Millisecond bidirectional protein translocation by a HSP100 machine

Hsp100 proteins, which belong to the AAA+ family, are abundant chaperones with a hexameric ring-like functional form that use the energy of ATP hydrolysis to translocate proteins through their lumen for quality control activities and for transportation between cellular compartments. A paradigmatic Hsp100 protein is ClpB, which rescues proteins from aggregation in bacteria. The mechanism of protein translocation by this machine and similar ones is under fierce debate. Recent structural work has proposed that ClpB and other Hsp100 proteins can form a spiral-like structure, suggesting the so-called ‘hand-over-hand’ translocation mechanism that involves sequential motion of subunits driven by ATP hydrolysis. However, this structure-based model does not agree with the results of functional studies, which demonstrate activity with only three active subunits, translocation that is much faster than ATP hydrolysis, and partial threading of looped polypeptide segments. To clarify these discrepancies, we directly study the translocation of an unstructured protein, κ-casein, by individual molecules of ClpB. Using a single-molecule FRET assay with a dye on the protein substrate and either one or two dyes on the ClpB machine, we demonstrate that in the presence of ATP, translocation events take just a few milliseconds. This ultrafast activity disappears when the slowly hydrolysable analog ATPγS replaces ATP. Our studies also indicate that complete substrate threading occurs only ∼40% of the time, and demonstrates partial substrate engagement events, as well as ‘backward threading’ starting from the C -terminus of the chaperone. Our studies indicate a fast stochastic mode of action of ClpB that is akin to a Brownian motor.