Mechanisms of Substrate Degradation by Energy-Dependent Proteases

Abstract

Energy-dependent proteases of the AAA+ family catalyze the highly specific protein degradation involved in cellular protein quality control and the regulation of numerous vital processes, yet the detailed mechanisms coupling ATP hydrolysis with mechanical substrate unfolding and translocation remain poorly understood.Our cryo-EM structural studies of the eukaryotic 26S proteasome show that its heterohexameric AAA+ ATPase ring adopts a conformation with pronounced spiral-staircase arrangement of subunits in the absence of substrate, but transitions into a translocation-competent conformation upon substrate engagement. This substrate-engaged ring conformation is characterized by uniform interfaces between the six ATPase subunit, a widened central channel coaxially aligned with the peptidase, and a rearranged, more planar spiral orientation of ATPase subunits that suggests a highly coordinated rapid progression of ATP-hydrolysis events around the ring.This coordinated ATP hydrolysis mechanism is further supported by our optical tweezers single-molecule studies of the related bacterial protease ClpXP and may be a general feature of AAA+ translocases. ClpXP translocates substrate polypeptides in steps with constant frequency but variable length, depending on the number of ATP-hydrolyzing subunits.