Topology and Grb2 interactions of the intrinsically disordered platform protein Gab1

• Applicants: Professor Dr. Jochen Balbach; Professor Stephan-Michael Feller, Ph.D.
• Subject Area: Biochemistry ; Biophysics
• Term: from 2017 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 374605181

• Final Report Year: 2024

Final Report Abstract

The Gab1 protein is a multifunctional platform for the formation of various signal protein complexes in cells and has existed for about 1 billion years in multicellular organisms with multiple cell types. Except for the small N-terminal PH domain, the nearly 700 amino acids of Gab1 are intrinsically disordered. Several binding sites for effector proteins have been mapped. Chemical and biological crowding leads to a bindingcompetent conformation for SHP2 at the C-terminus of Gab1 even without the canonical phosphorylations of Tyr 627 and 659. The binding of Grb2 via the N- and C-terminal SH3 domains with affinities in the low micromolar range leads to the formation of a network structure of Grb2 and Gab1 proteins, which was resolved using X-ray crystallography. Using NMR spectroscopy, the same Gab1 interactions were observed in solution for the N-terminal SH3 domain of Grb2 without altering its low thermodynamic stability. This network-like self-assembly leads to a liquid-liquid phase separation (LLPS) of the Grb2-Gab1 complex in solution. For this LLPS, an intact interface between the SH2 and cSH3 domains of Grb2 is necessary, which is disrupted by phosphorylation of Tyr160, thereby leading to the dissolution of the separated phases.

Publications

• In-Cell NMR: Analysis of Protein–Small Molecule Interactions, Metabolic Processes, and Protein Phosphorylation. International Journal of Molecular Sciences, 20(2), 378.
  Kumar, Amit; Kuhn, Lars & Balbach, Jochen
  
• Macromolecular Crowding Induces a Binding Competent Transient Structure in Intrinsically Disordered Gab1. Journal of Molecular Biology, 434(5), 167407.
  Gruber, Tobias; Lewitzky, Marc; Machner, Lisa; Weininger, Ulrich; Feller, Stephan M. & Balbach, Jochen
  
• Self-assembly of Grb2 meshworks revealed by Grb2-Gab1497-528complex structure. Cold Spring Harbor Laboratory.
  Breithaupt, Constanze; Gruber, Tobias; Mandel, Katharina; Lewitzky, Marc; Meister, Annette; Jochen, Balbach; Feller, Stephan M. & Stubbs, Milton T.