Role of Pore Loops in the Mechanism of Polypeptide Translocation by a AAA+ Protease Machine

Abstract

Proteases of the AAA+ family play crucial roles maintaining protein homeostasis in the cell by degrading misfolded/damaged proteins. ClpX is a homohexameric ring-shaped member of this family, which uses ATP to mechanically unfold native substrates and translocate them into the associated proteolytic chamber of ClpP. Polypeptide translocation is impelled by pore loops that protrude from every subunit into the lumen of ClpX pore, which contact the polypeptide through a highly-conserved motif: GYVG. We used single-molecule assays with optical tweezers to study the effect on the mechanochemistry of the motor of Y153A and V154F mutations, which decrease/increase the size of these loops. 1-3 ClpX WT subunits were substituted by either a Y153A or V154F mutant subunit, arranged in different positions relative to each other within the ClpX ring. Since the translocation cycle has two phases, a dwell and a burst, we analyzed the effect of these mutations on these phases. Y153A mutations decreased the dwell duration compared to WT, increasing translocation velocity, while V154F mutants had longer dwells, reducing translocation velocity. Interestingly, the burst-size remained unaffected. ATP-turnover analysis revealed that in Y153A mutants increased dramatically, while in V154F it was a smaller increase. We calculated the coupling coefficient (CC), the number of ATP hydrolysis cycles that are required for a productive power stroke. For WT the CC is one, Y153A mutants have a CC of two, while in V154F mutants is between one and two. Finally, we observed that mutant subunits either located across the ClpX ring or intercalated by a WT subunit had more effect in the duration of the dwell and in GFP unfolding capability. These results provide missing information about the spatial arrangement and inter-subunit coordination of pore loops during polypeptide translocation.