Quantitative Insights into Processivity of an Hsp100 Protein Disaggregase on Folded Proteins.

Abstract

The Hsp100 family of protein disaggregases play important roles in maintaining protein homeostasis in cells. E. coli ClpB is an Hsp100 protein that solubilizes protein 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 the work reported here we have used the approach 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 our measured 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 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 ATP coupling to enzyme catalyzed protein unfolding by E. coli ClpB and present a strategy that is broadly applicable to a variety of Hsp100 family members and AAA+ superfamily members.