Local and global mobility in the ClpA AAA+ chaperone detected by cryo-electron microscopy: functional connotations.

The ClpA chaperone combines with the ClpP peptidase to perform targeted proteolysis in the bacterial cytoplasm. ClpA monomer has an N-terminal substrate-binding domain and two AAA+ ATPase domains (D1 and D2). ClpA hexamers stack axially on ClpP heptamers to form the symmetry-mismatched protease. We used cryo-electron microscopy to visualize the ClpA-ATPgammaS hexamer, in the context of ClpAP complexes. Two segments lining the axial channel show anomalously low density, indicating that these motifs, which have been implicated in substrate translocation, are mobile. We infer that ATP hydrolysis is accompanied by substantial structural changes in the D2 but not the D1 tier. The entire N domain is rendered invisible by large-scale fluctuations. When deletions of 10 and 15 residues were introduced into the linker, N domain mobility was reduced but not eliminated and changes were observed in enzymatic activities. Based on these observations, we present a pseudo-atomic model of ClpAP holoenzyme, a dynamic proteolytic nanomachine.