In this study, the binding of multimodal chromatographic ligands to the
IgG1 FC domain were studied using nuclear magnetic resonance and
molecular dynamics simulations. Nuclear magnetic resonance experiments
carried out with chromatographic ligands and a perdeuterated 15N-labeled
FC domain indicated that while single mode ion exchange ligands
interacted very weakly throughout the FC surface, multimodal ligands
interacted with specific clusters of residues with relatively high
affinity, forming distinct binding regions on the Fc. The multimodal
ligand binding sites on the FC were concentrated in the hinge region and
near the interface of the CH2 and CH3 domains. Further, 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 FC 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-FC 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-FC interactions and
sets the stage for future analyses of even more complex biotherapeutics.