Proteome maintenance is underpinned by molecular motors from the AAA+ superfamily. E. coli ClpA is an AAA+ motor that assembles into hexameric rings upon nucleotide binding. Hexameric ClpA associates with the tetradecameric protease ClpP, forming the ATP-dependent protease, ClpAP. ClpA binds proteins targeted for degradation and couples ATP binding and hydrolysis to protein unfolding and translocation. ClpA translocates the newly unfolded polypeptide chain into the central chamber of ClpP, where the substrate encounters 14 serine protease active sites. However, when not associated with ClpP, ClpA uses its protein unfolding activity to noncovalently remodel proteins. We previously reported that ClpA and ClpAP exhibited different translocation mechanisms on unstructured substrates that report only on translocation and not protein unfolding. Here, we report results from single-turnover stopped-flow experiments to interrogate the ClpA catalyzed mechanism of protein unfolding. To this end, we use substrates containing tandem repeats of the Titin I27 domain. We show that both ClpA and ClpAP catalyze unfolding of the Titin I27 domain cooperatively in a single kinetic step. This cooperative unfolding step 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 -1 and (40 ± 1) aa s -1 , respectively. The complete ATP concentration dependence of the reaction indicates that ATP is differentially coupled to unfolding compared to translocation. These results reveal that ClpP not only allosterically impacts the translocation mechanism but also exerts its control over the reactions catalyzed by ClpA. More broadly, we advance the quantitative framework for deconvoluting the coupling of ATP to unfolding versus translocation, an approach that can be applied to a vast array of AAA+ motors that catalyze protein remodeling reactions.
Islam et al. (Sun,) studied this question.