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

Abstract

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 1H NMR chemical shifts are highly sensitive and accurate probes for the immediate chemical environment of protein‐ligand interfaces. By comparing the experimental ligand 1H 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 1H ligand chemical shift values. Overall, our findings highlight the usefulness of ligand 1H NMR chemical shift information in combination with a QM/MM MD workflow for generating protein‐ligand ensembles that accurately reproduce solution structural data.