Distinct Structural Features of the Lon Protease Drive Conserved Hand-over-Hand Substrate Translocation

Hand-over-hand translocation is emerging as the conserved mechanism by which ATP hydrolysis drives substrate translocation within the classical clade of AAA+ proteins. However, the operating principles of the distantly related HCLR clade, which includes the important quality control protease Lon, remains poorly defined. We determined a cryo-electron microscopy structure of Y. pestis Lon trapped in the act of processing substrate. This structure revealed that sequential ATP hydrolysis and hand-over-hand substrate translocation are conserved in this AAA+ protease. However, Lon processes substrates through a distinct molecular mechanism involving structural features unique to the HCLR clade. Our findings define a previously unobserved translocation mechanism that is likely conserved across HCLR proteins and reveal how fundamentally distinct structural configurations of distantly-related AAA+ enzymes can power hand-over-hand substrate translocation.