Mechanism of SOS PR-domain autoinhibition revealed by single-molecule assays on native protein from lysate

Young Kwang Lee, Shalini T. Low-Nam, Jean K. Chung, Scott D. Hansen, Hiu Yue Monatrice Lam, Steven Alvarez and Jay T. Groves ()
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Young Kwang Lee: University of California
Shalini T. Low-Nam: University of California
Jean K. Chung: University of California
Scott D. Hansen: University of California
Hiu Yue Monatrice Lam: University of California
Steven Alvarez: University of California
Jay T. Groves: University of California

Nature Communications, 2017, vol. 8, issue 1, 1-11

Abstract: Abstract The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) plays a critical role in signal transduction by activating Ras. Here we introduce a single-molecule assay in which individual SOS molecules are captured from raw cell lysate using Ras-functionalized supported membrane microarrays. This enables characterization of the full-length SOS protein, which has not previously been studied in reconstitution due to difficulties in purification. Our measurements on the full-length protein reveal a distinct role of the C-terminal proline-rich (PR) domain to obstruct the engagement of allosteric Ras independently of the well-known N-terminal domain autoinhibition. This inhibitory role of the PR domain limits Grb2-independent recruitment of SOS to the membrane through binding of Ras·GTP in the SOS allosteric binding site. More generally, this assay strategy enables characterization of the functional behaviour of GEFs with single-molecule precision but without the need for purification.

Date: 2017
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DOI: 10.1038/ncomms15061

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