Self-assembly of Grb2 meshworks revealed by Grb2-Gab1497-528 complex structure

The ubiquitously expressed adaptor protein Growth factor receptor bound protein 2 (Grb2) plays an essential role in signal transduction by binding to activated receptor tyrosine kinases through its SH2 domain and to downstream effectors via its N- and C-terminal SH3 domains (nSH3, cSH3). Here we present the first structure of ligand-bound full length Grb2. The crystal structure of Grb2 in complex with a bidentate nSH3-cSH3-binding peptide, derived from the multi-site docking protein Grb2- associated binder-1 (Gab1), provides molecular insight into effector recognition by Grb2 and reveals the assembly of a two-dimensional meshwork, consisting of multimeric filament-like Grb2 chains linked to each other by the bivalent bound Gab1497-528 peptide. Dominant contacts between Grb2 molecules in the multimer are provided by an intermolecular SH2/cSH3 domain interface that is also present in the closed dimer of ligand-free Grb2. We further show that Grb2 is able to self-assemble to form phase-separated condensates in solution. The Grb2 SH2 domain phosphotyrosine binding site is freely accessible in the multimeric assembly, and phase separation is fostered by addition of Gab1497- 528, as expected from the crystal structure. Multimeric assembly is also observed using a Grb2 SH2- cSH3 didomain construct, and suppressed using a Grb2 Tyr60Glu mutant, a mimic of the in vivo phosphorylated Tyr160 central to the SH2/cSH3 interface, demonstrating that an intact SH2/cSH3 interface is needed for Grb2 assembly in solution. ### Competing Interest Statement The authors have declared no competing interest. * SH2 : Src Homology 2 domain SH3 : Src Homology 3 domain PH : Pleckstrin homology domain PR region : proline rich region IDR : intrinsically disordered region rmsd : root mean square deviation RTK : receptor tyrosine kinase LLPS : liquid liquid phase separation