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

Abstract

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 mediumthroughput 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 a4 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 b2-b3 loop and helix a5 are involved in a novel conformational switch that operates through helix a4 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.

The Ras–Membrane Interface: Isoform-Specific Differences in the Catalytic Domain

“…The crystal structure of H-Ras in the presence of calcium acetate indicates an allosteric site pocket consisting of residues R97, D107, and Y137 that may have evolved to accompany the binding of a larger, membrane-related ligand capable of modulating catalytic residues in the effector lobe (21). Molecular dynamics simulations indicate that this allosteric pocket lies closer to the membrane in the GTP-bound orientation, which is consistent with the hypothesis of ligand-mediated promotion of intrinsic hydrolysis (21,52). An additional water-mediated communication network connects the helix 5 nucleotide sensor residues R161 and R164 to N85 at the N-terminal end of helix 3, which in turn connects to the active site ( Fig.…”

“…Lateral segregation is required for correct signaling activity (51), and effector proteins must be able to distinguish between the conformation of GTP-versus GDP-bound Ras with respect to the membrane. This is achieved through conserved nucleotide sensing residues from loop 3 (L3) on the effector lobe and helix 5 on the allosteric lobe near the membrane, providing a common mechanism through which the membrane can sense the state of the nucleotide at the opposite end of the molecule (52,53). Stabilization of the transient orientation of the catalytic domain with respect to the membrane is mediated by residues in the HVR and helix 4 regions of the allosteric lobe, the two regions of the protein that display the highest sequence divergence (9).…”

“…Active H-RasGTP signals from nonordered membrane domains, utilizing a membrane orientation that exposes the switch regions and effector lobe residues crucial for interactions with effectors, such as Raf and PI3K, as well as GAPs (52,54). The interaction between the G domain and the plasma membrane in this active orientation places positively charged helix 4 residues R128 and R135 close to the membrane, promoting specific electrostatic attraction to abundant negatively charged head groups or nonspecific interactions with phosphate groups in the plasma membrane (3,54).…”

Plasma membrane regulates Ras signaling networks

“…This mechanism of orientation-dependent function is called the "balance model". 20,67 Interaction of H-Ras with effectors, PI3K and Ras scaffolding protein galectin are sensitive to H-Ras reorientation at the membrane. For example, while PI3-K and galectin interact better with the R169A/K170A mutant of H-RasG12V, their interaction is impaired with the R128A/R135A mutant of H-RasG12V.…”

“…The C-terminal 22-25 amino acid-long portion of Ras is named the hypervariable region (HVR), since it differs significantly across isoforms in its amino acid sequence. 19 Although the N-terminal domains contribute to membrane binding, 20 Ras proteins primarily interact with the membrane through their HVRs. 21 The differences in amino acid sequence of Ras HVRs are augmented by different post-translational modifications.…”