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

Abstract

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

Multistep substrate binding and engagement by the AAA+ ClpXP protease

“…A highly specific AAA+ protease might be expected to bind tightly to a limited set of sequences, potentially limiting processivity, whereas a highly processive protease that can grip most sequences for translocation might be expected to have limited specificity. As outlined below, the kinetic studies presented here together with recent cryo-EM structures of substrate-bound ClpXP (28)(29)(30) suggest a mechanism by which ClpXP achieves specificity and processivity.…”

“…Positive cooperativity in the ATP activation of substrate binding to a ClpX mutant defective in ATP hydrolysis was reported previously (23), and likely reflects a requirement that multiple subunits in the ClpX hexamer must bind ATP to stabilize the substrate-binding conformation. In accord with this model, four or five subunits of the ClpX hexamer are ATP or ATPγS bound in substrate-bound cryo-EM structures (28)(29)(30). The finding that ATPγS supports substrate binding at lower concentrations and with a lower Hill constant than ATP suggests that faster hydrolysis of ATP at sub-saturating concentrations results in a lower population of nucleotide-bound intermediates that can bind substrate.…”

“…These results suggest that the rate of ATP/ATPγS hydrolysis is an important factor in determining the rates of the associated conformational transitions. Under the conditions of our experiments, the maximum steady-state rate of ATP hydrolysis by ClpXP is ~3.6 s -1 (28). The fitted values of the k 2 , k -2 , k 3 , and k -3 rate constants in experiments performed using ATP were all smaller than this hydrolysis rate (Table 1) Specific degron recognition by ClpXP and related AAA+ proteases is necessary to avoid uncontrolled protein destruction, as later steps including substrate unfolding and processive translocation through the axial channel have little specificity (18-19; 33).…”

“…ATP or ATPγS must bind to the AAA+ ring of ClpX to support binding to the ssrA tag, but hydrolysis of these nucleotides is not required for ssrA-tag recognition (15,(20)(21)(22)(23)(24). During degradation of multidomain substrates, ClpXP often releases a partially degraded product when a very stable native domain is encountered, and single-domain substrates are also likely to be released multiple times prior to degradation (16,19,(25)(26)(27)(28). Single-chain ClpX was labeled at residue 170 of one subunit with a fluorescent TAMRA dye, and a cysteine between native titin and the ssrA tag was labeled with a black-hole quencher.…”

“…This model suggests that ClpXP initially checks potential substrates for appropriate degrons and then transitions to a machine that can unfold, translocate, and degrade a wide range of proteins. We discuss these studies in light of recent cryo-EM structures of ClpXP bound to protein substrates (28)(29)(30).…”