Caseinolytic Proteases (Clp) use ATPase components such as the ClpY to mediate protein quality control within bacterial cells. The ring-shaped ATPase exerts repetitive mechanical forces onto substrate proteins (SPs) to unfold and translocate them through a narrow central pore. These nanomachines are able to degrade SPs with divers topology and local mechanical resistance near the terminus that experience the mechanical force. In this study, we use probe remodeling of the haloalkane dehydrogenase (HaloTag), which has a  helical structure in the C-terminal region, and wild-type (wt) dihydrofolate reductase (DHFR) and its variants obtained through cyclical permutation (CP Pro-25 and CP Lys-38), which have diverse terminal topologies. We perform Langevin dynamics simulations using an atomistic description of SPs in an unrestrained geometry that mimics in vivo force application by the ClpY central channel loops and a restrained geometry that mimics single-molecule force spectroscopy experiments through an additional opposing force applied onto the N-terminal. We find that in the unrestrained geometry the HaloTag core forms an unfolding intermediate with high mechanical resistance. By contrast, in the restrained geometry, alignment of the SP in the N-C direction facilitates SP reorientation which favors the degradation of this intermediate. Mechanisms of unfolding of wt DHFR, CP Lys-38 and CP Pro-25 in the unrestrained geometry are consistent with the mechanical resistance of their local structure near the pulled N-terminal and the direction of force application.

Computer simulations of direction-dependent mechanisms of substrate protein remodeling mediated by Clp biological nanomachines

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.