Hotspot Interactions between Two Fab Molecules in Molecular Dynamics Simulations Improve Predictive Models of Aggregation Kinetics

Protein-protein interactions (PPIs) are fundamental to numerous biological processes, and the identification of interaction hotspots is essential for understanding the mechanisms of protein aggregation and informing protein engineering efforts. Although various algorithms have been developed to predict hotspot regions for protein-protein interactions, little research has focused on understanding the relative roles of these largely protein surface-based interactions and the interactions between cross-β sheet-forming aggregation-prone regions (APRs) that are largely buried within proteins. This study uses all-atom molecular dynamics (MD) simulations to investigate the interactions between two Fab antibody fragments, focusing on the identification and characterization of the key interaction sites. Through frequency contact map analysis and principal component analysis, we identified specific residues that consistently formed stable contacts, distinguishing them from transient random interactions. Our findings revealed that while numerous contact points occurred throughout the simulations, relatively few sites acted as persistent hotspots based on the duration of their contacts. Comparison to single Fab simulations highlighted the influence of interfragment interactions on conformational dynamics. Inclusion of solvent accessibility for two surface hotspots, alongside one predicted APR, significantly improved models for predicting aggregation kinetics over 49 formulation conditions. The molecular-level insights gained will be important for guiding protein engineering strategies aimed at modulating these interactions to enhance product stability and retain therapeutic efficacy.