title: 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 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 alpha4 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 beta2-beta3 loop and helix alpha5 are involved in a novel conformational switch that operates through helix alpha4 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.
authors.affiliation: Molecular Cell Biology, Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia. d.abankwa@imb.uq.edu.au
authors.forename: Daniel
authors.initials: D
authors.lastname: Abankwa
authors.forename: Michael
authors.initials: M
authors.lastname: Hanzal-Bayer
authors.forename: Nicolas
authors.initials: N
authors.lastname: Ariotti
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Plowman
authors.forename: Alemayehu A
authors.initials: AA
authors.lastname: Gorfe
authors.forename: Robert G
authors.initials: RG
authors.lastname: Parton
authors.forename: J Andrew
authors.initials: JA
authors.lastname: McCammon
authors.forename: John F
authors.initials: JF
authors.lastname: Hancock
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane; D003001:Cloning, Molecular; D006224:Cricetinae; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras)