Identification of preferred multimodal ligand‐binding regions on IgG1 F_C using nuclear magnetic resonance and molecular dynamics simulations

Abstract

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 containing negatively charged and aromatic moieties 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. 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 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 π–π 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.

Probing IgG1 FC–Multimodal Nanoparticle Interactions: A Combined Nuclear Magnetic Resonance and Molecular Dynamics Simulations Approach

“…To facilitate the discussion, both the cartoon and surface representations of the protein surface are presented in the figure. As can be seen, the μM K D,app values obtained from NMR experiments with the NPs (Figure ) were 3 orders of magnitude smaller than those obtained previously with these ligands in solution . These results clearly indicate enhanced binding of the F C to MM-functionalized surfaces.…”

“…Briefly, residues that exhibited linear migration trajectories with saturation behavior were fit to the N-site binding model, as described in eq . As described in the Materials and Methods section, a geometric analysis was carried out to estimate the maximum number of Fc molecules that could bind to a single NP, assuming that the side face of the F C was the primary interacting binding region, as has been reported previously by our group . The results of this analysis indicated that there were at most 26 (N) binding sites (note: this calculation assumed that multiple ligand head groups could interact with a single residue/cluster of residues on the F C surface).…”

“…NMR experiments were performed with 15 N-labeled F C and “Capto ligand”- and “Nuvia ligand”-functionalized NPs at both high- and low-ligand densities. The CSP data at different NP concentrations for all F C residues were analyzed using the method described previously . Briefly, residues that exhibited linear migration trajectories with saturation behavior were fit to the N-site binding model, as described in eq .…”

“…This study was then extended to HCIC ligands on surfaces to evaluate ligand-density effects and to elucidate the binding mechanisms of F C in these systems at various pH conditions . We have recently shown that the MM ligands in free solution have preferential interaction sites in the hinge region and in the C H 2/C H 3 interface on the F C surface and that the binding affinities were in the mM range . While this study showed preferred MM interaction sites on the F C surface, it did not account for protein binding to MM surfaces.…”

Systematic workflow for studying domain contributions of bispecific antibodies to selectivity in multimodal chromatography

“…While these biophysical studies provided valuable insights into the nature of protein-ligand interactions in multimodal systems, the work had been limited to small model proteins. Recently, our group has employed NMR to identify preferred binding regions for IgG1 Fc interacting with multimodal cation exchange (MM CEX) and CEX resin systems (Gudhka et al, 2021).…”