Protein remodeling and translocation mediated by AAA+ nanomachines in the degradation and disaggregation pathways: computational studies

Protein degradation and disaggregation are essential quality control mechanisms that protect against cellular stresses. AAA+ nanomachines, such as the hexameric ring-shaped Clp (Caseinolytic protease) ATPases or the 26S eukaryotic proteasome, mediate these mechanisms by unfolding and translocating substrate proteins (SPs) through a narrow central channel. The primary remodeling action involves applying repetitive mechanical forces onto the substrate proteins through a set of ATPase loops that protrude into the channel. The fate of the substrate protein is largely dependent not on its global stability, but on the local mechanical strength near the pulled terminal, and on the direction of force application. To probe the effect of SP structure and force directionality, we used coarse-grained and atomistic, implicit solvent, modeling of diverse substrates, such as the I27 domain of the muscle protein titin, dihydrofolate reductase, green fluorescent protein (GFP), and knotted proteins.

We find that Clp surface plasticity modulates direction-dependent pulling mechanisms by favoring specific SP orientations. This action is complemented by the crowding effect of multiple SP domains, which yields slower rotational diffusion of the multidomain SP compared with monomeric domains in allosteric cycles of the ClpY ATPase. Our atomistic simulations of Clp-mediated degradation of knotted proteins reveal dependence of unknotting and translocation on tension propagation, sequence direction, non-native contacts and intermediates with strong local mechanical resistance. In coarse-grained models of protein degradation mediated by the 26S proteasome, we use machine learning approaches to characterize dynamic competition between GFP refolding and translocation in sequence direction-dependent (N-C and C-N) mechanisms. Simulations of the ClpB disaggregase, using an atomistic, explicit solvent description, reveal the networks of inter- and intraprotomer interactions that underlie dynamic stability of the ring structure. Relaxation times of the pore loop 1 are consistent with experimental single-molecule FRET values.