where fu and fb are the fractions of the unbound and bound protein, respectively, and δu and δb are the chemical shift values of the unbound and bound states of the protein. The linewidths were largely free of exchange broadening and had ligand-dependent chemical shift values similar to those without ligands. This was indicative of ‘fast-exchange’ behavior for protein-ligand interactions. The changes in combined (1H and 15N) chemical shift (∆δNH) were then calculated using equation 1.
An important first step towards binding analysis was to evaluate the CSPs for all the residues in the protein sequence. Accordingly, the plots of the observed CSPs at the highest concentrations of the SP Sepharose, Capto MMC and Nuvia cPrime ligands are presented in Figures 4a, b & c, respectively (note: the Fc residues 1-228 correspond to the IgG1 residues 220-447).The secondary structural elements of the FC sequence are also shown at the bottom of Figure 4a. The data were analyzed using the statistical method described by Schumann et al. (Schumann et al., 2007) to determine the corrected standard deviation σ0. The residues with CSPs smaller than σ0 were grouped as non-interacting (noise) and are presented as grey bars in the Figures. Residues exhibiting large CSPs are represented in red bars while residues with intermediate changes are shown in a red-grey color gradient. As can be seen in Figure 4a, for interactions of the SP Sepharose ligand to the FC, residues that experienced large changes in CSPs were spread across the entire protein sequence and did not group into a specific region. In addition, residues that exhibited intermediate changes were not localized around residues that showed large CSPs. In contrast to the single mode CEX ligand results, both the large and intermediate CSPs associated with the binding of MM CEX ligands were clearly clustered in specific regions in the FC sequence (Figures 4b & c). Interestingly, the residues experiencing these CSPs were primarily located in clusters associated with more flexible regions of the protein. For example, by comparing these data with the secondary structural elements of the FC, it can be seen that the clusters observed near residues 29-38 and 87-98 (Figures 4b & c) were associated with either loops or a combination of loops and α-helices. While the CSPs for the CEX and MM CEX systems were quite different, the changes within the MM CEX systems for Capto MMC and Nuvia cPrime were very similar. This was quantified by the Pearson correlation coefficient which was calculated to be 0.15 ± 0.03 for the CEX and each of the MM CEX systems and 0.85 for the Capto MMC and Nuvia cPrime systems (Figure S1).
To further visualize these ligand-induced changes in combined chemical shift, the CSPs for the residues were visualized on the protein surface using the PyMol viewer. A cartoon and a surface representation of these results for the three ligands studied are presented in Figures 5a and 6, respectively. To facilitate the discussion, the color schemes used in these figures are the same employed in Figure 4. As can be seen in Figure 5a, for interactions of the SP Sepharose ligand with the FC, residues that exhibited large and intermediate CSPs were spread across the entire protein surface. Further, as expected for a cation exchange ligand such as SP Sepharose, the residues that experienced CSPs were often associated with positively charged (blue colored) regions on the electrostatic potential (EP) map of the FC shown in Figure 5b.
In contrast, the results for binding of the Capto MMC and Nuvia cPrime ligands with the FC, indicated a more focused interaction region as highlighted by the dotted blue ellipses of Figures 6a and b, respectively. Interestingly, the residues that interacted with the MM ligands and were present in the flexible loops and alpha helices (as discussed above for Figure 4) were located in the interface of the CH2 and CH3 domains. Further, residues that experienced ligand-induced CSPs were mostly identified to be positively charged and/or aliphatic or aromatic in nature. In order to examine this in more detail, the focused interaction region for the MM ligands was also indicated on the EP and SAP maps presented in Figure 5b and c. As can be seen in the figures, this region was associated with both positive EP and some hydrophobicity which is expected for the interactions of multimodal ligands (Karkov, Woo, Krogh, Ahmadian, & Cramer, 2015).
Interpretation of the CSP data does come with a few caveats. For example, residues that are in close proximity of those directly interacting with the ligands may also undergo significant changes in chemical shift. In addition, residues interacting with a ligand may not experience significant changes in electronic environment and thus may not have a measurable CSP. In order to address these issues, the changes in combined chemical shift during the titration experiments were examined in more detail.
A few representative spectral overlays for the binding of the Nuvia cPrime ligand to FC are presented in Figure 7 and the corresponding CSPs as a function of ligand concentration are shown in Figure S2. As can be seen in the figures, different trajectories of the CSPs were observed with increasing ligand concentration for each of the residues. For example, residue Asp 182 (Figures 7a and S2a) did not exhibit any significant shift with increasing ligand concentration, indicating that it likely did not participate in binding. For residues that did show measurable CSPs, we observed primarily linear migrations with both saturated and unsaturated behavior. As can be seen in Figures 7b and S2b, Val 178 exhibited linear migration without saturation, which could be due to non-specific and/or weak interactions of the ligand with this residue. On the other hand, for His 66 (Figures 7c & S2c), a saturating linear trajectory was observed which was indicative of a simple two-state binding behavior that could be readily fit to the Langmuir equation (equation 2) to determine the binding dissociation constant (KD).
The CSP data at different ligand concentrations for all the FC residues were fit to equation 2 using Matlab R2019b with a maximum fitting error of 10% in a 95% confidence interval. For interactions of the SP Sepharose ligand with the FC, residues that showed saturation behavior exhibited high KD values (> 150 mM) which were indicative of very weak interactions. Further, the SP Sepharose ligand was observed to interact throughout the FC surface without clustering into specific regions (results not shown).
Due to solubility constraints with the Capto MMC ligand (≤ 3.2 mM at pH 5), the KD determination with the multimodal ligands was limited to the more soluble Nuvia cPrime ligand. The CSP data at different Nuvia cPrime ligand concentrations for all the FC residues were fit to equation 2 to determine the residue specific KD values which are presented in Table S1. Based on the maximum solubility of the Nuvia cPrime ligand (60 mM at pH 5), only KD values below 40mM were able to be accurately determined. As can be seen in the Table, the resulting KD values were up to two orders of magnitude smaller than those observed with the single mode SP Sepharose ligand. These results qualitatively agree with the marked differences observed in the elution salt concentrations, 0.12 and 0.63 M NaCl, for FC in SP Sepharose and Nuvia cPrime chromatographic systems, respectively.
The FC residues that exhibited saturation behavior for interactions with the Nuvia cPrime ligand were color coded based on their KD values and the resulting projections on the protein surface are presented in Figure 8. As can be seen in the Figure, residues interacting with a “high” binding affinity (1.2 ≤ KD ≤ 10 mM) were primarily located in the hinge region (green ellipse) and near the interface of the CH2 and CH3 domains (blue ellipse) (note: these regions are the same as those in Figure 6 which were based on the CSPs). The strong interactions with a cluster of positively charged (His 66, His 5, Lys 3 and Lys 55) and polar (Thr 4, Thr 6 and Cys 7) residues in the hinge region is indicative of this being a preferred binding region for the ligand. However, it is important to note that interactions observed near the flexible hinge region may be due to direct participation of the residues in ligand binding and/or NMR shifts due to ligand binding induced conformational changes in that region of the FC. Further, while the hinge region was identified by NMR as a preferred binding region for the ligand, steric affects would likely impact ligand interactions with the FC when present in an intact mAb and/or Fc fusion proteins.
As was observed with the CSP data (Figure 6), the KDresults also indicated that the interface of the CH2 and CH3 domains was an important preferred binding region for interacting with the Nuvia cPrime ligand (Figure 8). Residues involved in binding to the Nuvia cPrime ligand in this region are indicated in Table S1 with a #. As can be seen in the table, this contiguous MM ligand binding region was composed of positively charged (His 214, Arg 36, His 210 and His 91), polar (Asn 215, Gln 219, Thr 31, Thr 218, Thr 37, Gln 92, Ser 221, Ser 35 and Gly 166) and aliphatic (Leu 32, Ile 34, Ala 159, Val 160 and Met 33) residues. In this region, residue 214H interacted with one of the lowest KD values (3.94 ± 2.1 mM), indicating relatively strong binding to the ligand. Interestingly, this histidine residue has also been shown to be one of the most important residues for binding of the FCportion of IgG1 mAbs to Protein A resins (Idusogie et al., 2000; Moiani et al., 2009). As can be seen in Table S1, a few negatively charged residues (e.g. Glu 211 and Asp 93) also exhibited NMR shifts which may be due to their proximity to the clusters of positively charged and polar residues discussed above.
As was discussed above with the CSP results, this preferred binding region at the interface of the CH2 and CH3 domains (indicated by the blue ellipse) corresponded to an overlapping region of positive EP and hydrophobicity (Figures 5b and c). The fact that the KD results presented in Figure 8 are even more focused in this region lends further support to the importance of both types of interactions in MM systems.

Molecular Dynamics Simulations

The NMR experiments described so far provide a window into protein-multimodal ligand interactions on a molecular level. While NMR experiments yield molecularly-detailed data, they are time- and resource-intensive making it difficult to quickly apply these tools to a wide range of systems. In order to more deeply investigate the FC-ligand interactions at the molecular level and to explore some of the docked conformations of the Nuvia cPrime ligands, we carried out MD simulations. As described in the Methods section, simulations were performed with multiple ligand copies in free solution with the protein, and throughout the simulation ligands were free to reversibly interact from the protein surface. The free energy of a ligand binding to a given region of the protein surface was calculated as: