Molecular dynamics and machine learning stratify motion-dependent activity profiles of S-layer destabilizing nanobodies

Nanobody (Nb)-induced disassembly of surface array protein (Sap) S-layers, a two-dimensional paracrystalline protein lattice from <i>Bacillus anthracis</i>, has been presented as a therapeutic intervention for lethal anthrax infections. However, only a subset of existing Nbs with affinity to Sap exhibit depolymerization activity, suggesting that affinity and epitope recognition are not enough to explain inhibitory activity. In this study, we performed all-atom molecular dynamics simulations of each Nb bound to the Sap binding site and trained a collection of machine learning classifiers to predict whether each Nb induces depolymerization. We used feature importance analysis to filter out unnecessary features and engineered remaining features to regularize the feature landscape and encourage learning of the depolymerization mechanism. We find that, while not enforced in training, a gradient-boosting decision tree is able to reproduce the experimental activities of inhibitory Nbs while maintaining high classification accuracy, whereas neural networks were only able to discriminate between classes. Further feature analysis revealed that inhibitory Nbs restrain Sap motions toward an inhibitory conformational state described by domain-domain clamping and induced twisting of domains normal to the lattice plane. We believe these motions drive Sap lattice depolymerization and can be used as design targets for improved Sap-inhibitory Nbs. Finally, we expect our method of study to apply to S-layers that serve as virulence factors in other pathogens, paving the way forward for Nb therapeutics that target depolymerization mechanisms.