Time-Resolved Crystallography Reveals the Mechanisms of GTP hydrolysis for N-RAS and the Oncogenic Mutants G12C, G12V and Q61L

The RAS family of small GTPases are molecular switches that convey downstream signals regulating cell proliferation, differentiation, and apoptosis. The signaling competent GTP-bound RAS transitions to its inactive GDP-bound form through γ-phosphate hydrolysis. Oncogenic RAS mutations hamper GTP hydrolysis and are present in up to 30% of all human cancers. Structural studies of RAS proteins bound to non-hydrolysable GTP analogs have revealed snapshots of the enzyme in its possibly active form. Yet, the mechanism of GTP hydrolysis has not been structurally resolved. To visualize this reaction in real time, we performed time-resolved crystallographic experiments employing a photolabile caged-GTP substrate. Fifty-seven distinctive reaction intermediates were captured during hydrolysis of a live GTP for N-RAS, the oncogenic mutants G12C, G12V and Q61L; and Y32R, a fast hydrolytic mutant. The reaction mechanisms and rates for the native and each of the mutants differed significantly; however, they shared common elements: an initially catalytically-defective open state, which transitions into the closed Michaelis complex state with solvent-assisted O3B-Pγ bond lengthening and breaking, followed by the release of the Mg²⁺ stabilized PO3^-/PO4^-3 species and unfolding of the switch loops. Given the conserved nature of GTP- and ATP-ases active sites, this structural work lays the basis to understand the universal mechanism of γ-phosphate hydrolysis. Furthermore, search for cryptic binding sites during GTP hydrolysis in G12C, G12V, and Q61L mutants reveals the presence of distinctive state-dependent binding pockets that could be targets for structure-based drug discovery of experimentally resolved intermediates states.