Mechanism-Based Redesign of GAP to Activate Oncogenic Ras

Ras GTPases play a crucial role in cell signaling pathways. Mutations of the Ras gene occur in about one third of cancerous cell lines and are often associated with detrimental clinical prognosis. Hot spot residues Gly12, Gly13, and Gln61 cover 97% of oncogenic mutations, which impair the enzymatic activity in Ras. Using QM/MM free energy calculations, we present a two-step mechanism for the GTP hydrolysis catalyzed by the wild-type Ras.GAP complex. We found that the deprotonation of the catalytic water takes place via the Gln61 as a transient Bronsted base. We also determined the reaction profiles for key oncogenic Ras mutants G12D and G12C using QM/MM minimizations, matching the experimentally observed loss of catalytic activity, thereby validating our reaction mechanism. Using the optimized reaction paths, we devised a fast and accurate procedure to design GAP mutants that activate G12D Ras. We replaced GAP residues near the active site and determined the activation barrier for 190 single mutants. We furthermore built a machine learning for ultrafast screening, by fast prediction of the barrier heights, tested both on the single and double mutations. This work demonstrates that fast and accurate screening can be accomplished via QM/MM reaction path optimizations to design protein sequences with increased catalytic activity. Several GAP mutations are predicted to re-enable catalysis in oncogenic G12D, offering a promising avenue to overcome aberrant Ras-driven signal transduction by activating enzymatic activity instead of inhibition. The outlined computational screening protocol is readily applicable for designing ligands and cofactors analogously.

Automated summary

This study reveals the mechanism of GTP hydrolysis catalyzed by the Ras.GAP complex and determines the reaction pathways of oncogenic Ras mutants. A fast and accurate program is designed to design GAP mutants that activate G12D Ras, and a machine learning model is built for ultrafast screening. This work provides a method for designing protein sequences with enhanced catalytic activity and predicts that some GAP mutants can reactivate oncogenic G12D.