COSMIC-Linked Ras Mutations at the Interface Between H-Ras and PI3Kγ_RBD Frequently Generate Affinity Increases

The three conventional isoforms of the Ras G-protein (H-, K-, N-Ras) function as molecular on-off switches that regulate a wide array of signaling pathways, including the Ras-PI3K-PIP₃-PDK-AKT pathway that is central to innate immunity and normal cell growth and is dysregulated in many disease states. Activation of the pathway by Ras requires adequate Ras-PI3K binding affinity. Here we focus on the interface of known structure in the H-Ras:PI3Kγ co-complex essential to multiple pathways including directed pseudopod growth in leukocyte chemotaxis. At this interface 10 H-Ras residues, all 100% conserved between the H-, K-, and N-Ras isomers, contact the Ras binding domain of PI3Kγ (PI3Kγ_RBD). To investigate the degree to which the native H-Ras:PI3Kγ_RBD interface is optimized by evolution for maximal binding affinity, 8 interfacial Ras mutations selected from the COSMIC database and the literature were introduced at the contact positions. All 8 Ras mutations were observed to alter the H-Ras:PI3Kγ_RBD binding affinity, with 4 mutations yielding significant affinity increases and 4 yielding significant affinity decreases. These findings indicate that the native H-Ras:PI3Kγ_RBD interface provides intermediate, rather than maximal, binding affinity. Such intermediate affinity is consistent with the substantial binding plasticity of the conserved H-, N-, K-Ras effector docking surface, which has evolved to bind a diverse array of effectors. Furthermore, the findings provide evidence that COSMIC-linked mutations at the H-Ras:PI3Kγ_RBD interface frequently generate affinity increases (not just the affinity decreases typical of random interfacial mutations) with potential implications for molecular mechanisms of disease and for tool development in cell biology.