ANALYSIS OF THE INTERACTION BETWEEN THE GRB2-SH2 DOMAIN AND PEPTIDES FROM THE DNA REPAIR PATHWAY

ANALYSIS OF THE INTERACTION BETWEEN THE GRB2-SH2 DOMAIN AND PEPTIDES FROM THE DNA REPAIR PATHWAY

Renan P. Pedro1 2, Jéssica A. Tedesco1 2, Zamal Ahmed3, Raphael V. R. Dias1 2, Ícaro P. Caruso1 2, Fernando A. de Melo1 2

1Department of Physics, Institute of Biosciences, Humanities and Exact Sciences (IBILCE), São Paulo State University “Júlio de Mesquita Filho” (UNESP), São José do Rio Preto, SP, Brazil ([email protected]); 2Multiuser Center for Biomolecular Innovation (CMIB), São Paulo State University “Júlio de Mesquita Filho” (UNESP), São José do Rio Preto, SP, Brazil; 3Departments of Molecular and Cellular Oncology and Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, USA.

Homology-directed repair (HDR) is a highly precise mechanism for DNA damage correction, with histone H2AX serving as a crucial marker in this process. When double-strand breaks occur in the DNA, H2AX undergoes autophosphorylation at serine-139 and tyrosine-142 residues, facilitating the recruitment of essential proteins, including MRE11, for DNA repair. The interaction between H2AX and MRE11 is intermediated by the SH2 domain of the adaptor protein GRB2. In this study, we investigated the structural interaction between the GRB2-SH2 domain and peptides derived from H2AX (seven residues containing phosphoserine and phosphotyrosine) and MRE11 (22 residues without phosphorylated residues) using nuclear magnetic resonance (NMR) and computational simulations. The GRB2-SH2 domain was expressed in E.coli BL21(DE3) and purified through affinity chromatography and size exclusion. The interaction with H2AX and MRE11 peptides was evaluated using 1H-15N-HSQC spectroscopy, combined with chemical shift perturbation (CSP) analysis and molecular dynamics (MD) simulations. NMR results revealed no interaction between the GRB2-SH2 domain and the H2AX_pSpY peptide. In contrast, an interaction was observed with the MRE11_22 peptide. This finding suggests that after histone H2AX autophosphorylation, the information is transmitted to the MRE11 protein, but not to GRB2. Consequently, MRE11 recruits GRB2 through its SH2 domain, allowing GRB2 to transport MRE11 to the HDR pathway and initiate DNA repair. CSP analysis of the GRB2-SH2/MRE11_22 complex revealed conformational changes in residues K64, R67, Q77, F83, E87, E89, S90, L111, and T138, identifying regions involved in peptide binding. Computational results supported experimental findings, providing insights into the mode and stability of the interaction. This study enhances our understanding of the molecular microenvironment governing peptide interaction and its structural implications for the protein. Notably, the peptide with phosphorylated serine and tyrosine did not interact, while the unphosphorylated peptide demonstrated interaction. CSP-based mapping of the GRB2-SH2/MRE11_22 interaction suggests the involvement of recognition sites I and II.

Keywords: Homology-Directed Repair, H2AX histone, MRE11 protein, SH2 domain, NMR.

This work was supported by The National Council for Scientific and Technological Development (CNPq: 140306/2020-0), and by The São Paulo Research Foundation (FAPESP: 2019/24974-0; 2019/08967-4; 2017/20642-8; 2014/17630-0).