Evaluation of AlphaFold modeling for elucidation of nanobody-peptide epitope interactions

Models of antibody (Ab)-antigen complexes can be used to understand interaction mechanisms and for improving affinity. This study evaluates the use of the protein structure prediction algorithm AlphaFold (AF) for exploration of interactions between peptide epitope tags and the smallest functional Ab fragments, nanobodies (Nbs). Although past studies of AF for modeling Ab-target (antigen) interactions suggested modest algorithm performance, those were primarily focused on Ab-protein interactions, while the performance and utility of AF for Nb-peptide interactions, which are generally less complex because of smaller antigens, smaller binding domains, and fewer chains, is less clear. In this study, we evaluated the performance of AF for predicting the structures of Nbs bound to experimentally validated, linear, short peptide epitopes (Nb-tag pairs). We expanded the pool of experimental data available for comparison through crystallization and structural determination of a previously reported Nb-tag complex (Nb₁₂₇). Models of Nb-tag pair structures generated from AF were variable with respect to consistency with experimental data, with good performance in just over half (four of six) of cases. Even among Nb-tag pairs successfully modeled in isolation, efforts to translate modeling to more complex contexts failed, suggesting an underappreciated role of the size and complexity of inputs in AF modeling success. Finally, the model of an Nb-tag pair with minimal previous characterization was used to guide the design of a peptide-electrophile conjugate that undergoes covalent crosslinking with Nb upon binding. These findings highlight the utility of minimized Ab and antigen structures to maximize insights from AF modeling.