Quantitative Insights into Processivity of an Hsp100 Protein Disaggregase on Folded Protein Substrates

The Hsp100 family of proteins play important roles in maintaining protein homeostasis in cells. E. coli ClpB is an Hsp100 protein that remodels misfolded proteins or aggregates. ClpB is proposed to couple the energy from ATP binding and hydrolysis to processively unfold and translocate protein substrates through its axial channel in the hexameric ring structure. However, many of the details of this reaction remain obscure. We have recently developed a transient state kinetics approach to study ClpB catalyzed protein unfolding and translocation. In this work we have used this approach to begin to examine how ATP is coupled to the protein unfolding reaction. Here we show that at saturating [ATP], ClpB induces the cooperative unfolding of a complete TitinI27 domain of 98 amino acids, which is represented by the kinetic step-size m ∼100 amino acids. This unfolding event is followed by rapid and undetected translocation up to the next folded domain. At sub-saturating [ATP], ClpB still induces cooperative unfolding of a complete TitinI27 domain but translocation becomes partially rate-limiting, which leads to an apparent reduced kinetic step-size as small as ∼ 50 amino acids. Further, we show that ClpB exhibits an unfolding processivity of P = (0.74 ± 0.06) independent of [ATP]. These findings advance our understanding of the elementary reactions catalyzed by E. coli ClpB but are broadly applicable to a variety of Hsp100 family members.