title: Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence.
abstract: The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly (15)N-labeled Ras as well as [(13)C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions.
authors.affiliation: From the Manchester Institute of Biotechnology and Faculty of Life Sciences, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom and.
authors.forename: Uybach
authors.initials: U
authors.lastname: Vo
authors.affiliation: Discovery Sciences, AstraZeneca, Mereside, Alderley Park, Cheshire SK10 4TF, United Kingdom.
authors.forename: Navratna
authors.initials: N
authors.lastname: Vajpai
authors.affiliation: Discovery Sciences, AstraZeneca, Mereside, Alderley Park, Cheshire SK10 4TF, United Kingdom.
authors.forename: Liz
authors.initials: L
authors.lastname: Flavell
authors.affiliation: Discovery Sciences, AstraZeneca, Mereside, Alderley Park, Cheshire SK10 4TF, United Kingdom.
authors.forename: Romel
authors.initials: R
authors.lastname: Bobby
authors.affiliation: Discovery Sciences, AstraZeneca, Mereside, Alderley Park, Cheshire SK10 4TF, United Kingdom.
authors.forename: Alexander L
authors.initials: AL
authors.lastname: Breeze
authors.affiliation: Discovery Sciences, AstraZeneca, Mereside, Alderley Park, Cheshire SK10 4TF, United Kingdom. Electronic address: Kevin.Embrey@astrazeneca.com.
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Embrey
authors.affiliation: From the Manchester Institute of Biotechnology and Faculty of Life Sciences, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom and. Electronic address: A.Golovanov@manchester.ac.uk.
authors.forename: Alexander P
authors.initials: AP
authors.lastname: Golovanov
mesh_terms: D000495:Allosteric Site; D020134:Catalytic Domain; D005453:Fluorescence; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020839:Son of Sevenless Protein, Drosophila / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: C117307:KRAS protein, human; D020839:Son of Sevenless Protein, Drosophila; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)
keywords: Ras protein; Sos protein; allosteric regulation; nuclear magnetic resonance (NMR); protein-protein interaction; small GTPase