Open and Closed States of the AAA+ Protease Lon Provide the Structural Basis for Distinct Operational Modes

Abstract

Substrate-bound structures of AAA+ protein translocases support sequential ATP hydrolysis cycle as the main driver for hand-over-hand substrate translocation. However, the operating principles of these enzymes remain controversial, as biochemical studies are not all consistent with this strictly sequential model. We determined the structures of Y. pestis Lon AAA+ protease in the absence and presence of substrate using cryo-electron microscopy, which reveal the structural basis for two distinct operational modes. In the absence of substrate, Lon adopts a left-handed, “open” spiral organization whose subunits appear bound to ADP. Upon the addition of substrate, Lon undergoes a substantial reorganization to assemble the right-handed “closed” spiral staircase that is similar to those observed for many other substrate-bound AAA+ enzymes. While this closed conformer confirms conservation of hand-over-hand translocation in this essential protein quality control enzyme, our structure reveals a distinct mechanism for nucleotide-dependent allosteric communication between subunits. Further, the mechanisms involved in transitioning between these two states reconcile sequential and stochastic models for AAA+ activity. We define the mechanistic principles encompassing substrate engagement, translocation, and release, and provide a structural basis for the operational plasticity required to process protein substrates in distinct fold states.