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

Abstract

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

Fig. 1

Cartoon model of substrate recognition and degradation by the ClpXP protease. In an initial recognition step, a peptide tag in a protein substrate binds in the axial pore of the ClpX hexamer. In subsequent ATP-dependent steps, ClpX unfolds the substrate and translocates the unfolded polypeptide into the degradation chamber of ClpP for proteolysis, where it is cleaved into small peptide fragments. Adapted with permission from ref. [113].

Fig. 2

Domain structure of ClpX. (A) Arrangement of domains and characteristic functional motifs with respect to the linear sequence are shown for E. coli ClpX. Motifs are colored blue for ssrA-tag binding, orange for ATP binding and hydrolysis (orange), or purple for ClpP binding. The pore-2 loop is also involved in ClpP binding. (B) Structure of the N-domain dimer (1OVX) [16]. Spheres represent zinc atoms. (C) Structure of a AAA+ module in a single ClpX subunit (3HWS) [18]. Nucleotide binds in the cleft between the large and small AAA+ domains. Motif colors correspond to those in panel A.

Fig. 3

ClpX hexamer structures (3HWS). (A) Face view (substrate side) colored by subunits. Nucleotide loadable (L) and unloadable (U) subunits are marked. (B) Side view (substrate side up) colored by subunit. (C) Face view (substrate side) colored by rigid-body units. (D) Side view (substrate side up) colored by rigid-body units. (E) Close up of nucleotide-binding site and hinge between two rigid-body units. In each panel, the polypeptide backbone of the hinge region and ADP are shown in CPK representation, whereas most of the protein is shown in cartoon representation.

Fig. 4

C-terminal and N-terminal sequence tags that target substrates for ClpXP degradation [39]. Red dots – α-carboxyl group. Blue dots – α-amino group of mature protein.

Fig. 5

ClpX pore loops mediate binding to several degradation tags. (A) Cartoon of three types of pore loops. (B) Human ClpX containing the RKH and pore-2 loops from E. coli ClpX supports robust ClpP degradation of an ssrA-tagged substrate, but human ClpX or variants containing just the transplanted E. coli RKH loop or just the pore-2 loop do not [41]. (C) E. coli ClpXP degrades an ssrA-tagged protein with a 10-fold lower K_M than the same protein with a λO tag [44]. (D) AKH ClpXP degrades the λO-tagged substrate with a 30-fold lower K_M than the ssrA-tagged substrate [44]. Adapted with permission from ref. [44].

Fig. 6

SspB delivers ssrA-tagged substrates to ClpXP. (A) SspB (surface representation; 1OU8) binds to the AANDENY portion of the ssrA tag [37,52]. The C-terminal AA-COOH of the tag binds ClpX. (B) When SspB is disulfide bonded to the ssrA tag of a GFP substrate, both the adapter and substrate are degraded, requiring concurrent translocation of 3 polypeptides through the ClpX pore. Adapted with permission from ref. [58].

Fig. 7

Adaptor-mediated degradation. (A) A bacterial cell expresses a substrate with a weak ClpXP-degradation tag constitutively and expresses the SspB adaptor under conditional IPTG control. (B) Western blots show that the tagged substrate accumulates over time when SspB is not induced but is degraded rapidly upon induction of SspB synthesis. Adapted with permission from ref. [62].

Fig. 8

Structures of ClpP. (A) ClpP (1YG6) contains 14 subunits, arranged as two stacked heptameric rings [77,86]. Interactions between residues 125–146 (blue) in the handle region help stabilize the 14-mer. (B) The axial pore in free ClpP (1YG6) is very narrow, allowing entry of only small peptides into the internal proteolytic chamber. The pore dimensions are established by residues 1–21 (red), which form stem-loop structures. (C) Active site of a ClpP subunit (magenta; 2ZL2) with a bound peptide product (cyan) [81]. Residues of the Ser-His-Asp catalytic triad are labeled and the oxyanion hole is marked. The P1 side chain of the substrate (phenylalanine) sits in a hydrophobic cavity. (D) Acyldepsipeptide binding increases the size of the axial pore (3MT6), allowing degradation of unfolded proteins by ClpP alone [,–90]. (E) ClpP can exist in a more compact structure (3HLN) in which the active-site residues assume non-functional conformations are disordered and a portion of the handle region is disordered [91,94].

Fig. 9

Interaction of ClpX and ClpP. (A) The ClpXP complex is stabilized by peripheral interactions between the IGF loops of ClpX and hydrophobic clefts on ClpP, as well as by axial interactions between the pore-2 loops of ClpX and the N-terminal stem-loop of ClpP. Adapted with permission from ref. [24]. (B) Structure of an acyldepsipeptide (ADEP1; stick representation; 3MT6) bound in one of the ClpP clefts (surface representation) [89]. (C) Model of the ClpX IGF peptide binding in the ClpP cleft in a manner analogous to ADEP1.

Fig. 10

Singe-chain ClpX^ΔN hexamers [23]. (A) Two, three, or six ClpX^ΔN subunits can be connected by peptide linkers [23]. (B) SDS-PAGE of purified ClpX^ΔN proteins containing 1, 2, 3, or 6 linked subunits. (C) Unlinked ClpX^ΔN and linked proteins chromatograph at the position expected for hexamers or pseudo hexamers in gel-filtration chromatography. (D) ClpP-mediated degradation of an ssrA-tagged substrate by single-chain hexamers with different numbers of wild-type subunits (W) and/or subunits defective in ATP hydrolysis (E or R). Note that the RWE/RWE hexamer is far more active that the EWR/EWR isomer.

Fig. 11

Expansion and contraction of the axial pore by a snake-jaws model in which pore size is controlled by the size of the substrates and the conformation of the hinge region and flanking structure of the unloadable (U) ClpX subunits.

Fig. 12

Single-molecule unfolding and translocation of a multi-domain filamin substrate assayed by optical trapping nanometry [36]. Beads attached to substrate or ClpXP are trapped in laser beams. The distance between beads changes as ClpXP denatures or translocates a domain. In the trace shown in the center of the panel, horizontal movements to the right correspond to highly cooperative unfolding in single domains. Subsequent diagonal movements back to the left correspond to translocation. The inset shows that translocation occurs in steps of ~10 Å. In the dwell time between completion of translocation of one domain and unfolding of the next domain, the length of the substrate does not change. Adapted with permission from ref. [36].