ClpA- and ClpAP-catalyzed unfolding and translocation are differentially coupled to ATP binding.

Proteome maintenance is underpinned by molecular motors from the AAA+ superfamily. E. coli ClpA is a representative AAA+ motor that associates with the tetradecameric serine protease ClpP forming the ATP-dependent protease, ClpAP. ClpA unfolds substrates targeted for degradation and translocates them into the central channel of ClpP where the substrate is degraded. However, when ClpA is not associated with ClpP, the motor uses its unfolding activity to noncovalently remodel protein substrates. Although a large body of work exists on the mechanisms of ClpAP-catalyzed protein unfolding and degradation, much less is known about the mechanisms of protein remodeling reactions. In fact, there is a dearth of mechanistic information to complement the emerging static structural information on many AAA+ family members that remodel proteins without covalent modification. Here, we report results from single-turnover stopped-flow experiments to interrogate the ClpA-catalyzed mechanisms of protein unfolding, both alone and when associated with ClpP. To this end, we used substrates containing tandem repeats of the Titin I27 domain. We show that both ClpA and ClpAP catalyze cooperative protein unfolding of the Titin I27 domain in a single kinetic step. This cooperative unfolding is followed by repeated rounds of translocation on the newly unfolded polypeptide. At saturating [ATP], ClpA and ClpAP catalyze protein unfolding and translocation at (12.0 ± 0.4) aa s