Untangling the translocation mechanism of the protein machine ClpB
Remi Casier
Biological and Chemical Physics
Weizmann Institute of Science, Rehovot, Israel
Wednesday, December 4, 2024
10 a.m.
In-person: C2-361
Abstract: Hsp100 proteins are hexameric barrel shaped machines capable of reversing protein aggregation, making them vital for cell homeostasis and a target for therapeutic development battling neurodegenerative disorders. Utilizing the energy of ATP hydrolysis, these machines liberate misfolded proteins by active translocation through a large central pore. However, the mechanism utilized by Hsp100 proteins, such as ClpB, to convert hydrolysis into translocation activity, including the timescale at which this occurs, remains uncertain. While structural studies suggest translocation is driven by slow sequential motions of subunits directly coupled to ATP hydrolysis, other functional studies suggest translocation occurs much more quickly. To address this inconsistency, we use single-molecule FRET (smFRET) spectroscopy to directly observe the translocation of the model protein, casein, by individual molecules of ClpB. The real-time monitoring of the efficiency of energy transfer between fluorescently tagged casein and ClpB revealed that, in the presence of ATP, casein is fully translocated through the lumen of ClpB in only a few milliseconds. These fast translocation events disappeared in the presence of the slowly hydrolysable analog ATPγS, demonstrating their ATP dependence. Additionally, we find that complete translocation occurs in a bi-directional manner starting from both the N- and C-termini of ClpB, along with the existence of partial threading events. Together, these features point towards a complex mode of action of ClpB where translocation itself is diffusive in nature and is commensurate with a Brownian ratchet mechanism.