Ligand ¹ H NMR Chemical Shifts as Accurate Reporters for Protein-Ligand Binding Interfaces in Solution

The availability of high-resolution 3D structural information is crucial for investigating guest-host systems across a wide range of fields. In the context of drug discovery, the information is routinely used to establish and validate structure-activity relationships, grow initial hits from screening campaigns, and to guide molecular docking. For the generation of protein-ligand complex structural information, X-ray crystallography is the experimental method of choice, however, with limited information on protein flexibility. An experimentally verified structural model of the binding interface in the native solution-state would support medicinal chemists in their molecular design decisions. Here we demonstrate that protein-bound ligand ¹ H NMR chemical shifts are highly sensitive and accurate probes for the immediate chemical environment of protein-ligand interfaces. By comparing the experimental ligand ¹ H chemical shift values with those computed from the X-ray structure using quantum mechanics methodology, we identify significant disagreements for parts of the ligand between the two experimental techniques. We show that quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) ensembles can be used to refine initial X-ray co-crystal structures resulting in a better agreement with experimental ¹ H ligand chemical shift values. Overall, our findings highlight the usefulness of ligand ¹ H NMR chemical shift information in combination with a QM/MM MD workflow for generating protein-ligand ensembles that accurately reproduce solution structural data.