Mimotope-Guided Molecular Dynamics Enables Precise Functional Epitope Mapping.

Mimotopes selected by phage display have been used for antibody epitope mapping for nearly four decades, but residue-level recovery of functional epitopes has remained unreliable. This is not a problem of algorithms but of assumptions: mimotopes recapitulate native epitope recognition through compensatory interaction networks that preserve binding energetics, without conserving sequence or surface geometry. Predictors trained on sequence similarity or static interface geometry therefore miss the residues that drive recognition. We present a physics-based workflow that refines antibody-bound mimotope conformations by microsecond-scale molecular dynamics and maps the resulting structures onto the cognate antigen using Folddisco. Across four antibody-antigen systems with crystallographically defined epitopes, the workflow reaches 60 to 100% precision in epitope localization and recovers 100% of mutagenesis- or structurally validated functional hotspots, compared with at most 25% for three widely used benchmarks (EpiSearch, ClusPro, SEPPA-mAb). For the clinically relevant ChiLob 7/4 system, it further identifies a minimal tetrapeptide (PWVP) sufficient for antibody recognition, validated by Western blot and immunoprecipitation. By grounding epitope prediction in experimentally selected binding information and energy-resolved conformational refinement, the workflow connects phage display to mechanistic structural insight.