Computational rescue of antibody binding to monkeypox virus E8L: from electrostatic repulsion to salt bridge-enhanced...

The ongoing Mpox (monkeypox) outbreak underscores the need for effective therapeutic antibodies. Antibody VACV-66 binds strongly to the vaccinia virus D8 protein but shows markedly reduced cross-reactivity to the MPXV homolog E8L, despite >95% sequence identity. In this study, we combined AlphaFold3 modeling, molecular dynamics simulations, and ASGB-IE free energy calculations to investigate this mechanism and rationally design high-affinity antibody mutations targeting E8L. Our calculations revealed that the single N18D mutation in E8L (where N18 in D8 corresponds to D18 in E8L) introduces electrostatic repulsion with antibody residue D34_L, triggering local conformational changes that displace key residue R20 from the aromatic cage formed by W106_H and Y33_L. Based on this mechanism, saturation mutation of D34_L identified three optimal mutations (D34_LR, D34_LK, and D34_LN) that enhance binding free energy by removing electrostatic repulsion, restoring R20 insertion into the aromatic cage, and forming new favorable interactions. Quantitative analysis of the binding free energy contributions of residues N18 and R20 across different systems provided additional support for this mechanism. This work elucidates how a single viral mutation dampens antibody cross-reactivity and provides a general computational strategy for mechanism-guided antibody optimization against emerging orthopoxviruses. The identified D34_LR, D34_LK, and D34_LN mutants represent promising candidates for experimental validation and therapeutic development.