Dynamics and Allosteric Information Pathways of Unphosphorylated c-Cbl.

Abstract

Human c-Cbl is a RING-type ligase and plays a central role in the protein degradation cascade. To elucidate its conformational changes related to substrate binding, we performed molecular dynamics simulations of different variants/states of c-Cbl for a cumulative time of 68 μs. Our simulations demonstrate that before the substrate binds, the RING domain samples a broad set of conformational states at a biologically relevant salt concentration, including the closed, partially open, and fully open states, whereas substrate binding leads to a restricted conformational sampling. Phe378 and the C-terminal region play an essential role in stabilizing the partially open state. To visualize the allosteric signal transmission pathways from the substrate-binding site to the 40 Å apart RING domain and identify the critical residues for allostery, we have created a subgraph from the optimal and suboptimal paths. Redundant paths are seen in the SH2 domain where the substrate binds, while the major bottlenecks are found at the junction between the SH2 domain and the linker helix region as well as that between the SH2 domain and the 4H bundle. These bottlenecks separate the paths into two overall routes. The nodes/residues at the bottlenecks on the subgraph are considered allosteric hot spots. This subgraph approach provides a general tool for network visualization and determination of critical residues for allostery. The structurally and allosterically critical residues identified in our work are testable and would provide valuable insights into the emerging strategies for drug discovery, such as targeted protein degradation.