Multistep substrate binding and engagement by the AAA+ ClpXP protease

Abstract

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. 3 Significance AAA+ 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.

Structural basis of ClpXP recognition and unfolding of ssrA-tagged substrates

“…Structures, by themselves, can suggest but do not establish order in a kinetic pathway. However, a recent study of the kinetics of ClpXP association with a substrate bearing a 20-residue ssrA degron similar to the one studied here provides evidence for three sequentially occupied substrate-bound conformations ( Saunders et al, 2020 ). The first two are likely to correspond to our recognition and intermediate complexes.…”

“…The GFP portion of this substrate consisted of residues 1–229, which includes only the C-terminal amino acids observed in a crystal structure ( Ormö et al, 1996 ). An ATPγS/ATP mixture was used to allow rapid substrate binding, while slowing translocation and unfolding ( Burton et al, 2003 ; Martin et al, 2008a ; Saunders et al, 2020 ). Samples were blotted and vitrified within 15 s of final mixing.…”

Structural basis of ClpXP recognition and unfolding of ssrA-tagged substrates

“…The GFP portion of this substrate consisted of residues 1-229, which includes only the C-terminal amino acids observed in a crystal structure (Ormö et al, 1996). An ATPγS/ATP mixture was used to allow rapid substrate binding, while slowing translocation and unfolding (Burton et al, 2003;Martin et al 2008b;Saunders et al, 2020). Samples were blotted and vitrified within 15 s of final mixing.…”

The AAA+ superfamily: a review of the structural and mechanistic principles of these molecular machines

“…Two HslU hexamers sandwich two HsIV hexamers to form a cylindrical hetero-24mer (Bochtler et al 2000). ClpX forms a hexamer as well (Glynn et al 2009) and associates with its conjugate protease ClpP (Wang et al 1997) to form the ClpXP protease (Gatsogiannis et al 2019;Fei et al 2020;Ripstein et al 2020), which recognizes and degrades proteins that have a degradation signal in a multistep binding and engagement process (Saunders et al 2020). Some of these degradation signals are added to the C-terminus of an incomplete protein from a stalled ribosome, which directs the incomplete protein to ClpXP for degradation (Flynn et al 2001).…”

“…This eukaryotic protein family is a key regulatory in spindle assembly. TRIP13, with its adaptor protein p31 comet , loads substrate protein MAD2 in a spiral conformation (Alfieri et al 2018) similar to other AAAþ proteins (Zhao et al 2015;Puchades et al 2017;White et al 2018;Fei et al 2020;Ripstein et al 2020;Saunders et al 2020).…”