Mechanism Of Substrate Protein Remodeling By Molecular Chaperones PDF Download

Proteins are large complex molecules that play important roles in cellular activities such as DNA replication, molecular transport, cell immunity, and regulatory activities. Based on these roles, the half-lives of proteins within cells vary widely, but when the time comes they need to be degraded regardless. To address this issue, cell utilizes a protein quality control (PQC) system that unfolds and degrades proteins. One of the pathways used by PQC is selective degradation via molecular chaperones. The focus of this study is on the molecular chaperones that assist in unfolding and the mechanism through which they interact with their substrate proteins (SP). Unfolding chaperones are members of the AAA+ (ATPases Associated with various cellular Activities) family that form oligomers, typically hexamers, that enclose a central pore. It has been suggested that the conserved hydrophobic loop located at the entrance to the pore engages the SP and remodels it by exerting mechanical forces via a paddling motion. However the mechanism of SP processing is not known yet. Here I am presenting four studies regarding this issue: (1) Mechanism of transient binding and release of the SP during allosteric cycle of p97 (a group II AAA+ machine). (2) Mechanism of SP unfolding and translocation by p97. (3) Asymmetric processing of SP protein in sequential allosteric cycles. (4) The role of I-domain in unfolding and translocation of SulA by ClpY (group I) nanomachine. I use molecular dynamics simulations to probe the interaction of p97 and a long extended peptide as the SP. Mechanical pulling of the substrate through the p97 pore reveals that less work is required for translocation from the D1 (ring I) towards the D2 (ring II) compartment than in the opposite direction. Also based on simulations of SP binding to p97, I found that transient binding inside the pore happens through a lever mechanism in which SP binding to Glu554 supersede Arg599 as the pore closes during the allostric cycle. I perform coarse-grained (one bead per amino acid) Langevin dynamics simulations of a tagged four-helix bundle protein introduced to p97. The results indicate that interaction with both binding site inside D2 pore (Arg599), and the hydrophobic loop of D2 (Trp551, Phe551), is required for unfolding and translocation of the SP. I conduct further simulations of the same system with both clockwise and counterclockwise directionality of sequential intra-ring allosteric motion. I find that in both directions the SP conformational changes take place along the same pathway. However, the rates and yields of SP unfolding and translocation are higher in the clockwise direction due to better SP handling between adjacent subunits. Finally I introduced an improved coarsed-grained model (two bead per amino acid) parametrized using a statistical potential. I carried out Langevin dynamics simulations of the ClpY nanomachine and its natural substrate SulA. The results show that the absence of Clpy I-domain induces a deficiency in SP unfolding and therefore affects translocation. In this case, the I-domain binds SulA and facilitates the unfolding process by exerting additional forces on SP.