STRUCTURAL ANALYSIS OF THE INTERACTION BETWEEN GRB2-SH2 DOMAIN AND MRE11 PEPTIDE: IMPLICATIONS FOR HOMOLOGY-DIRECTED REPAIR

Homology-directed repair (HDR) is a highly precise mechanism for correcting DNA damage, with histone H2AX playing a pivotal role as a marker in this process. Upon the occurrence of DNA double-strand breaks, H2AX undergoes phosphorylation at serine-139 and tyrosine-142, which facilitates the recruitment of essential repair proteins, including MRE11. The interaction between H2AX and MRE11 is mediated by the SH2 domain of the adaptor protein GRB2. This study investigates the structural interaction between the GRB2-SH2 domain and peptides derived from H2AX (seven residues containing phosphorylated serine and tyrosine) and MRE11 (22 residues without phosphorylation) using nuclear magnetic resonance (NMR) spectroscopy and computational simulations. The GRB2-SH2 domain was expressed in E.coli BL21(DE3) and purified via affinity and size exclusion chromatography. Interactions with H2AX and MRE11 peptides were assessed using
1H-15N HSQC spectroscopy, complemented by chemical shift perturbation (CSP) analysis and molecular dynamics (MD) simulations. NMR results indicated no interaction between the GRB2-SH2 domain and the H2AX-pSpY peptide. Conversely, an interaction was observed with the MRE11-22 peptide, suggesting that post-phosphorylation of histone H2AX, the signal is conveyed to the MRE11 protein rather than directly to GRB2. This implies that MRE11 recruits GRB2 through its SH2 domain, enabling GRB2 to transport MRE11 to the HDR pathway, thereby initiating DNA repair. CSP analysis of the GRB2-SH2/MRE11-22 complex revealed significant conformational changes in residues K64, R67, Q77, F83, E87, E89, S90, L111, and T138, pinpointing regions crucial for peptide binding. Computational results corroborated experimental findings, providing detailed insights into the interaction's mode and stability. This study advances our understanding of the molecular mechanisms underpinning peptide interactions and their structural implications for protein function. Notably, the phosphorylated H2AX peptide did not exhibit interaction, while the non-phosphorylated MRE11 peptide did, highlighting specific recognition sites involved in binding as suggested by CSP-based mapping. This work was supported by the National Council for Scientific and Technological Development (CNPq: 140306/2020-0) and the São Paulo Research Foundation (FAPESP: 2023/01744-5; 2019/24974-0; 2019/08967-4; 2017/20642-8).