Probing multimodal ligand binding regions on ubiquitin using nuclear magnetic resonance, chromatography, and...

Site-directed mutagenesis, nuclear magnetic resonance (NMR) chemical shift perturbation experiments, and molecular dynamics (MD) simulations are employed in concert with chromatographic experiments to provide insight into protein-ligand interactions in multimodal chromatographic systems. In previous studies, a preferred binding region was identified on the surface of the protein ubiquitin for binding with a multimodal ligand. In this study, site-directed mutagenesis is used to enable direct NMR evaluation of the mutant protein as compared to the wild type. It is found that reversing the charge of a key residue (K6E) in the proposed preferred binding region results in substantial decreases in the magnitude of the ligand-induced NMR chemical shift perturbations relative to those detected for the wild type protein, particularly for residues located within the preferred binding region. These NMR results also indicate a decrease in ligand affinity, consistent with the weaker chromatographic retention observed for the mutant as compared to the wild type on a multimodal cation exchange resin. MD simulation results provide additional insight at a molecular level and demonstrate that many residues located within the preferred binding region exhibit weaker binding interactions due to the mutation. The analysis suggests that multimodal ligand binding consists of initial localization of the ligand by long-ranged electrostatic interactions followed by multiple short-ranged synergistic interactions to attain high affinities of the ligand to specific residues.