A novel switch region regulates H‐ras membrane orientation and signal output

The plasma membrane nanoscale distribution of H‐ras is regulated by guanine nucleotide binding. To explore the structural basis of H‐ras membrane organization, we combined molecular dynamic simulations and medium‐throughput FRET measurements on live cells. We extracted a set of FRET values, termed a FRET vector, to describe the lateral segregation and orientation of H‐ras with respect to a large set of nanodomain markers. We show that mutation of basic residues in helix α4 or the hypervariable region (HVR) selectively alter the FRET vectors of GTP‐ or GDP‐loaded H‐ras, demonstrating a critical role for these residues in stabilizing GTP‐ or GDP‐H‐ras interactions with the plasma membrane. By a similar analysis, we find that the β2–β3 loop and helix α5 are involved in a novel conformational switch that operates through helix α4 and the HVR to reorient the H‐ras G‐domain with respect to the plasma membrane. Perturbation of these switch elements enhances MAPK activation by stabilizing GTP‐H‐ras in a more productive signalling conformation. The results illustrate how the plasma membrane spatially constrains signalling conformations by acting as a semi‐neutral interaction partner.