Rozza, R. | bioRxiv Science

bioRxiv2026

Glutamine Tautomerization Drives RhoGAP-Aided GTP Hydrolysis in Small Rho GTPases

Rho GTPases promote GTP hydrolysis aided by specific GTPase-activating proteins (GAPs). By alternating between an active GTP-bound and an inactive GDP-bound state, Rho GTPases function as molecular switches regulating cytoskeletal dynamics and cell motility. Despite their biological relevance, the detailed molecular mechanism underlying Rho GTPases catalysis remains contentious. Here, using classical and hybrid quantum-classical molecular dynamics, we resolve the mechanism of GTP hydrolysis in the RhoGAP-RhoA complex. We reveal that GTP hydrolysis proceeds through a dissociative nucleophilic substitution mechanism, driven by an amide [->] imide tautomerization of Gln63, which aids in delivering a proton from the nucleophilic water to the leaving phosphate group. The Gln63 imide tautomer also loosens RhoGAP-RhoA interfacial contacts, allowing solvent molecules to enter and drive a water-mediated reverse tautomerization of Gln63 that restores the catalytically-competent configuration of the RhoA active site. Conservation of key interface residues across Rho/Rho GAP family members suggests that this mechanism may be shared by most Rho GTPases.

biophysics↗

bioRxiv2025

The Covalent Binding of Splicing Inhibitors occurs via a Zinc- Assisted Enzymatic-like mechanism

Branch-site recognition is a pivotal event in spliceosome assembly, and branch-site antagonists disrupt this process, inducing a splicing rewiring that underlies much of their antitumor activity. Antagonists from the spliceostatin family covalently bind the SF3b complex nearby the branch-site pocket, forming an adduct with Cys26 of the PHF5A zinc finger while remarkably preserving Zn2+ coordination - an unconventional mode of covalent inhibition. Yet the molecular features that activate this normally protected ZnCys4 cysteine and initiate spliceostatins epoxide ring opening have remained obscure. Here, we combine extensive classical and QM/MM molecular dynamics simulations to map the full reactive trajectory of spliceostatin attachment to PHF5A. We identify two alternative noncovalent spliceostatin binding poses and show that only its thermodynamically favoured conformation pre-organizes the epoxide for nucleophilic attack. The local distortion of the ZnCys4 coordination sphere weakens the Zn-Cys26 bond, enabling water-Cys26 exchange and generating a highly reactive nucleophilic thiolate. Covalent bond formation is subsequently accelerated by an Asp34-Lys29 proton relay that activates the epoxide leaving group. Free-energy calculations confirm that the overall reaction is fast and strongly exergonic. Together, these findings provide a complete mechanistic framework for zinc-assisted inhibition of early spliceosomes by branch-site antagonists and advance our fundamental understanding of zinc finger reactivity.

biochemistry↗