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<p>Growth factor receptor–bound protein 2 (Grb2) is a modular adaptor that links activated receptor tyrosine kinases to downstream signaling pathways through its SH3–SH2–SH3 architecture. Previous studies showed that perturbations in the SH2 domain can influence ligand binding at the C-terminal SH3 domain, suggesting directional allosteric communication within the protein. Here, we tested whether the flanking SH3 domains, in turn, modulate SH2-mediated ligand recognition. Using a parallel mutational scan of the isolated SH2 domain and full-length Grb2, combined with stopped-flow kinetics and double-mutant-cycle analysis, we found that SH2 binding energetics remain essentially unchanged in the presence of the SH3 domains. Only three of twenty-one variants displayed measurable coupling, and none exceeded the threshold typically associated with significant allosteric effects. Pre-binding of the C-SH3 domain to a Gab2-derived peptide likewise produced no detectable influence on SH2–ligand binding. These results reveal a marked asymmetry in interdomain communication in Grb2 and show that the SH2 domain functions as a robust, largely autonomous module within the full-length protein.</p>

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Robust SH2 binding affinity indicates minimal SH3-to-SH2 communication in Grb2

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年1月23日

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  • Eduarda Santos Ventura,
  • Mariana Di Felice,
  • Julian Toso,
  • Valeria Pennacchietti,
  • Angelo Toto,
  • Stefano Gianni

摘要

Growth factor receptor–bound protein 2 (Grb2) is a modular adaptor that links activated receptor tyrosine kinases to downstream signaling pathways through its SH3–SH2–SH3 architecture. Previous studies showed that perturbations in the SH2 domain can influence ligand binding at the C-terminal SH3 domain, suggesting directional allosteric communication within the protein. Here, we tested whether the flanking SH3 domains, in turn, modulate SH2-mediated ligand recognition. Using a parallel mutational scan of the isolated SH2 domain and full-length Grb2, combined with stopped-flow kinetics and double-mutant-cycle analysis, we found that SH2 binding energetics remain essentially unchanged in the presence of the SH3 domains. Only three of twenty-one variants displayed measurable coupling, and none exceeded the threshold typically associated with significant allosteric effects. Pre-binding of the C-SH3 domain to a Gab2-derived peptide likewise produced no detectable influence on SH2–ligand binding. These results reveal a marked asymmetry in interdomain communication in Grb2 and show that the SH2 domain functions as a robust, largely autonomous module within the full-length protein.