Biotechnology and Bioengineering, Vol.118, No.2, 809-822, 2021
Identification of preferred multimodal ligand-binding regions on IgG1 F-C using nuclear magnetic resonance and molecular dynamics simulations
In this study, the binding of multimodal chromatographic ligands to the IgG1 F-C domain were studied using nuclear magnetic resonance and molecular dynamics simulations. Nuclear magnetic resonance experiments carried out with chromatographic ligands and a perdeuterated N-15-labeled F-C domain indicated that while single-mode ion exchange ligands interacted very weakly throughout the F-C surface, multimodal ligands containing negatively charged and aromatic moieties interacted with specific clusters of residues with relatively high affinity, forming distinct binding regions on the F-C. The multimodal ligand-binding sites on the F-C were concentrated in the hinge region and near the interface of the C(H)2 and C(H)3 domains. Furthermore, the multimodal binding sites were primarily composed of positively charged, polar, and aliphatic residues in these regions, with histidine residues exhibiting some of the strongest binding affinities with the multimodal ligand. Interestingly, comparison of protein surface property data with ligand interaction sites indicated that the patch analysis on F-C corroborated molecular-level binding information obtained from the nuclear magnetic resonance experiments. Finally, molecular dynamics simulation results were shown to be qualitatively consistent with the nuclear magnetic resonance results and to provide further insights into the binding mechanisms. An important contribution to multimodal ligand-F-C binding in these preferred regions was shown to be electrostatic interactions and pi-pi stacking of surface-exposed histidines with the ligands. This combined biophysical and simulation approach has provided a deeper molecular-level understanding of multimodal ligand-F-C interactions and sets the stage for future analyses of even more complex biotherapeutics.