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Structure-based mutagenesis reveals distinct functions for Ras switch 1 and switch 2 in Sos-catalyzed guanine

B E Hall1, S S Yang, P A Boriack-Sjodin

  • 1Department of Molecular Genetics and Microbiology and the Graduate Programs in Molecular Pharmacology and Molecular and Cellular Biology, State University of New York at Stony Brook, New York 11794-5222, USA.

The Journal of Biological Chemistry
|May 3, 2001
PubMed

Insights

Site-directed mutagenesis reveals how the guanine nucleotide exchange factor Sos activates Ras GTPases. Sos binding to Ras switch 2 anchors it, while interactions with switch 1 disrupt nucleotide binding, promoting GDP dissociation.

Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Protein Structure-Function Relationships

Background:

  • Ras GTPases are critical binary switches regulating cell growth and differentiation.
  • The guanine nucleotide exchange factor Sos activates Ras in response to external signals.
  • Previous structural studies revealed Sos induces conformational changes in Ras switch 1 and switch 2 regions.

Purpose of the Study:

  • To investigate the functional significance of Sos-induced conformational changes in Ras.
  • To elucidate the specific roles of Ras switch 1 and switch 2 interactions with Sos in Ras activation.

Main Methods:

  • Site-directed mutagenesis of key residues in Ras and Sos.
  • Analysis of Ras-Sos binding affinity and catalytic function.
  • Structure-function analysis based on previous crystallographic data.

Main Results:

  • Mutagenesis of Ras switch 2-Sos interface residues showed limited impact on binding, with Tyr64 being crucial.
  • Mutations in Ras switch 2 residues interacting with the nucleotide-binding site did not affect Sos catalytic function.
  • Mutations in Ras switch 1 residues disrupted Sos binding and increased nucleotide dissociation rates.

Conclusions:

  • Sos interaction with Ras switch 2 primarily mediates stable anchoring to the guanine nucleotide exchange factor.
  • Sos interaction with Ras switch 1 is critical for disrupting the nucleotide-binding site and facilitating GDP release.
  • Distinct functional roles of switch 1 and switch 2 interactions explain Sos-mediated Ras activation.

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