Mechanistic insights into the effect of phosphorylation on Ras conformational dynamics and its interactions with cell signaling proteins

Ras undergoes interconversion between the active GTP-bound state and the inactive GDP-bound state. This GTPase cycle, which controls the activities of Ras, is accelerated by Ras GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (SOS). Oncogenic Ras mutations could affect the GTPase cycle and impair Ras functions. Additionally, Src-induced K-Ras Y32/64 dual phosphorylation has been reported to disrupt GTPase cycle and hinder Ras downstream signaling. However, the underlying mechanisms remain unclear. To address this, we performed molecular dynamics simulations (similar to 30 mu s in total) on unphosphorylated and phosphorylated K-Ras4B in GTP-and GDP-bound states, and on their complexes with GTPase cycle regulators (GAP and SOS) and the effector protein Raf. We found that K-Ras4B dual phosphorylation mainly alters the conformation at the nucleotide binding site and creates disorder at the catalytic site, resulting in the enlargement of GDP binding pocket and the retard of RasGTP intrinsic hydrolysis. We observed phosphorylation-induced shift in the distribution of Ras-GTP inactive-active sub-states and recognized potential druggable pockets in the phosphorylated Ras-GTP. Moreover, decreased catalytic competence or signal delivery abilities due to reduced binding affinities and/or distorted catalytic conformations of GAP, SOS and Raf were observed. In addition, the allosteric pathway from Ras/Raf interface to the distal Raf L4 loop was compromised by Ras phosphorylation. These results reveal the mechanisms by which phosphorylation influences the intrinsic or GAP/SOS catalyzed transformations between GTP-and GDP-bound states of Ras and its signal transduction to Raf. Our findings project Ras phosphorylation as a target for cancer drug discovery. (C) 2021 The Author(s). Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.

Automated summary

The dual phosphorylation of K-Ras induces structural changes affecting nucleotide and catalytic sites, reducing the catalytic competence and signal delivery abilities of GAP, SOS, and Raf. Phosphorylation also disrupts the Ras/Raf interface allosteric pathway, altering the distribution of Ras-GTP sub-states and identifying potential druggable pockets. These findings highlight the impact of phosphorylation on Ras function and signal transduction, suggesting it as a target for cancer drug discovery.