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 1 . 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 2 . 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 3 . 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 Mg 2+ 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 4 . 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.