for catalysis cannot be achieved41.

We previously observed an analogous discrepancy between proteolytically competent conformations in our comparison of the bacterial and human Lon protease30. Substrate interaction with the AAA+ module of bacterial Lon promotes rearrangement of the protease component to an active conformation but is insufficient for protease activation in human LONP1. We previously showed that the proteolytically active conformation of LONP1 could be obtained by introducing the peptide mimetic bortezomib30, and we surmised that hClpP activation may similarly require substrate peptide interaction with the peptidase to promote the active/extended conformation. To test this, we used negative staining and found that covalent binding of bortezomib indeed promotes heptameric hClpPs to assemble into tetradecamers (Supplementary Fig. 4f). We next determined a cryo-EM structure of bortezomib-bound hClpP to an overall resolution of ~2.3 Å, which was sufficient to resolve the covalent linkage between bortezomib and the catalytic S153 residue at the protease site (Fig. 5b and Supplementary Fig. 19, Supplementary Table 1). Notably, bortezomib binding induced hClpP to adopt an active, extended tetradecamer, confirming our hypothesis (Fig. 5b).

The similarity between our bortezomib-bound hClpP structure and the previous X-ray crystal structure of apo hClpP maybe explained by the presence of an unknown density linked to the catalytic serine residue in the crystal structure, which may have contributed to adoption of the active conformation19. The bortezomib density in our structure also resembles that of a putative tripeptide identified in the protease active site of Thermus thermophilus ClpP (TtClpP), which also crystallized in the active conformation42. Analogous to what has been described for Thermus thermophilus ClpP (TtClpP)42 and Mycobacterium tuberculosis ClpP1ClpP243 (MtClp1ClpP2), bortezomib mimics peptide binding at the active sites, interacting with the handle region to stabilize an extended/active conformation. Upon bortezomib binding, the catalytic H178 residue shifts towards S153 while the neighboring Q179 residue shifts equatorially to establish an interaction network stabilizing the extended conformation (Supplementary Fig. 16d,e,f). The previously described “oligomerization sensors10,” corresponding to E225 and R226 in hClpP, contribute to the stabilization of the handle region in the extended state by interacting with N187 and T189 residues, which are located in a loop that is disordered in the compact state. These additional structural observations—coupled with our degradation assays on the E225A and R226A constructs—further confirm the conserved role of these residues in hClpP (Supplementary Fig. 9b, 16f).

Collectively, our structural data clarify the interpretation of prior crystallographic studies and demonstrate a role for substrate peptide interaction with the proteolytic active site in protease activation in various species. The conformational impact of substrate interaction is also consistent with findings from another cryo-EM study of hClpP that was performed while this manuscript was under preparation39.

Methods
Protein expression and purification
The genes encoding hClpX (residues 66-633) and hClpP (residues 58-277) were cloned into a PET28a expression vector containing a His6-SUMO tag at the N-terminus. Various hClpX variants (hClpXI146-dCTE, hClpXI146-dCTE-dE, hClpXI146-dCTE-E396Q, and hClpXI146-dCTE-E396A) and hClpP variants (hClpPdCTE and hClpPdCTE-S153A) were generated using the Q5 Site-Directed Mutagenesis Kit from New-England Biolabs. The hClpX and hClpP constructs were transformed into BL21 Rosetta competent cells and grown overnight in LB medium supplemented with 50 μg/ml kanamycin and 25 μg/ml chloramphenicol. The overnight culture was then diluted 1:100 in fresh LB medium and incubated at 37°C with a shaking speed of 225 rpm. When OD600 reached 0.6, protein expression was induced by adding isopropyl beta-D-thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM, and the culture was shaken overnight at 16°C. The following day, cells were harvested by centrifugation at 4500 rpm for 30 minutes, and the pellet was resuspended in lysis buffer (25 mM HEPES, pH 7.5, 100 mM KCl, 400 mM NaCl, 10% glycerol, 0.1 mM PMSF protease inhibitor). The cell suspension was sonicated, and the lysate was clarified by centrifugation at 35,000 rpm for 30 minutes. The supernatant containing the proteins was purified using Ni-NTA beads. The eluted proteins were cleaved with Ulp1 protease and dialyzed overnight in a buffer containing 25 mM HEPES, pH 7.5, 300 mM KCl, and 10% glycerol. To remove the His6-SUMO tag and Ulp1 protease, the cleaved proteins were passed through Ni-NTA beads again. The proteins were subsequently further purified using size exclusion chromatography (SEC) on a Superdex 200 10/300 GL column. For hClpX, the SEC buffer contains 25 mM HEPES pH 7.5, 300 mM KCl, 10% glycerol, 2 mM magnesium, and 1 mM DTT. hClpP was purified using SEC buffer containing 25 mM HEPES pH 7.5, 150 mM KCl, 5% glycerol, and 1 mM DTT. Purified proteins were concentrated, aliquoted, flash frozen in liquid nitrogen, and stored at -80°C.

Sample preparation for electron microscopy
For all three cryo-EM structures presented in this paper, 4 μl of sample was applied onto 300 mesh R1.2/1.3 UltraAuFoil Holey Gold grids (Quantifoil), which were plasma cleaned for 15 seconds using a Pelco glow discharge cleaning system (TED PELLA, INC.) with atmospheric gases at 15 mA. The grids were manually blotted using Whatman #1 blotting paper for 6 seconds before being plunged frozen in liquid ethane in a 4°C cold room with 90% humidity (all subsequently described vitrification of samples were performed using the same methodology).

To prepare hClpXPX-linked sample, hClpXI146-dCTE-E396Q (50 μM) and hClpPdCTE-S153A (8 μM) were incubated in a buffer containing 25 mM HEPES (pH 7.5), 150 mM KCl, 5 mM ATP, and 5 mM MgCl2 for 5 minutes at room temperature. The sample was then crosslinked with 160 μM disuccinimidyl suberate (DSS) (Thermo Fisher A39267) for 30 minutes at room temperature, and the reaction was quenched with 40 μM Tris (pH 7.5). The crosslinked sample was concentrated and injected onto a Superose 6 increase 10/300 GL column in SEC buffer (25 mM HEPES, 150 mM KCl, 5% glycerol, 2 mM MgCl2, 2 mM ATP). Fractions corresponding to the crosslinked hClpXP complexes were collected and concentrated 15-fold. The concentrated sample was then applied to the cryo-EM grid and manually blotted before being plunged frozen in liquid ethane. For the uncrosslinked hClpXP sample, a similar concentration of full-length hClpX and hClpP (with the mitochondrial targeting sequence removed) was incubated in a buffer containing 25 mM HEPES (pH 7.5), 150 mM KCl, 5 mM ATPγS, and 5 mM MgCl2 for 30 minutes at room temperature. The sample was then applied to the cryoEM grid and manually blotted before being plunged frozen in liquid ethane.

For the apo hClpP cryo-EM, 4 μl of 15 mg/ml of hClpP (WT) in a buffer containing 25 mM HEPES, 150 mM KCl, 2 mM DTT, and a final 4 mM fluorinated fos-choline or 0.08% n-Decyl-beta-Maltoside (DM) detergent was applied onto the cryo-EM grid and immediately blotted before being plunged into liquid ethane. For the bortezomib-bound hClpP structure, a 10X molar excess of bortezomib (Sigma-Aldrich) was added to 15 mg/ml of hClpP and incubated on ice for 30 minutes. Just before applying the sample to the cryo-EM grid, detergent at a final 0.08% DM concentration was added to the sample to help mitigate the preferred orientation of hClpP in vitrified ice.

Cryo-EM data collection and processing
For all datasets, cryo-EM data were collected on a Thermo-Fisher Talos Arctica transmission electron microscope operating at 200 kV with parallel illumination conditions57,58. Micrographs were acquired with a Gatan K2 Summit direct electron detector with a total electron exposure of 50 e-/Å2. All subsequent image processing was performed using cryoSPARC59. Detailed processing workflows and dataset-specific parameters are provided in Supplementary Figs. 6, 12, 17, 19 and Supplementary Table 1.

For the hClpXPX-linked complex cryo-EM dataset, Leginon data collection software60 was used to collect 10,333 micrographs at 45K nominal magnification (0.91 Å/pixel-1), with a nominal defocus range from -0.23 μM to -3.3 μm. Stage movement was used to target the center of either 4 or 16 1.2 μm holes for focusing, and image shift was utilized to acquire high magnification images. The Appion image processing wrapper61 was used to run MotionCor262 for micrograph frame alignment and dose-weighting in real-time during data collection. Subsequent image processing was done in cryoSPARC. The contrast transfer function (CTF) parameters were estimated using patch CTF estimation (multi). Template-based particle picking, using representative 2D class averages generated from 1,175 particles selected with blob picking using a diameter of 200 Å to 320 Å, resulted in approximately 2.07 million particles. Particles were extracted from the motion-corrected and dose-weighted micrographs with a box size of 352 pixels. These particles were classified into 100 2D classes using default parameters, and 2D classes (containing 1,940,578 particles) displaying high-resolution secondary features were selected to generate four reference-free 3D Ab-initio models. The initial volumes generated from ab initio were used for heterogeneous refinement to classify the particles into four classes with C1 symmetry. The class containing well-resolved structural features, corresponding to 1,321,170 particles, was chosen for non-uniform refinement of hClpP with C1 symmetry, resulting in a final reconstruction with a reported GSFCS at 2.91 Å. For hClpX, the same particle set underwent additional 3D classification (six classes) with a mask corresponding to hClpX (O-EM learning rate init: 0.2, initialization mode: PCA; PCA/simple: particles per reconstruction: 750; PCA: number of reconstructions: 75; Force hard classification; class similarity: 0.1). Two good classes (513,188 particles total) were combined and subjected to a final round of non-uniform refinement with C1 symmetry. Subsequently, the 3D volume was re-aligned centering on hClpX, and the particles re-extracted with the centered coordinates were locally refined with a mask around hClpX, resulting in a final resolution of 3.11 Å based on the FSC at a cutoff of 0.143. A composite map was generated using individual high-resolution maps of hClpX and hClpP in ChimeraX.

For the uncrosslinked hClpXP complex cryo-EM dataset, Leginon data collection software60 was used to collect 3298 micrographs at 36K nominal magnification (1.15 Å/pixel-1), with a nominal defocus range from -0.2 μM to -3.6 μm. Stage movement was used to target the center of either 4 or 16 1.2 μm holes for focusing, and image shift was utilized to acquire high magnification images. The Appion image processing wrapper61 was used to run MotionCor262 for micrograph frame alignment and dose-weighting in real-time during data collection. Subsequent image processing was done in cryoSPARC. The contrast transfer function (CTF) parameters were estimated using patch CTF estimation (multi). Template-based particle picking, using representative 2D class averages generated from 2,205 particles selected with blob picking using a diameter of 200 Å to 320 Å, resulted in approximately ~1.1 million particles. Particles were extracted from the motion-corrected and dose-weighted micrographs with a box size of 352 pixels (Fourier cropped to a box size of 176 pixels). These particles were classified into 100 2D classes using default parameters, and 2D classes (containing 70,623 particles) displaying high-resolution secondary features were selected to generate four reference-free 3D Ab-initio models. The initial volumes generated from ab initio were used for heterogeneous refinement to classify the particles into four classes with C1 symmetry. The class containing 26,401 particles of hClpXPIA was re-extracted with full box size and used as input for non-uniform refinement with C1 symmetry, resulting in a final reconstruction with a reported GSFCS at 4.4 Å. Focused-refinement of hClpXIA and hClpPIA using individual masks led to final resolution of 4.3 Å and 4.0 Å, respectively. The class containing 23,084 particles of hClpXP was re-extracted and chosen for homogenous refinement and symmetry expansion (D1), followed by local refinement of the top half of hClpXP and 3D classification (5 classes). The three classes of particles with good hClpX density were combined for local refinement, while the other 2 classes were combined for homogenous refinement. The particles from local refinement and homogenous refinement were then combined for local refinement with an hClpX mask, leading to a reconstruction of hClpX resolved to ~4.4 Å resolution. Further local refinement with an hClpP mask resulted in hClpP density w than overall resolution of ~3.5 Å. A composite map of uncrosslinked hClpXP was generated using individually local refined maps of hClpX and hClpP in ChimeraX.

For the apo hClpP (WT) structure, a total of 14,989 micrographs were collected at 150K nominal magnification (0.94 Å/pixel-1) with a nominal defocus range from -0.3 μm to -3.5 μm using the EPU software. A maximum image shift of 2 µm was used to acquire high magnification images. Dose-weighting, motion correction, and CTF estimation were performed within cryoSPARC live. Template-based particle picking, using representative 2D class averages generated from 4,710 particles selected with blob picking (diameter of 70 Å to 100 Å), resulted in ~2.7 million particles. Two rounds of 2D classification into 100 class averages using default parameters were performed to remove junk particles, with 935,410 particles selected for Ab-initio and iterative hetero-refinement (four classes). The class with the best-resolved structural features (corresponding to 501,826 particles) was used for non-uniform refinement (C1 symmetry), yielding a hClpP reconstruction at 3.63 Å resolution. Subsequent global and local CTF refinement, and another round of non-uniform refinement, t slightly improved the resolution to 3.61 Å. 3D classification into 10 classes was performed, with particles from eight good classes used for the final round of non-uniform refinement (C1 symmetry), resulting in a final reported GSFCS resolution of 3.52 Å.

For the bortezomib-bound-hClpP dataset, 3972 micrographs were collected using EPU at 150K nominal magnification (0.94 Å/pixel-1) with a nominal defocus range from -0.1 μm to -3.28 μm. Image shift was used to acquire high magnification images as for the prior dataset. Dose-weighting, motion correction, and CTF estimation were performed within cryoSPARC live. Blob picking of the CTF estimated images using a particle diameter of 100 Å to 150 Å resulted in ~1.9 million particles. Two rounds of 2D classification were performed to remove junk particles, leaving 545,529 particles for Ab-initio and subsequent hetero-refinements (4 classes). The class with the best-resolved structural features (corresponding to 224,782 particles) was used for non-uniform refinement (C1 symmetry), producing a hClpP reconstruction at 2.67 Å resolution. Further global and local CTF refinement slightly increased the resolution to 2.63 Å with C1 symmetry and 2.28 Å with D7 symmetry.

Atomic model building and refinement
For the hClpXPx-linked complex, the AlphaFold model of hClpX and the hClpP crystal structure (PDB 1TG6) were used as starting models. For the uncrosslinked hClpXP complex, the refined models from the crosslinked hClpXP were used as initial references. For the bortezomib-bound hClpP, the hClpP crystal structure (PDB 6BBA) served as a starting model, whereas for the hClpP apo structure, the hClpX-bound hClpP model was used. The models were docked into the 3D reconstruction using UCSF ChimeraX63. Manual model building and real-space structural refinement were performed with Coot64 and Phenix65, respectively. Phenix real-space refinement includes global minimization, rigid body, local grid search, atomic displacement parameters, and morphing for the first cycle. It was run for 100 iterations, five macro-cycles, with a target bonds RMSD of 0.01 and a target angles RMSD of 1.0. The refinement settings also include secondary structure restraints and Ramachandran restraints. ChimeraX plug-in ISOLDE66 was used to manually fix any Ramachandran outliers, rotamers, and clashes not fixed by Phenix. For the bortezomib-bound hClpP, a covalent linkage between bortezomib and hClpP S153 was made using Phenix. Figures for publication were prepared using UCSF ChimeraX. Supplementary Table 1 includes data collection, refinement, and validation statistics.

ATPase activity assay
The ATPase activity experiments of different hClpX variants were carried out in a black flat-bottom 96-well plate in a final volume of 100 μl. 0.3 μM of hClpX proteins were pre-incubated at 37°C for 5 min in the activity buffer (50 mM HEPES pH 7.5, 75 mM KCl, 5 mM MgCl2, and 1 mM DTT, 1.6 mM NADH (Caymen Chemical; 606-69-8), 4 mM phosphoenolpyruvate (1 Plus Chem; 5541-93-5), 120 U/ml lactate dehydrogenase (Worthington Chemical; LS002755), 40 U/ml pyruvate kinase (Sigma; 10128155001). The reaction was initiated by adding 5 mM ATP. For ATP titration assays, increasing final concentrations (0.05 mM, 0.1 mM, 0.15 mM, 0.2 mM, 0.3 mM, 0.8 mM, 2 mM, 5 mM, and 8 mM) of ATP were added to initiate the reaction. Absorbance at 340 nm was measured at 37°C using a TECAN plate reader.

Degradation assays
The ClpXP-mediated degradation of FITC-casein was carried out in a black flat-bottom 96-well plate in a final volume of 100 μl. 1 μM ClpX and 0.5 μM ClpP were pre-incubated at 37°C for 5 min in activity buffer containing 50 mM HEPES pH 7.5, 75 mM KCl, 5 mM MgCl2, 5 mM ATP, and 1 mM DTT. 1 μM FITC-casein (Sigma Aldrich; C3777), which had also been pre-incubated at 37°C, was added to initiate the degradation reaction. For substrate titration assays, increasing final concentrations (0.125 μM, 0.250 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM, 10 μM, and 16 μM) of FITC-casein was added to initiate the reaction. For the hClpP or hClpPdCTE titration assay, 0.5 μM of hClpX and increasing concentrations of hClpP or hClpPdCTE (0.03 μM, 0.06 μM, 0.125 μM, 0.25 μM, 0.5 μM) were used in the reaction. 2 μM FITC-casein was added to initiate the degradation reaction. An increase of fluorescence resulting from FITC molecules was measured (excitation 485 nm, emission 535 nm) using a TECAN plate reader.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this revised manuscript by Chen, Lander and Yang, the authors have addressed my major concerns with additional data and descriptions. For one of these specific concerns, demonstrating the role of the CTE of hClpP in reducing hClpX interactions with hClpP, I do not think the data in Extended Data Figure 4 is consistent with their conclusion that the hClpPdCTE has a higher binding affinity with hClpX. In fact, because the Kactivation (mistakingly called the KM) they report is the same for WT and dCTE, it seems that affinity to hClpX is not affected. If anything, the data is consistent with the hClpP-CTE restricting enzymatic activity of hClpP itself (either by limiting product release or substrate hydrolysis) as the maximum rate of activity is the only parameter different. This minor point should be corrected in their manuscript.\nThe authors have also added an additional analysis of a new state of hClpXP, where hClpX is bound to a heptamer of a hClpP in a likely inactive state, which they name the initial assembly complex as they speculate this is the first step of encounter of hClpX with hClpP. Importantly, there is a rotation of the hClpX relative to hClpP in these structures not seen with the fully assembled hClpXP complex, consistent with some models in the field where 6:7 mismatches of the I/LGF loops of ClpX with ClpP sites results in a switching of loop binding to neighboring binding sites. This is a nice addition to the paper and leads to a great deal of possible speculation for future studies that, in this reviewer's opinion, goes beyond the scope of this current work.\nOverall, the authors have addressed my initial concerns with only minor comments remaining.\nMinor comments:\n1. Discuss ClpP heptamer asymmetry for IA complex in the lines 283-302.\n2. Extended Fig 15. Legends are only for a and b, but extra results about IA complex are now present and should be discussed.", "reviewer_2": "The paper by Chen et al. reports the first Cryo-EM structure of human ClpXP. The authors made a strong effort to determine additional structures in response to reviewers’ questions. However, the major issue that remains unaddressed is the biological significance of these structures—specifically, whether they represent real biological conformations or are artifacts introduced by sample preparation, detergent use, or other modifications. Additionally, the fact that some structures could be obtained without extensive mutations or cross-linking (such as the uncrosslinked maps) raises the concern that such efforts may not have been necessary. I am not suggesting additional experiments, but I do recommend that the authors remove or at least deemphasize some of the earlier structures obtained with extensive mutations, as well as those where their own data indicate that detergents led to inactivation of the complex. Below, I outline my major and minor comments:\n1. While the authors emphasize the need for extensive sample preparation, their uncrosslinked dataset using full-length ClpX and ClpP (without the mitochondrial signal sequence) already yielded several structures. This observation suggests that the claim of requiring “excessive” sample preparation may not be fully justified, especially since the number of movies collected was not substantially larger than in their other datasets.\n2. In Extended Figure 20, the docking of the crosslinked model into the uncrosslinked map does not convincingly demonstrate ClpXP fitting. A more rigorous approach would be to refine the model into the map and then provide quantitative validation, such as the RMSD between the crosslinked and uncrosslinked models, together with map–model correlation coefficients. Alternatively, both maps could be low-pass filtered to the same resolution and compared using Fourier cross-correlation. Also, I could not find the model or map corresponding to Extended Figure 20 in Table 1. Are the authors not planning to deposit this map?\n3. All reconstructions of ClpXP (other than the uncrosslinked sample) were performed with the C-terminal extension removed. Although the authors show in their revision that this truncation does not influence ATP binding, they also demonstrate that it slows hClpX ATPase activity and reduces degradation of unfolded substrates. They then argue that this modification stabilizes the complex for Cryo-EM characterization. While this rationale may satisfy a structural audience focused on optimization, it does not resolve the concern that the reported structures may therefore lack biological relevance.\n4. The authors note that detergents such as DM alter the conformation and activity of ClpXP. However, their final asymmetric heptameric ClpP structure was also determined using Fos-choline-8 detergent. This raises confusion about which conformations are biologically meaningful versus those that are artifacts of detergent-dependent sample preparation.\n5. The comparison between the substrate-free hClpXP complex and the substrate-bound form is problematic. How can the authors be certain that the so-called “substrate-free” map is not an artifact arising from preferred orientation or partial denaturation of the ClpX channel at the air–water interface? Given the reported sphericity score, this seems a strong possibility.\n6. The authors state: “Consistent with previous observations, crosslinking hClpX to hClpP limits the mobility of the hClpX LGF loops, thereby preventing substrate unfolding and translocation, abolishing FITC-casein degradation activity.” The crosslinker used by the authors appears less specific — it could target multiple regions of the ClpX or ClpP complexes, rather than being restricted to the LGF loops. Also, Kim et al., eLife 2020, demonstrated that crosslinked ClpAP remain active in degrading unstructured substrates, contradicting the structural model previously described (ref. 11). This suggests that full rotation of ClpX or ClpA on ClpP is not required. I suggest revising this statement.\n7. While structural models can be informative, Figure 6 is problematic because the structures were obtained under disparate conditions. I strongly recommend removing this figure. In particular, the depiction of partially unfolded proteins is misleading, as it could give general readers the false impression that such structure were directly determined by the authors.\nMinor Points\n1. In Extended Figure 19, right panel: the mask should correspond to hClpP, not hClpX.\n2. The following statement is confusing. If the resolution is insufficient, no claim should be made. If the authors rely on indirect evidence (e.g., Arg finger), that must be stated explicitly; otherwise, the speculation should be removed:\n“Although the resolution of the reconstruction is insufficient to unambiguously identify the nucleotide states of each pocket, they generally appear to be more consistent with ADP than ATP.”\n3.I highly recommend that the authors carefully review the extended figures and ensure that all captions clearly indicate the specific mutations introduced into hClpXP complex.", "reviewer_4": "The authors have made a major effort to carefully address all the reviewer’s concerns. The newly added experiments, including the low-resolution cryo-EM map of uncrosslinked hClpXP complex have strongly strengthened the study."}, "author_response": "We thank the reviewers for carefully assessing our revised manuscript and providing constructive and critical comments. We have taken all the reviewers’ comments into earnest consideration, and we provide point-by-point response to these comments below. Our responses are in blue, in-line with the reviewers’ original comments.\nREVIEWER #1.\nReviewer #1 Remarks to Authors. “In this revised manuscript by Chen, Lander and Yang, the authors have addressed my major concerns with additional data and descriptions. For one of these specific concerns, demonstrating the role of the CTE of hClpP in reducing hClpX interactions with hClpP, I do not think the data in Extended Data Figure 4 is consistent with their conclusion that the hClpPdCTE has a higher binding affinity with hClpX. In fact, because the Kactivation (mistakingly called the KM) they report is the same for WT and dCTE, it seems that affinity to hClpX is not affected. If anything, the data is consistent with the hClpP-CTE restricting enzymatic activity of hClpP itself (either by limiting product release or substrate hydrolysis) as the maximum rate of activity is the only parameter different. This minor point should be corrected in their manuscript.\nThe authors have also added an additional analysis of a new state of hClpXP, where hClpX is bound to a heptamer of a hClpP in a likely inactive state, which they name the initial assembly complex as they speculate this is the first step of encounter of hClpX with hClpP. Importantly, there is a rotation of the hClpX relative to hClpP in these structures not seen with the fully assembled hClpXP complex, consistent with some models in the field where 6:7 mismatches of the I/LGF loops of ClpX with ClpP sites results in a switching of loop binding to neighboring binding sites. This is a nice addition to the paper and leads to a great deal of possible speculation for future studies that, in this reviewer's opinion, goes beyond the scope of this current work.\nOverall, the authors have addressed my initial concerns with only minor comments remaining.”\nWe thank reviewer for their overall positive comments regarding our manuscript. We address the remaining comments from this reviewer in this revised submission, as described below:\n“For one of these specific concerns, demonstrating the role of the CTE of hClpP in reducing hClpX interactions with hClpP, I do not think the data in Extended Data Figure 4 is consistent with their conclusion that the hClpPdCTE has a higher binding affinity with hClpX. In fact, because the Kactivation (mistakingly called the KM) they report is the same for WT and dCTE, it seems that affinity to hClpX is not affected. If anything, the data is consistent with the hClpP-CTE restricting enzymatic activity of hClpP itself (either by limiting product release or substrate hydrolysis) as the maximum rate of activity is the only parameter different. This minor point should be corrected in their manuscript.”\nWe thank the reviewer for the helpful clarification. In our revised manuscript we replace K with K in M activation Extended Data Figure 3c and updated the corresponding text (lines 90-93) to more accurately reflect our interpretation:\n“Titration experiments comparing wild-type hClpP and hClpP∆CTE suggest that both proteins bind and activate hClpX similarly, but hClpP∆CTE exhibits a modestly higher substrate turnover rate, indicating that the C-terminal extension negatively regulates hClpP activity (Extended Data Fig. 3c).”\nWe agree with the reviewer that Extended Data Fig.3c does not suggest an increased affinity of hClpPdCTE for hClpX. It is plausible that the increased number of hClpXP particles observed with the ClpPdCTE-S153A construct in Extended Data Figure 4b is due to the combined effect of the CTE deletion and the S153A mutation. We have clarified this point and added the relevant citations in the revised text (lines 87-90):\n“Previous studies showed that removing the C-terminal extension (CTE) (Kang et al., 2004; Kang et al., 2002), and introducing a proteolytically inactivating S153A mutation (Gatsogiannis et al., 2019) stabilize ClpXP complex assembly. Accordingly, we generated an hClpP∆CTE-S153A construct, which exhibited improved binding to hClpX as observed by negative stain EM (Extended Data Figs. 4a,b, 5c).”\n“Discuss ClpP heptamer asymmetry for IA complex in the lines 283-302.”\nWe thank the reviewer for the comment. We added a discussion of the hClpP heptamer asymmetry for IA complex (lines 276-278):\n“The heptameric hClpP in this newly identified hClpXP complex exhibits slight asymmetry, representing an intermediate state between asymmetric hClpP heptamer and the symmetric hClpX-bound hClpP tetradecamer (Extended Data Fig.13b).”\n“Extended Fig 15. Legends are only for a and b, but extra results about IA complex are now present and should be discussed.”\nWe thank the reviewer for pointing this out. We updated the figure legend in the revised text (previously Extended Data Fig 15, now Extended Data Fig 13) to extend and clarify our discussion of the hClpXPIA complex:\n“a, Seven-fold symmetry parameters were used to overlay individual subunits of ClpP from one ClpP ring of the apo hClpP heptamer, which was refined without imposed symmetry. The apo hClpP exhibits distinct subunit positions, indicating an asymmetric arrangment. b, The same analysis was applied to hClpP subunits of the hClpXPIA complex. Here, hClpP adopts an intermediate conformation between the asymmetric apo hClpP and the symmetric hClpX-bound hClpP, showing increased but incomplete symmetry. c, The same operation was performed on hClpP from the hClpXPx-linked complex refined without imposed symmetry.In this case, the subunits overlay perfectly, demonstrating a fully symmetric arrangement of the subunits in the context of the tetradecamer.”\nREVIEWER #2.\nReviewer #2 Remarks to Author.“The paper by Chen et al. reports the first Cryo-EM structure of human ClpXP. The authors made a strong effort to determine additional structures in response to reviewers’ questions. However, the major issue that remains unaddressed is the biological significance of these structures—specifically, whether they represent real biological conformations or are artifacts introduced by sample preparation, detergent use, or other modifications. Additionally, the fact that some structures could be obtained without extensive mutations or cross-linking (such as the uncrosslinked maps) raises the concern that such efforts may not have been necessary. I am not suggesting additional experiments, but I do recommend that the authors remove or at least deemphasize some of the earlier structures obtained with extensive mutations, as well as those where their own data indicate that detergents led to inactivation of the complex. Below, I outline my major and minor comments:”\nWe thank the reviewer for their thoughtful comments. For the wild-type uncrosslinked samples, we encountered substantial challenges in expressing and purifying full-length human ClpX (without the mitochondrial targeting sequence). Specifically, the wild-type protein was markedly less stable, and the yield was significantly lower relative to the optimized construct. At the outset of this project, we focused on preparing wild-type hClpX and hClpP samples for high-resolution structural determination but were unsuccessful due to the intrinsic instability and extremely low yield of human ClpX. As a result, we were only able to resolve a low-resolution map of the wild-type uncrosslinked hClpXP complex. To overcomes these limitations in pursuit of a high-resolution structure, we turned to construct optimization. Notably, the mutations and deletions introduced to hClpX and hClpP were guided by previous studies of bacterial ClpXP. Similar modifications in bacterial ClpXP have been shown to either not affect structural conformation or enhance ClpXP complex formation and stability (lines 72-78; lines 87-90). Accordingly, we have revised the manuscript to clarify these points and, per the reviewer’s suggestion, deemphasized construct optimization as follows:\na. The title in line 68 has been updated.\nb. The section “purification of the hClpXP sample for cryo-EM structural studies” (lines 69-97) has been rewritten to clarify that the hClpX and hClpP construct designs are based on well-established precedents from bacterial ClpXP studies (with corresponding citations).\nc. We expanded our discussion of the biochemical analyses to better explain how deletion of the hClpX and hClpP C-terminal extensions influences enzymatic activity, revealing their potential regulatory roles (lines 82- 84; lines 90-93).\nPer the reviewer’s suggestion, we have also removed the data related to the detergent-induced inactive ClpP complex. Specifically, the previous Extended Data Figures 13 and 14, as well as Extended Data Figure 9c and the corresponding text describing this complex, have been deleted from the revised manuscript.\n1. While the authors emphasize the need for extensive sample preparation, their uncrosslinked dataset using full-length ClpX and ClpP (without the mitochondrial signal sequence) already yielded several structures. This observation suggests that the claim of requiring “excessive” sample preparation may not be fully justified, especially since the number of movies collected was not substantially larger than in their other datasets.\nWe have now addressed this point in our response above. Specifically, we de-emphasized our previous statements regarding construct optimization in the revised manuscript.\nNotably, a quantitative comparison between the crosslinked and uncrosslinked hClpXP datasets highlights the significant challenges in obtaining high-quality data from the uncrosslinked samples. Specifically, based on the total number of micrographs and final hClpX particle counts, even if we were to collect three times the number of micrographs for the uncrosslinked dataset, the number of usable hClpX particles would still be approximately four-fold lower than the crosslinked dataset. While we were able to determine the structures of the uncrosslinked hClpXP complex and an intermediate state, the corresponding hClpX density were much lower in resolution compared to those from the crosslinked dataset. This reduced resolution limited our ability to confidently build atomic models and key conformational features, such as E-domain movements and nucleotide states.\n2. In Extended Figure 20, the docking of the crosslinked model into the uncrosslinked map does not convincingly demonstrate ClpXP fitting. A more rigorous approach would be to refine the model into the map and then provide quantitative validation, such as the RMSD between the crosslinked and uncrosslinked models, together with map–model correlation coefficients. Alternatively, both maps could be low-pass filtered to the same resolution and compared using Fourier cross-correlation. Also, I could not find the model or map corresponding to Extended Figure 20 in Table 1. Are the authors not planning to deposit this map?\nAs suggested, we have generated a new model of uncrosslinked hClpXP refined into the uncrosslinked hClpXP composite map (Extended Data Fig. 18a). Quantitative validation for the hClpX and hClpP is summarized in Table 1. The refined model and the associated composite map have been deposited in PDB and EMDB.\nThe two structures are almost identical overall, and an overlay of the hClpXP and hClpXPx-linked models reveals an RMSD of 0.5 Å (Extended Data Fig. 18b). The hClpP tetradecamers share a consistent conformation, and only subtle rigid-body shifts are observed in hClpX subunits. This analysis suggests that the crosslinked hClpXP structures are representative of the uncrosslinked full-length hClpXP structures (lines 261- 267 in manuscript).\n3. All reconstructions of ClpXP (other than the uncrosslinked sample) were performed with the C-terminal extension removed. Although the authors show in their revision that this truncation does not influence ATP binding, they also demonstrate that it slows hClpX ATPase activity and reduces degradation of unfolded substrates. They then argue that this modification stabilizes the complex for Cryo-EM characterization. While this rationale may satisfy a structural audience focused on optimization, it does not resolve the concern that the reported structures may therefore lack biological relevance.\nWe thank the reviewer for raising this important point. According to AlphaFold predictions, the C-terminal extension (CTE) of hClpX or hClpP is intrinsically disordered and projects outward from the ClpXP complex (see below), making it unlikely to affect the overall architecture, including subunit oligomerization or the fundamental assembly of the complex. We thus expect our cryo-EM structures to faithfully represent the native organization and capture the mechanistic principles of ClpXP. However, as both our data and the reviewer’s comments suggest, the CTE may indeed modulate enzymatic activity through mechanisms that cannot be structurally characterized and remain to be fully understood. We view this as an exciting avenue for future investigation and now explicitly acknowledge this point in the revised manuscript (lines 82- 84).\n“While removal of the hClpX C-terminal extension reduces its enzymatic activity and stabilizes the complex for cryo-EM characterization, the underlying mechanisms by which this modification alters activity remain to be elucidated in future studies.”\n4. The authors note that detergents such as DM alter the conformation and activity of ClpXP. However, their final asymmetric heptameric ClpP structure was also determined using Fos-choline-8 detergent. This raises confusion about which conformations are biologically meaningful versus those that are artifacts of detergent-dependent sample preparation.\nWe apologize for the confusion we have caused by not writing this section more clearly. When we determined the structure of hClpP in the presence of DM (n-decyl-β-D-maltoside), we observed additional EM density at residue Y118 in the hydrophobic pocket of hClpP (see below), that was not observed when ClpP was determined in the absence of the detergent. This led us to speculate that this density may correspond to a DM detergent molecule. Further, this extra density suggests a direct interaction between DM and hydrophobic pockets of hClpP that may have promoted hClpP tetradecamer formation at a high concentration of 15 mg/ml used during sample preparation. Since, we did not observe any density corresponding to Fos-Choline-8 in the cryo-EM map of asymmetric hClpP heptamer, this is suggests that Fos-choline-8 does not interact with hClpP and is therefore unlikely to induce structural changes. To avoid potential confusion about detergent-dependent effects and per the reviewer’s suggestion, we have nowremoved all DM-related data in the manuscript.\n5. The comparison between the substrate-free hClpXP complex and the substrate-bound form is problematic. How can the authors be certain that the so-called “substrate-free” map is not an artifact arising from preferred orientation or partial denaturation of the ClpX channel at the air–water interface? Given the reported sphericity score, this seems a strong possibility.\nWe thank the reviewer for raising this important point regarding the substrate-free hClpXP complex. We carefully considered the possibility that the observed “initial assembly” state could arise from preferred particle orientation or partial denaturation of the ClpX channel at the air–water interface. However, several lines of evidence strongly argue against this explanation and instead support the biological relevance of this conformation:\n1. The substrate-free hClpXP complex was resolved from the same dataset as the substrate-bound complex under identical grid preparation, imaging, and processing conditions. Despite this, the substrate-free map exhibits clear and reproducible structural differences, including a ~10° rotation between the ATPase and protease rings and a distinct rearrangement of the LGF loops, with the E subunit loop engaging a neighboring hydrophobic pocket. These well-defined conformational changes are inconsistent with partial denaturation, which would typically result in poorly defined or heterogeneous density rather than specific and reproducible structural rearrangements.\n2. We previously determined that crosslinked hClpXP structure fits into the uncrosslinked hClpXP density with high fidelity (Extended Data. Fig.18), indicating that the global architecture of the complex remains intact.\n3. The substrate-free state was only observed in the uncrosslinked hClpXP dataset but not in the crosslinked, substrate-trapped datasets suggests that it represents a bona fide intermediate in the assembly pathway rather than a grid-induced artifact.\n4. Similar substrate-free conformations with an open ATPase ring and ADP-like nucleotide states have been reported for other AAA+ unfoldases and proteases, including ClpA and Lon(Shin et al., 2021; Shin, 2020), where they represent pre-engagement or pre-activation intermediates poised for substrate loading. The features we observe — including a widened central pore, a flattened ATPase spiral, and ADP-like nucleotide density — are consistent with this interpretation. We note that Reviewer #1 also highlighted the significance of this finding, stating: “The authors have also added an additional analysis of a new state of hClpXP, where hClpX is bound to a heptamer of a hClpP in a likely inactive state, which they name the initial assembly complex as they speculate this is the first step of encounter of hClpX with hClpP. Importantly, there is a rotation of the hClpX relative to hClpP in these structures not seen with the fully assembled hClpXP complex, consistent with some models in the field where 6:7 mismatches of the I/LGF loops of ClpX with ClpP sites results in a switching of loop binding to neighboring binding sites. This is a nice addition to the paper and leads to a great deal of possible speculation for future studies that, in this reviewer's opinion, goes beyond the scope of this current work.”\n6. The authors state: “Consistent with previous observations, crosslinking hClpX to hClpP limits the mobility of the hClpX LGF loops, thereby preventing substrate unfolding and translocation, abolishing FITC-casein degradation activity.” The crosslinker used by the authors appears less specific — it could target multiple regions of the ClpX or ClpP complexes, rather than being restricted to the LGF loops. Also, Kim et al., eLife 2020, demonstrated that crosslinked ClpAP remain active in degrading unstructured substrates, contradicting the structural model previously described (ref. 11). This suggests that full rotation of ClpX or ClpA on ClpP is not required. I suggest revising this statement.\nWe appreciate the reviewer’s point regarding the work of Kim et al. (eLife 2020), which demonstrated that crosslinked ClpAP complexes retain the ability to degrade unstructured substrates. We have now cited this study and revised our statement in lanes 130-134:\n“However, recent studies show that the crosslinked ClpAP complex remains capable of degrading unstructured substrates, suggesting that a rotational motion may not require for proteolysis(Kim et al., 2020). Our crosslinked hClpXP abolished FITC-casein degradation activity (Extended Data Fig. 9a), which may result from impaired substrate unfolding and translocation caused by the promiscuity of the DSS crosslinker.”\n7. While structural models can be informative, Figure 6 is problematic because the structures were obtained under disparate conditions. I strongly recommend removing this figure. In particular, the depiction of partially unfolded proteins is misleading, as it could give general readers the false impression that such structure were directly determined by the authors.\nPer the reviewer’s suggestion, we have removed Figure 6 from the revised manuscript.\nMinor Points\n1. In Extended Figure 19, right panel: the mask should correspond to hClpP, not hClpX.\nWe thank the reviewer for catching this typo, we have updated the figure (now Extended Data Fig.17).\n2. The following statement is confusing. If the resolution is insufficient, no claim should be made. If the authors rely on indirect evidence (e.g., Arg finger), that must be stated explicitly; otherwise, the speculation should be removed:\n“Although the resolution of the reconstruction is insufficient to unambiguously identify the nucleotide states of each pocket, they generally appear to be more consistent with ADP than ATP.”\nWe have removed this speculation.\n3.I highly recommend that the authors carefully review the extended figures and ensure that all captions clearly indicate the specific mutations introduced into hClpXP complex.\nWe thank the reviewer for the recommendation and have thoroughly reviewed the extended figures and ensured that specific mutations are included in all captions.\nREVIEWER #4.\nReviewer #4 Remarks to Author. “The authors have made a major effort to carefully address all the reviewer’s concerns. The newly added experiments, including the low-resolution cryo-EM map of uncrosslinked hClpXP complex have strongly strengthened the study.”\nWe thank the reviewer for their support of our manuscript!\nReferences cited in this response:\nGatsogiannis, C., Balogh, D., Merino, F., Sieber, S. A., & Raunser, S. (2019). Cryo-EM structure of the ClpXP protein degradation machinery. Nat Struct Mol Biol, 26(10), 946-954. https://doi.org/10.1038/s41594-019-0304-0\nKang, S. G., Maurizi, M. R., Thompson, M., Mueser, T., & Ahvazi, B. (2004). Crystallography and mutagenesis point to an essential role for the N-terminus of human mitochondrial ClpP. J Struct Biol, 148(3), 338-352. https://doi.org/10.1016/j.jsb.2004.07.004\nKang, S. G., Ortega, J., Singh, S. K., Wang, N., Huang, N. N., Steven, A. C., & Maurizi, M. R. (2002). Functional proteolytic complexes of the human mitochondrial ATP-dependent protease, hClpXP. J Biol Chem, 277(23), 21095-21102. https://doi.org/10.1074/jbc.M201642200\nKim, S., Zuromski, K. L., Bell, T. A., Sauer, R. T., & Baker, T. A. (2020). ClpAP proteolysis does not require rotation of the ClpA unfoldase relative to ClpP. Elife, 9. https://doi.org/10.7554/eLife.61451\nShin, M., Watson, E. R., Song, A. S., Mindrebo, J. T., Novick, S. J., Griffin, P. R., Wiseman, R. L., & Lander, G. C. (2021). Structures of the human LONP1 protease reveal regulatory steps involved in protease activation. Nat Commun, 12(1), 3239. https://doi.org/10.1038/s41467-021-23495-0\nShin, M. P., C.; Asmita, A.; Puri, N.; Adjei, E.; Wiseman, R.L.; Karzai, A.W.; Lander G.C.;. (2020). Structural basis for distinct operational modes and protease activation in AAA protease Lon. Sci. Adv., 6(21). https://doi.org/10.1126/sciadv.aba8400"}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors have addressed all of my previous concerns. I have no further comments."}, "author_response": ""}]

Decision letter