Coarse grained molecular dynamics simulations of the coupling between the allosteric mechanism of the ClpY nanomachine and threading of a substrate protein

Protein quality control is critical in maintaining cell viability. Recognitionand degradation of aberrant proteins in the cellular environment is essential,as many neurodegenerative diseases are linked to protein misfolding andaggregation. Powerful AAA+ ATPases, such as ClpY and p97, play avital role in the degradation pathway of PQC. These nanomachines form hexameric macromolecularassemblies which selectively recognize and thread proteins tagged fordegradation through their narrow central pore to be delivered to sequesteredprotease components for degradation. Flexible loops with aconserved G-hydrophobic-aromatic-G sequence reside within the central pore andundergo a large scale paddling motion resulting from ATPhydrolysis. The loops impart mechanical forces onto substrateproteins (SP) which results in unfolding and translocation. While the overallmechanism of these macromolecular machines isgenerally understood, molecular details of SP processing have not been established. The focus of this study is on the molecular details of SPprocessing by the single ring ClpY ATPase, whichhas been well characterized crystallographically in multiple nucleotide boundstates. Here, I am presenting studies based on four aspects regarding this problem : (1) Unfolding and translocation pathway of substrate protein controlled by structure in repetitive allostericcycles of the ClpY ATPase, (2) Asymmetric processing of a substrate protein insequential allosteric cycles of the ClpY nanomachine, (3) Thedependence of SP topology on unfolding and translocation mechanisms by theClpY nanomachine, and (4) Structure mediated unfolding and translocationpathways of SulA by pulling through non-allosteric AAA+ pores. I used coarse-grained Langevindynamics simulations to probe the unfolding and translocation of a four-helix bundlemodel SP using the allosteric transitions of ClpY. My results indicate thatunfolding occurs by unraveling from the SP's tagged C-terminus, resulting in athree helix bundle. This minimally unfolded, obligatory unfolding intermediate,is competent for translocation. Translocation occurs through sharpstepped transitions indicating a powerstroke mechanism. To further understandthe coupling between intra-ring motions of ClpY and mechanical action applied tothe SP, I performed additional simulations of clockwise,counterclockwise and random allosteric mechanisms. Myresults indicate that the SP unfolding and translocation mechanism is independent of thedirectional allosteric mechanism. The work provided by single SP-loopinteractions allows the SP to sample identical conformational states; However,the rates and yields are affected, with clockwise allostery emerging as the mostefficacious due to the favorable torque applied onto the SP. Next, I have probedthe dependence of SP topology by examining ClpY processing of a SP withalpha/beta topology. Comparison with the all-alpha SP indicate a wellpreserved unfolding event by unraveling at the C-terminus and translocationvia a powerstroke mechanism. The effective forces for unfolding andthe kinetics depend on topology. Last, I have probed theunfolding and translocation of a natural SP of ClpY, SulA, by performingminimalistic simulations pulling through non-allosteric AAA+ pores. The results indicatethat initial unfolding near the C-terminus is required for initiation oftranslocation. Interactions with the surface of the pore facilitate unfoldingand translocation by decreasing the energy barriers.