Conformational plasticity of the ClpAP AAA+ protease couples protein unfolding and proteolysis

Abstract

The ClpAP complex functions as a "bacterial proteasome" that simultaneously unfolds and degrades proteins targeted for destruction. ClpA utilizes two AAA+ domains per protomer to power substrate unfolding and translocation into the ClpP proteolytic chamber. To understand this mechanism, we determined high-resolution structures of wildtype E. coli ClpAP in distinct substrate-bound states.ClpA forms a spiral with substrate contacts across both AAA+ domains, while protomers at the seam undergo nucleotide-specific rearrangements indicating a conserved rotary mechanism. ClpA IGL loops extend flexibly to bind the planar, heptameric ClpP surface and support a large ClpA-P rotation that reorients the translocation channel. The symmetry mismatch is maintained at the spiral seam through bind and release states of the IGL loops, which appear precisely coupled to substrate translocation. Thus, ClpA rotates around the apical surface of ClpP to processively translocate substrate into the protease. MainThe Hsp100 (Clp) family of proteins, widely present in bacteria and eukaryotes, function as protein unfoldases and disaggregases 1,2 . Some family members can assemble into large proteolytic machines homologous to the 26S proteasome and serve critical roles in targeted protein degradation and quality control [3][4][5][6][7] . Proteolysis requires substrate recognition and ATP hydrolysis-driven unfolding by a AAA+ Hsp100 complex, which unfolds and translocates the substrate into a proteolytic chamber 8-12 . The highly conserved serine protease, ClpP forms this chamber as a double ring of heptamers 13,14 which partner with 1-2 ClpX or ClpA AAA+ hexamers in bacteria, assembling into single and double-capped complexes [15][16][17] . To promote client degradation, ClpXP and ClpAP are aided by SspB 18,19 and ClpS 20,21 , specificity adaptors that promote recognition of substrates including those containing the ssrA degron 22,23 and N-end rule substrates 24 , respectively. Other substrates, such as the RepA DNA-binding protein, recognized by ClpA, are remodeled or degraded in support of specific cellular functions 3,25 ..

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

“…Moreover, in approximately half of the crosslinked enzymes, it would not be possible to have two empty ClpP clefts. Hence, the proposal that this intermediate is a requisite step in translocation (Lopez et al, 2020) is inconsistent with our results. We conclude that rotation of the ClpA or ClpX rings with respect to the ClpP ring is not required for degradation.…”

“…The sequential hand-over-hand mechanism posits that each subunit in a AAA+ hexamer cycles through each spiral position during processive translocation (Puchades et al, 2020). Further, in ClpAP cryo-EM structures, a single empty ClpP cleft is always located between the clefts occupied by the IGL loops of the lowest and second-lowest subunits (S6 and S5 in Figure 1A); when a second empty cleft is observed, it results from disengagement of the IGL loop of the S5 subunit (Lopez et al, 2020). Very similar structural results have been observed for ClpXP complexes (Fei et al, 2020;Ripstein et al, 2020) If ClpA or ClpX subunits cycle through each spiral position, then the empty ClpP cleft would rotate relative to the unfoldase ( Figure 4A-B).…”

“…The penultimate C-terminal residue of ClpP forms part of the binding cleft for the IGL loops of ClpA and appears close enough to allow chemical crosslinking between the IGL loops of ClpA and the C-terminal region of ClpP (Figure 2A) (Lopez et al, 2020). We introduced an E613C mutation into the IGL loop of a cysteine-free ClpA ‡ variant (C47S, C203S, C243S) to generate E613C ClpA ‡ .…”

“…Recent cryo-EM structures of ClpAP and ClpXP reveal six flexible loops protruding from the bottom face of the ClpPproximal AAA+ ring. Each loop contains a conserved tripeptide motif (IGL in ClpA; IGF in ClpX) that docks into five or six of the seven hydrophobic binding clefts on the "top" of the ClpP ring (Fei et al, 2020;Ripstein et al, 2020;Lopez et al, 2020). Intriguingly, the unoccupied ClpP clefts are always located between two specifically positioned subunits in the ClpA or ClpX hexamers ( Figure 1A).…”

“…Intriguingly, the unoccupied ClpP clefts in ClpAP and ClpXP are always located between the IGL/IGF loops of the second lowest and lowest subunits within the spiral (S5 and S6 in Figure 1A). If subunits in the ClpA or ClpX hexamers pass sequentially through each position in the spiral and the empty clefts in ClpP are always between two specific subunits, then the AAA+ ring should rotate with respect to the ClpP ring during protein translocation ( Figure 1B; Ripstein et al, 2020;Lopez et al, 2020).…”