Recent Advances in the Structural Studies of the Proteolytic ClpP/ClpX Molecular Machine

Abstract

AAA+ ATPases are ring-shaped hexameric protein complexes that operate as elaborate macromolecular motors, driving a variety of ATP-dependent cellular processes. AAA+ ATPases undergo large-scale conformational changes that lead to the conversion of chemical energy from ATP into mechanical work to perform a wide range of functions, such as unfolding and translocation of the protein substrate inside a proteolysis chamber of an AAA+-associated protease. Despite extensive biochemical studies on these macromolecular assemblies, the mechanism of substrate unfolding and degradation has long remained elusive. Indeed, until recently, structural characterization of AAA+ protease complexes remained hampered by the size and complexity of the machinery, harboring multiple protein subunits acting together to process proteins to be degraded. Additionally, the major structural rearrangements involved in the mechanism of this complex represent a crucial challenge for structural biology. Here, we report the main advances in deciphering molecular details of the proteolytic reaction performed by AAA+ proteases, based on the remarkable progress in structural biology techniques. Particular emphasis is placed on the latest findings from high-resolution structural analysis of the ClpXP proteolytic complex, using crystallographic and cryo-EM investigations. In addition, this review presents some additional dynamic information obtained using solution-state NMR. This information provides molecular details that help to explain the protein degradation process by such molecular machines.

Keywords: AAA+ ATPase; ClpXP; Cryo-EM; NMR; X-ray crystallography; protease; structural biology; unfoldase.

Figure 1

Timeline of the major biostructural advances leading to an improved mechanistic description of the substrate translocation and degradation by the bacterial AAA+ protease ClpXP [11,12,13,18,19,20,21,22,23,24,25,26,27,28,29,30]. Insert: number of AAA+ protease structures published over years using either X-ray (gray) and cryo-EM (blue) methods (from PDB data bank https://www.rcsb.org/ accessed on 1 January 2025).

Figure 2

Summary of substrate degradation by ClpXP. The ssrA degron is located on C-Ter of the substrate protein. The substrate is represented in gray with a green background, the ssrA degron in yellow, and a selected region as the indicator in green. AAA+ ClpX unfoldase is represented with different shades of blue and ClpP protease in gray, with their catalytic sites represented as yellow crosses.

Figure 3

Structure of the ClpP tretradecamer of E. coli (PDB code: 1YG6) [14]. (a) Side view. A ClpP protomer has been highlighted from the complex with distinct colors corresponding to the three regions of ClpP. Gray: handle; purple: head; light green: location of the N-ter region. The lack of electronic density highlights the critical flexibility of the N-ter domain. The two upper-left protomers of the upper ring have been set transparent to reveal the different regions within a single protomer. (b) Top view of the ClpP tetradecamer. (c) Top view of the surface representation. The surface is color-coded according to hydrophobicity (purple: most hydrophilic; beige: most hydrophobic) to highlight the hydrophobic pockets involved in the interaction with ClpX.

Figure 4

Structure of the Z-LY-CMK inhibitor bound to E. coli ClpP. Multiple bonding interactions between Z-LY-CMK (yellow) and the 3rd ClpP protomer of a heptamer are shown (PDB code: 2FZS) [17]. Hydrogen bonds between Z-LY-CMK and ClpP are shown as dashed lines. Gly68, Ile70, and Leu125 create four hydrogen bonds with the peptide backbone of the inhibitor, while the backbone amide nitrogens of Gly68 and Met98 form an “oxyanion hole” and make hydrogen bonds to the hemiketal oxyanion of the inhibitor. The inhibitor is attached to ClpP by two covalent bonds (green circle), one between the carbonyl carbon of the inhibitor and Oγ of Ser97 and the other between the methylene group of the inhibitor and Nε2 of His122. The so-called “oligomerization sensor” Arg170 form an inter-ring salt bridge that further help to correctly position the catalytic Asp171. The third catalytic residue, Asp171, forms a hydrogen bond with Nδ1 of His122, and an additional hydrogen bond is formed with Nε2 of His138 from the adjacent monomer (yellow dotted lines).

Figure 5

Crystal structure of E. coli ClpP in the apo form (PDB code: 1YG6) [14] and bound to acyldepsipeptide or ADEP (PDB code: 3MT6) [21]. (a) Side view of the crystal structures. Ribbon representation of the N-terminal region of ClpP protomer without ADEP (left) or bound to ADEP (right). The N-terminal region in the “up” conformation is shown in blue. (b) Top view of the crystal structure of ClpP (ADEP-free) (left) or bound to ADEP (right), leading to an opening of the pore of around 20 Å. (c) Close-up view of the residues 1–18 of two N-terminal regions shown as ribbons. The residues E14 and R15, which are involved in intermolecular interactions stabilizing the open conformation of the axial channel, are displayed in balls and sticks and labeled accordingly.

Figure 6

Crystal structures of S. aureus ClpP extended state (left) (PDB code: 3STA) [72,73,75,78,79], compact state (middle) (PDB code: 4EMM) [73], and compressed state (right) (PDB code: 3QWD) [72].

Figure 7

Structure of the monomer and hexamer of E. coli ClpX (PDB code: 3HTE). (a) Top view of a ClpX hexamer. The protomer #B is depicted in cartoon visualization with the large and small AAA+ domains, respectively, colored in blue and gray. (b) Cartoon visualization of a ClpX protomer. The poorly defined axial RHH, pore-1, and pore-2 are highlighted in yellow. The poorly defined IGF loop is highlighted in green. (c,d) Side view of a ClpX hexamer. In (c), the protomer #B is depicted as in panel (b) with the same orientation. The loops of the large domain are highlighted in the structure (IGF loop in green; RKH, pore-1, and pore-2 loops in yellow). In (d), the protomers #D and #E are set to 100% transparent, and protomers #A, #C, and #F are set to 50% transparent to allow for a proper view of the loops of protomer #B lining the axial pore.

Figure 8

Crystal structure of ClpX^ΔN protomer bound to ATP. (a) The ATP binding pocket is located at the hinge between the large and small domains (PDB code: 3HWS). (b) Details of the ATP binding pocket in the ClpX hexamer (PDB code: 6WRF) with the conserved motifs and residues involved in the ATP binding including the sensor-II, BoxII, Walker-A, and Walker-B from a given protomer (#D in this example) and the arginine finger from the adjacent subunit (#E in this example). (c) Schematic of the ATP binding pocket involving key residues from the large (blue) and small (gray) domains of a given protomer, as well as residue R307 from the arginine finger from the adjacent subunit.

Figure 9

(a) Composite cryo-EM structure of ClpX bound to ClpP and protein substrate (PDB codes: 6PPE and 6PP6). The insets show a close-up view of the IGF sequence of ClpX bound deeply in a ClpP cleft. ClpP is represented as a surface and colored with respect to (b) hydrophobicity and (c) polarity. (d) Axial view of ClpP (PDB code: 6PPE) bound to ClpX showing an open axial pore, with the clefts that serve as docking sites for the ClpX IGF loops (displayed in ribbon). The ClpP protomer with an empty cleft missing an IGF loop is colored in blue, and the cleft is highlighted in yellow. (e) Structure alignment of the IGF tip loops, suggesting the flexibility adopted by the 6 loops to dock the ClpX asymmetric ring on top of the flat ClpP barrel.

Figure 10

Cryo-EM structure of substrate-free and substrate-bound ClpX. (a) Flat substrate-free ClpX hexamer in surface representation colored by ClpX protomer (PDB code: 6SFW). (b) Substrate-engaged ClpX hexamer in surface representation colored by ClpX protomer (PDB code 6WRF). (c) Structure details of a ClpX protomer (#A) with substrate engaged. The large and small domains are colored in blue and gray, respectively. The axial loops (RKH, pore-1, and pore-2) are highlighted in yellow. The IGF loop is highlighted in green. The substrate is depicted in surface representation. (d) Side view of ClpX hexamer with substrate engaged. The colors are conserved for ClpX protomer (#A). Protomers (#D) and (#E) are set transparent to facilitate the observation of the inner part of the axial pore. (e) Top view of the ClpX hexamer. One of the rigid-body subunits involving the small domain of protomer (#D) and the large domain of protomer (#E) is circled with dotted line. (f) Representation of the six ClpX protomers interacting with the substrate, as observed in the cryo-EM structure of the substrate-engaged ClpX hexamer. The six protomers are represented with the same orientation as protomer (#A) in panel (c). The height of the substrate has been kept constant for the six protomers to highlight the differences in the positions of the axial loops from the six protomers.

Figure 11

Recognition complex. Substrate-engaged ClpX hexamer in surface representation colored by ClpX protomer (PDB code: 6WRF). The two protomers from the front of the ring are set transparent to allow for a view of the recognized substrate. The substrate is displayed in yellow in the surface and atom representations. Insert: zoom on the molecular details of the ssrA degron recognition by ClpX involving the RKH and pore-1 loops of various protomers from the ring. The axial loops of the ClpX protomers are colored by protomers and displayed as ribbons.

Figure 12

Helical staircase organization along the engaged substrate. (a) Intermediate complex: substrate-engaged ClpX hexamer in surface representation colored by ClpX protomer (PDB code: 6WSG). The two protomers from the front of the ring are set transparent to allow for a view of the recognized substrate. The substrate is displayed in yellow in the surface and atom representations. Insert: zoom on the molecular details of the interaction of engaged substrate with the ClpX protomers organized as a helical staircase around the substrate through the Tyr153 and Val154 residues of the pore-1 loop of each protomer. (b–d) Example of the helical staircase conformation conserved through the AAA+ ATPase family observed in the cryo-EM structure of substrate-engaged complex of (a) ClpB (PDB code: 6RN2), (b) 26S proteasome (PDB code: 6MSD), and (c) Vsp4 (PDB code: 6BMF). The amino acids inserted between the β-carbons of every two residues of the substrates are displayed in a ball and stick representation.

Figure 13

Structural variation of ClpX loops in recognition and intermediate complexes bound to substrates (PDB code: 6WRF and 6WSG). (a) The distance (dotted line) between the Tyr153 and the IGF loop/ClpP interface plan (gray plan) has been measured for each protomer in the recognition and intermediate complexes. (b) Six-amino-acid translocation step undergone by the substrate between the two ClpXP transient states. Tyr153 of each subunit is represented in balls and sticks, with Tyr153 of subunits #F and #A disengaged from the substrate, respectively, in the recognition and intermediate structures. (c) and (d) are representations of the height of Tyr153 with respect to the IGF/ClpP interface for all subunits in the recognition (c) and intermediate (d) complexes. The white, green, and dashed circles on top of the bars indicate if the ClpX protomer is ATP-free (dashed), ATP-loaded (white), or ADP-loaded (green).

Figure 14

Substrate translocation driven by ATP hydrolysis. (a) Top view of superimposition of an ATP-free and ATP-loaded ClpX protomer from the intermediate complex ClpX ring after structural alignment of the large domains of the protomers (PDB code:6WSG). The dotted lines give the orientation of the small domain of the protomer in the ATP-free and ATP-loaded configurations. The curved arrow depicts the horizontal rotation of the small domain with respect to the large domain upon ATP binding. (b) Mechanism for processive substrate translocation driven by a complete cycle of ATP hydrolysis among the AAA+ ATPase family. Upon release of Pi, the green subunit, the substate-pore-1 loop interactions, as well as the inter-subunits interactions mediated by the Arg fingers and ISS loops are disrupted, leading to a disengagement of the subunit from the complex. Additionally, this release is combined with a 30° to 40° vertical rotation to reposition itself on top of the spiral, while the other subunits undergo a 5° to 10° rotation to follow the progression of the substate towards the protease chamber. The ATP-free subunit previously disengaged joins the helical staircase assembly upon the binding of a new ATP [91].

Figure 15

(a) Representation of the six ClpX protomers in interaction with the substrate, as observed in the cryo-EM structure of the substrate-engaged ClpX hexamer. The six protomers are represented with the same orientation as protomer #A in panel (c). The height of the substrate has been kept constant for the six protomers to highlight the differences in the positions of the axial loops from the six protomers. (b) Sequential model of substrate translocation triggered by a sequential ATP hydrolysis of the protomer located at the bottom of the helical staircase. Each hydrolysis event allows to pull the substrate by 2 amino acids. (c) Probabilistic model of substrate translocation, where the translocation can be triggered by any of the ClpX protomers. Upon ATP hydrolysis, the ATP-bound protomer position itself at the bottom of the staircase, thus pulling the substrate. The length of the translocation step then depends on the initial position of the protomer compared to the bottom of the staircase.