The structures of Q61 Ras mutants solved in a new space group reveals insights into the mechanism of intrinsic GTPase actitvity

Ras is the canonical member of a large superfamily of small monomeric GTPase proteins that act as “molecular switches” in a number of cellular signaling pathways. Mutations in codons 12, 13 or 61 convert ras into a constitutively active, oncogenic protein and these mutations are found in a variety of human tumors. In particular, substitutions of Q61 for any of the other amino acids all decrease the GTPase activity of Ras about 10‐fold, but the transformation efficiency of the mutant proteins vary by a factor of 1000. To explore the idea that conformational properties of the Ras Q61 mutants might have a role in the potency of its oncogenic phenotype, we have solved the structures of wild type Ras, and a number of Q61 Ras mutants from crystals having the symmetry of space group R32. In this crystal form, the conformation of switch II is undistorted by direct crystal contacts and the switch I residue Tyr32 is found in the closed conformation predicted to exist by 31P NMR spectroscopy and observed in the Raps/Raf complex. Comparison of Q61 mutant structures with wild type Ras‐GppNHp, solved in the same crystal form, predict a novel role for Tyr32 and a bridging water molecule in intrinsic, substrate assisted GTP hydrolysis. Conversely, the oncogenic mutants stabilize the ground state of the GTPase reaction by stabilizing a direct H‐bond between Tyr32 and the gamma phosphate and by stabilizing Wat175 in an “anticatalytic” position. This project was supported by a grant from the National Institutes of Health.