Modular and coordinated activity of AAA+ active sites in the double-ring ClpA unfoldase of the ClpAP protease

Significance Understanding of how ClpA and other double-ring AAA+ enzymes perform mechanical work is limited. Using site-specific cross-linking and mutagenesis, we introduced ATPase-inactive AAA+ modules at alternating positions in individual ClpA rings, or in both rings, to investigate potential active-site coordination during ClpAP degradation. ClpA variants containing alternating active/inactive ATPase modules processively unfolded, translocated, and supported ClpP degradation of protein substrates with energetic efficiencies similar to, or higher than, completely active ClpA. These results impact current models describing the mechanisms of AAA+ family enzymes. The cross-linking/mutagenesis method we employed will also be useful for answering other structure-function questions about ClpA and related double-ring enzymes.
ClpA is a hexameric double-ring AAA+ unfoldase/translocase that functions with the ClpP peptidase to degrade proteins that are damaged or unneeded. How the 12 ATPase active sites of ClpA, 6 in the D1 ring and 6 in the D2 ring, work together to fuel ATP-dependent degradation is not understood. We use site-specific cross-linking to engineer ClpA hexamers with alternating ATPase-active and ATPase-inactive modules in the D1 ring, the D2 ring, or both rings to determine if these active sites function together. Our results demonstrate that D2 modules coordinate with D1 modules and ClpP during mechanical work. However, there is no requirement for adjacent modules in either ring to be active for efficient enzyme function. Notably, ClpAP variants with just three alternating active D2 modules are robust protein translocases and function with double the energetic efficiency of ClpAP variants with completely active D2 rings. Although D2 is the more powerful motor, three or six active D1 modules are important for high enzyme processivity, which depends on D1 and D2 acting coordinately. These results challenge sequential models of ATP hydrolysis and coupled mechanical work by ClpAP and provide an engineering strategy that will be useful in testing other aspects of ClpAP mechanism.