The non-dominant AAA+ ring in the ClpAP protease functions as an anti-stalling motor to accelerate protein unfolding and translocation

ATP-powered unfoldases containing D1 and D2 AAA+ rings play important roles in protein homeostasis, but uncertainty about the function of each ring remains. Here we use single-molecule optical-tweezers to assay mechanical unfolding and translocation by a variant of the ClpAP protease containing an ATPase-inactive D1 ring. This variant displays substantial mechanical defects both in unfolding and translocation of protein substrates. Notably, when D1 is hydrolytically inactive, ClpAP often stalls for times as long as minutes, and the substrate can \"back-slip\" through the enzyme when ATP concentrations are low. The inactive D1 variant also has substantially more difficulty traveling in the N-to-C direction on a polypeptide track than moving C-to-N. These results indicate that D1 normally functions as an auxiliary/regulatory motor to promote uninterrupted enzyme advancement that is fueled largely by the D2 ring.