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 ..

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AbstractThe 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 re-orients 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.

Heat shock protein (Hsp) 104 is a hexameric ATPases associated with diverse cellular activities motor protein that enables cells to survive extreme stress. Hsp104 couples the energy of ATP binding and hydrolysis to solubilize proteins trapped in aggregated structures. The mechanism by which Hsp104 disaggregates proteins is not completely understood but may require Hsp104 to partially or completely translocate polypeptides across its central channel. Here, we apply transient state, single turnover kinetics to investigate the ATP-dependent translocation of soluble polypeptides by Hsp104 and Hsp104 A503S , a potentiated variant developed to resolve misfolded conformers implicated in neurodegenerative disease. We establish that Hsp104 and Hsp104 A503S can operate as nonprocessive translocases for soluble substrates, indicating a ''partial threading'' model of translocation. Remarkably, Hsp104 A503S exhibits altered coupling of ATP binding to translocation and decelerated dissociation from polypeptide substrate compared to Hsp104. This altered coupling and prolonged substrate interaction likely increases entropic pulling forces, thereby enabling more effective aggregate dissolution by Hsp104 A503S .

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