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

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Listed:
  • Young Kwang Lee

    (University of California
    Lawrence Berkeley National Laboratory)

  • 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
    Lawrence Berkeley National Laboratory)

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.

Suggested Citation

  • Young Kwang Lee & Shalini T. Low-Nam & Jean K. Chung & Scott D. Hansen & Hiu Yue Monatrice Lam & Steven Alvarez & Jay T. Groves, 2017. "Mechanism of SOS PR-domain autoinhibition revealed by single-molecule assays on native protein from lysate," Nature Communications, Nature, vol. 8(1), pages 1-11, April.
    • Handle: RePEc:nat:natcom:v:8:y:2017:i:1:d:10.1038_ncomms15061
    DOI: 10.1038/ncomms15061
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