Role of Diffusion in Unfolding and Translocation of Multidomain Titin I27 Substrates by a Clp ATPase Nanomachine.

Degradation of multidomain substrate proteins (SPs) by AAA+ nanomachines takes place processively from the tagged terminal. Ring-shaped ATPase components, such as ClpY, apply repetitive mechanical forces to effect domain unfolding and translocation of polypeptide segments through a narrow central channel. We study these mechanisms through atomistic Langevin dynamics simulations of C-terminal-tagged SPs in allosteric ClpY cycles. We find that monomeric SPs are processed through single unfolding pathways and fast timescales, whereas multimeric SPs involve branched pathways and slower timescales. These distinct mechanisms are attributed to the slower rotational diffusion of the C-terminal domain in multidomain SPs that hinders access to the soft mechanical direction. In the geometry specific to laser optical tweezers experiments, involving a restrained SP N-terminal, a single unfolding pathway is found for both monomeric and tetrameric SPs as pulling is applied along the N-C direction. Non-native interactions modulate unfolding of unrestrained monomers by weakening the C-terminal interface but do not contribute significantly to unfolding of restrained SPs. On the basis of these results, we propose that the interplay of restricted SP dynamics and ATPase kinetics underlies partitioning of multidomain SPs into completely degraded products and undegraded fragments comprising folded domains.