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Phosphorylation induces altered protonation states and allosterically regulates Rac1-RhoGDI complex.

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|December 22, 2025
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Summary
This summary is machine-generated.

Site-specific phosphorylation of Rho Guanine nucleotide Dissociation Inhibitor (RhoGDI) controls Rac1 GTPase release. Dual phosphorylation at Ser101/Ser174 allosterically weakens Rac1-RhoGDI binding by altering protonation states and disrupting communication pathways.

Keywords:
GTPaseMD simulationRhoGDIallosteryconstant pH MDphosphorylation

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Area of Science:

  • Molecular Cell Biology
  • Biochemistry
  • Computational Biology

Background:

  • Rho GTPases regulate essential cellular functions, including cell motility.
  • Rho Guanine nucleotide Dissociation Inhibitor (RhoGDI) sequesters Rho GTPases in an inactive GDP-bound state.
  • Site-specific phosphorylation of RhoGDI is known to trigger selective GTPase release, but the underlying mechanisms are not fully understood.

Purpose of the Study:

  • To elucidate the molecular mechanisms by which dual phosphorylation of RhoGDI at Ser101 and Ser174 facilitates Rac1 GTPase dissociation.
  • To contrast the effects of dual phosphorylation (SP101/174) with inert phosphorylation at Ser96 on Rac1-RhoGDI interaction.
  • To investigate the role of altered histidine protonation states and allosteric communication in phosphorylation-mediated regulation.

Main Methods:

  • Constant pH molecular dynamics simulations were employed to model RhoGDI phosphorylation.
  • Analysis of histidine protonation states, electrostatic environments, and binding affinity changes.
  • Structural clustering, conformational free energy landscape analysis, and allosteric network analysis were performed.

Main Results:

  • SP101/174 phosphorylation induced distinct shifts in RhoGDI histidine protonation states, altering the local electrostatic environment.
  • A significant reduction in Rac1-RhoGDI binding affinity was observed due to SP101/174, linked to disrupted hydrogen bonds and interfacial contacts.
  • SP101/174 promoted increased conformational variability and displacement of key regions in both Rac1 and RhoGDI, disrupting allosteric communication pathways essential for complex stability.

Conclusions:

  • Site-specific phosphorylation of RhoGDI, particularly dual phosphorylation at Ser101/Ser174, allosterically regulates Rac1 interaction.
  • Phosphorylation modulates RhoGDI's protonation states, weakens binding affinity, induces conformational changes, and disrupts allosteric networks.
  • These findings provide a mechanistic basis for the selectivity of the phosphorylation code in controlling Rac1 GTPase activity.