Multistep substrate binding and engagement by the AAA+ ClpXP protease

E. coli ClpXP is one of the most thoroughly studied AAA+ proteases, but relatively little is known about the reactions that allow it to bind and then engage specific protein substrates before the ATP-fueled mechanical unfolding and translocation steps that lead to processive degradation. Here, we employ a fluorescence-quenching assay to study the binding of ssrA-tagged substrates to ClpXP. Polyphasic stopped-flow association and dissociation kinetics support the existence of at least three distinct substrate-bound ClpXP complexes. These kinetic data fit well to a model in which ClpXP and substrate form an initial binding complex, followed by an intermediate complex, and then an engaged complex that is competent for substrate unfolding. The initial association and dissociation steps do not require ATP hydrolysis, but subsequent forward and reverse kinetic steps are accelerated by faster ATP hydrolysis. Our results, together with recent cryo-EM structures of ClpXP bound to substrates, support a model in which the ssrA degron initially binds in the top portion of the axial channel of the ClpX hexamer and then is translocated deeper into the channel in steps that eventually pull the native portion of the substrate against the channel opening. Reversible initial substrate binding allows ClpXP to check potential substrates for degrons, potentially increasing specificity. Subsequent substrateengagement steps allow ClpXP to grip a wide variety of sequences to ensure efficient unfolding and translocation of almost any native substrate. SignificanceAAA+ proteases play key regulatory and quality-control roles in all domains of life. These destructive enzymes recognize damaged, unneeded, or regulatory proteins via specific degrons and unfold them prior to processive degradation. Here, we show that E. coli ClpXP, a model AAA+ protease, recognizes ssrA-tagged substrates in a multistep binding and engagement reaction. Together with recent cryo-EM structures, our experiments reveal how ClpXP transitions from a machine that checks potential substrates for appropriate degrons to one that can unfold and translocate almost any protein. Other AAA+ proteases in organelles and bacteria are likely to use similar mechanisms to specifically identify and then destroy their target proteins.