[NMR paper] Monitoring Ras Interactions with the Nucleotide Exchange Factor Sos using Site-specific NMR Reporter Signals and Intrinsic Fluorescence.

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Monitoring Ras Interactions with the Nucleotide Exchange Factor Sos using Site-specific NMR Reporter Signals and Intrinsic Fluorescence.

Related Articles Monitoring Ras Interactions with the Nucleotide Exchange Factor Sos using Site-specific NMR Reporter Signals and Intrinsic Fluorescence.

J Biol Chem. 2015 Nov 12;

Authors: Vo U, Vajpai N, Flavell L, Bobby R, Breeze AL, Embrey KJ, Golovanov AP

Abstract
The activity of Ras is controlled by the inter-conversion 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 [15N]-labeled Ras, as well as [13C-methyl-M,I]-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 supports 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.


PMID: 26565026 [PubMed - as supplied by publisher]



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