Coarse-Grained Simulations of Topology-Dependent Mechanisms of Protein Unfolding and Translocation Mediated by ClpY ATPase Nanomachines

Clp ATPases are powerful ring shaped nanomachines which participate in the degradation pathway of the protein quality control system, coupling the energy from ATP hydrolysis to threading substrate proteins (SP) through their narrow central pore. Repetitive cycles of sequential intra-ring ATP hydrolysis events induce axial excursions of diaphragm-forming central pore loops that effect the application of mechanical forces onto SPs to promote unfolding and translocation. We perform Langevin dynamics simulations of a coarse-grained model of the ClpY ATPase-SP system to elucidate the molecular details of unfolding and translocation of an α/β model protein. We contrast this mechanism with our previous studies which used an all-α SP. We find conserved aspects of unfolding and translocation mechanisms by allosteric ClpY, including unfolding initiated at the tagged C-terminus and translocation via a power stroke mechanism. Topology-specific aspects include the time scales, the rate limiting steps in the degradation pathway, the effect of force directionality, and the translocase efficacy. Mechanisms of ClpY-assisted unfolding and translocation are distinct from those resulting from non-allosteric mechanical pulling. Bulk unfolding simulations, which mimic Atomic Force Microscopy-type pulling, reveal multiple unfolding pathways initiated at the C-terminus, N-terminus, or simultaneously from both termini. In a non-allosteric ClpY ATPase pore, mechanical pulling with constant velocity yields larger effective forces for SP unfolding, while pulling with constant force results in simultaneous unfolding and translocation.Author Summary: Cell survival is critically dependent on tightly regulated protein quality control, which includes chaperone-mediated folding and degradation. In the degradation pathway, AAA+ nanomachines, such as bacterial Clp proteases, use ATP-driven mechanisms to mechanically unfold, translocate, and destroy excess or defective proteins. Understanding these remodeling mechanisms is of central importance for deciphering the details of essential cellular processes. We perform coarse-grained computer simulations to extensively probe the effect of substrate protein topology on unfolding and translocation actions of the ClpY ATPase nanomachine. We find that, independent of SP topology, unfolding proceeds from the tagged C-terminus, which is engaged by the ATPase, and translocation involves coordinated steps. Topology-specific aspects include more complex unfolding and translocation pathways of the α/β SP compared with the all-α SP due to high stability of β-hairpins and interplay of tertiary contacts. In addition, directionality of the mechanical force applied by the Clp ATPase gives rise to distinct unfolding pathways.