Methyl probes in proteins for determining ligand binding mode in weak protein-ligand complexes

Structures of protein-ligand complexes provide critical information for drug design. Most protein-ligand complex structures are determined using X-ray crystallography, but where crystallography is not able to generate a structure for a complex, NMR is often the best alternative. However, the available tools to enable rapid and robust structure determination of protein-ligand complexes by NMR are currently limited. This leads to situations where projects are either discontinued or pursued without structural data, rendering the task more difficult. We previously reported the NMR Molecular Replacement (NMR2) approach that allows the structure of a protein-ligand complex to be determined without requiring the cumbersome task of protein resonance assignment. Herein, we describe the NMR2 approach to determine the binding pose of a small molecule in a weak protein-ligand complex by collecting sparse protein methyl-to-ligand NOEs from a selectively labeled protein sample and an unlabeled ligand. In the selective labeling scheme all methyl containing residues of the protein are protonated in an otherwise deuterated background. This allows measurement of intermolecular NOEs with greater sensitivity using standard NOESY pulse sequences instead of isotope-filtered NMR experiments. This labelling approach is well suited to the NMR2 approach and extends its utility to include larger protein-ligand complexes.

Automated summary

The structures of protein-ligand complexes are crucial for drug design, and are mainly determined using X-ray crystallography. However, when crystallography fails, NMR is often used as an alternative method. Unfortunately, the tools available for rapid and robust structure determination of protein-ligand complexes by NMR are currently limited. In this study, the NMR2 approach is described, which allows the determination of the binding pose of a small molecule in a weak protein-ligand complex by collecting sparse protein methyl-to-ligand NOEs from a selectively labeled protein sample and an unlabeled ligand. This approach extends the utility of NMR2 to larger protein-ligand complexes.