Phosphorylation induces altered protonation states and allosterically regulates Rac1– <scp>RhoGDI</scp> complex

Rho GTPases, critical for cellular functions like motility, are sequestered in an inactive GDP-bound state by RhoGDI. While site-specific RhoGDI phosphorylation is known to trigger selective GTPase release, the precise mechanisms remain elusive. This study dissects how dual phosphorylation at Ser101 (SP101) and Ser174 (SP174) in RhoGDI facilitates Rac1 dissociation, contrasting it with the inert Ser96 phosphorylation. Using constant pH molecular dynamics simulations, we demonstrate that unlike Ser96, Ser101/Ser174 phosphorylation (SP101/174) induces distinct shifts in RhoGDI histidine protonation states, reflecting altered local electrostatic environments. These changes correlate with a significant reduction in Rac1-RhoGDI binding affinity, attributed to disrupted hydrogen bonds and key interfacial contacts. Structural clustering and analysis of the conformational free energy landscape reveal that SP101/174 promotes increased conformational variability and displacement of crucial regions, including Rac1's Switch I and polybasic region, and RhoGDI's N-terminal helix. Allosteric network analysis corroborates these findings, showing that SP101/174 critically disrupts communication pathways essential for the Rac1-RhoGDI complex stability, an effect minimally observed with Ser96 phosphorylation. Collectively, these results illuminate how site-specific phosphorylation allosterically orchestrates Rac1-RhoGDI interaction by modifying protonation states, weakening binding affinity, inducing conformational shifts, and rewiring allosteric communication, thereby providing a mechanistic basis for the selectivity of the phosphorylation code in Rac1 regulation.