Desvendando epítopos virais via RMN de interações antígeno-anticorpo

Abstract

Antibodies are glycoproteins of the humoral immune response that, due to their remarkable diversity of primary structures, can recognize, bind, and neutralize antigens derived from pathogens and eradicate microorganisms by opsonization. Because of this ability to selectively recognize and bind a given antigen, some studies aim to experimentally identify the epitope, the region in contact with an antibody, which is a process called epitope mapping. Identifying and characterizing these binding sites aid in developing new therapeutics, vaccines, and diagnostics. The COVID-19 pandemic has underscored the need to identify new targets for treatments and diagnostics. In this study, we used nuclear magnetic resonance (NMR) spectroscopy to map the epitopes of the immunogenic SARS-CoV-2 nucleocapsid RNA binding site domain (N-NTD) protein that interacts with polyclonal antibodies from patient sera from the Belo Horizonte metropolitan region. Observing changes in the NMR spectra 1H-15N HSQC of the antigen upon binding to the antibodies and calculating the CSP, we determine specific residues involved in the interaction. To gain further insight into the dynamics and conformational properties, we conducted paramagnetic relaxation enhancement experiments (PRE) with gadolinium (Gd) on both the free protein and the protein-antibody complex, elucidating solvent accessibility. We observed that residues Leu16, Lys25, Ser38, Asp63, Leu64, and Thr126, mainly located in a loop region, have been described as having significant CSP when the protein interacts with viral RNA. These residues are also observed in protein-antibody interaction and in the PRE experiment. This suggests that the formation of the complex protein-antibody would prevent the RNA genome packaging and the assembly into virus particles. Residues Lys25, Asp63, and Thr126 have also been observed to be important for the initial contact of the protein with ligands. Additionally, three epitopes have been proposed that could be used for COVID-19 diagnosis. Thus, the results of this study will contribute to a better understanding of antibody-antigen binding, stability, and functional properties, providing valuable information for vaccine development and potential therapeutic interventions.