GRB2 Y160F: A VERSATILE MUTATION TO STUDY OLIGOMERIC TRANSITIONS AND SIGNALING REGULATION

The Growth factor receptor-bound protein 2 (Grb2) regulates normal vs oncogenic proliferation through a monomer-dimer equilibrium. The dimer inhibits signal transduction whereas the monomer promotes signaling downstream by recruiting the guanine exchange factor SOS to the membrane, resulting in an increase in RAS active GTP-bound form. Phosphorylation of residue Y160 is a well-known mechanism of dimer dissociation due to the disruption of the Y160 and E87 hydrogen bond between protomers. Grb2Y160F is a functional mutation in cells that has been reported as an adjuvant, aiming to demonstrate the Y160 importance for dimer dissociation. However, Grb2Y160F oligomeric state has not been studied yet, even though the hydrogen bond between the protomers is also disrupted. Therefore, the goal of this study was to determine if the tyrosine to phenylalanine mutation on residue 160 would interfere with the monomer-dimer transition in solution through biophysical experimental and computational techniques. Wild-type and Y160F Grb2 were expressed in E. coli BL21 (DE3). Proteins were purified by affinity chromatography followed by size exclusion chromatography (SEC). SEC was performed on a XK16/70 column packed with Superdex 75 resin and each protein was loaded separately on the column. The final buffer was 20 mM NaPi (pH 7.0), 50 mM NaCl and 1 mM β-Mercaptoethanol. DLS measurements were carried out using a Zetasizer Nano ZS90 with 1.5 mg/mL concentrated samples of Grb2WT and Grb2Y160F at 20 ºC. Molecular Dynamics simulations were performed using GROMACS and rendered by VMD. Adaptive Biasing Force MD simulations were performed on NAMD with charmm36 force field with the distance between monomers as a colvar of 20 Å (divided on 5 windows). The computational analyses were based on PDB Id 1GRI. SEC chromatogram superposition revealed that Grb2Y160F has two populations, with the minor one eluting in the same volume as Grb2WT and the major one 10 mL later. The major population of Grb2Y160F was selected and analyzed by DLS. The hydrodynamic diameter values obtained were 4.949 nm (MW ~ 28 kDa) for Grb2Y160F and 6.789 nm (MW ~ 58.7 kDa) for Grb2WT. In addition, the computational analysis compared the structures of Grb2WT, Grb2Y160F, and Grb2Y160E (which was selected because it provides an irreversible monomer in solution). The hydrogen bonds profile revealed that residue 160 of Grb2WT establishes up to 3 interactions with the other protomer, while Grb2Y160F and Grb2Y160E establish none or only 1 interaction in a hypothetical dimer. However, Grb2Y160F presented an intermediate potential mean force to dissociate the protomers when compared to the other two proteins, but more similar to the wild-type. Thus, considering Grb2Y160F particle size and the similar hydrogen bond profile with Grb2Y160E, we propose that the Grb2Y160F prevails as a monomer in solution, being also capable to dimerize. Therefore, this mutant is an excellent alternative to structural studies focused on Grb2 oligomeric state transitions. Moreover, Grb2Y160F also has a monomer-dimer equilibrium that regulates cell signaling and the dimerization of this mutant corroborates with the existence of other dimerization interfaces for Grb2 that may regulate signaling pathways in different manners.