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

Abstract

In this study, NMR and molecular dynamics simulations were employed to study IgG1 F C binding to multimodal surfaces. Gold nanoparticles functionalized with two multimodal cation-exchange ligands (Capto and Nuvia) were synthesized and employed to carry out solutionphase NMR experiments with the F C . Experiments with perdeuterated 15 N-labeled F C and the multimodal surfaces revealed micromolar residuelevel binding affinities as compared to millimolar binding affinities with these ligands in free solution, likely due to cooperativity and avidity effects. The binding of F C with the Capto ligand nanoparticles was concentrated near an aliphatic cluster in the C H 2/C H 3 interface, which corresponded to a focused hydrophobic region. In contrast, binding with the Nuvia ligand nanoparticles was more diffuse and corresponded to a large contiguous positive electrostatic potential region on the side face of the F C . Results with lower-ligand-density nanoparticles indicated a decrease in binding affinity for both systems. For the Capto ligand system, several aliphatic residues on the F C that were important for binding to the higher-density surface did not interact with the lower-density nanoparticles. In contrast, no significant difference was observed in the interacting residues on the F C to the high-and low-ligand density Nuvia surfaces. The binding affinities of F C to both multimodal-functionalized nanoparticles decreased in the presence of salt due to the screening of multiple weak interactions of polar and positively charged residues. For the Capto ligand nanoparticle system, this resulted in an even more focused hydrophobic binding region in the interface of the C H 2 and C H 3 domains. Interestingly, for the Nuvia ligand nanoparticles, the presence of salt resulted in a large transition from a diffuse binding region to the same focused binding region determined for Capto nanoparticles at 150 mM salt. Molecular dynamics simulations corroborated the NMR results and provided important insights into the molecular basis of F C binding to these different multimodal systems containing clustered (observed at high-ligand densities) and nonclustered ligand surfaces. This combined biophysical and simulation approach provided significant insights into the interactions of F C with multimodal surfaces and sets the stage for future analyses with even more complex biotherapeutics.

Evaluation of preferred binding regions on ubiquitin and IgG1 FC for interacting with multimodal cation exchange resins using DEPC labeling/mass spectrometry

“…Figure 8 presents the DEPC results superimposed (highlighted in yellow) on previously published data obtained with NMR (Gudhka, Vats, et al, 2021). In that work, we employed isotopically labeled F C and gold nanoparticles immobilized with Capto MMC ligands as a surrogate to study F C binding domains to solid MM surfaces.…”

“…Figure 8 presents the DEPC results superimposed (highlighted in yellow) on previously published data obtained with NMR (Gudhka, Vats, et al, 2021). In that work, we employed isotopically labeled identified as preferred binding regions by NMR, which was also the case when using the DEPC labeling approach.…”

“…While the structural model of Ubiquitin was used from the protein database (PDB code: 1 Ubq), the structure of F C was generated by homology modeling as previously described in Gudhka, Bilodeau, et al (2021) Gudhka, Vats, et al (2021). The electrostatic potential (EP) maps for both Ubiquitin and F C were generated at pH 5.5 using the APBS linear Poisson Boltzmann Solver (APBS) (Baker et al, 2001).…”

“…Kittelmann et al developed QSAR models to capture the binding behavior of proteins on single mode charged surfaces and employed descriptors based on this work to further probe these protein binding orientations (Kittelmann et al, 2017a, 2017b). Previous work in our lab has involved the use of several biophysical techniques such as NMR (Chung, Freed, et al, 2010; Gudhka, Bilodeau, et al, 2021; Gudhka, Vats, et al, 2021; Holstein, Chung, et al, 2012; Holstein, Parimal, et al, 2012; Srinivasan et al, 2014), ITC (Srinivasan et al, 2014), atomic force microscopy (AFM) (Srinivasan et al, 2017), QSM (Srinivasan et al, 2018) as well as molecular dynamic simulations (Banerjee et al, 2017; Freed & Cramer, 2011) to study protein–ligand interactions. A combination of AFM and MD simulations has been used to evaluate hypothesized binding regions and to quantify interaction energies.…”

“…A combination of AFM and MD simulations has been used to evaluate hypothesized binding regions and to quantify interaction energies. NMR studies with isotopically labeled ubiquitin (Srinivasan et al, 2014) and IgG1 F C proteins (Gudhka, Vats, et al, 2021) were also carried out using MM ligand functionalized gold nanoparticles to identify binding regions and to evaluate the impact of avidity and cooperativity effects in these systems. Although NMR has been successful in identifying protein binding regions, there are several challenges associated with this approach such as the availability of isotopically labeled proteins and the need for prior protein backbone assignments.…”