Differential Epitope Mapping by STD NMR Spectroscopy To Reveal the Nature of Protein–Ligand Contacts

Abstract

Saturation transfer difference (STD) NMR spectroscopy is extensively used to obtain epitope maps of ligands binding to protein receptors, thereby revealing structural details of the interaction, which is key to direct lead optimization efforts in drug discovery. However, it does not give information about the nature of the amino acids surrounding the ligand in the binding pocket. Herein, we report the development of the novel method differential epitope mapping by STD NMR (DEEP‐STD NMR) for identifying the type of protein residues contacting the ligand. The method produces differential epitope maps through 1) differential frequency STD NMR and/or 2) differential solvent (D2O/H2O) STD NMR experiments. The two approaches provide different complementary information on the binding pocket. We demonstrate that DEEP‐STD NMR can be used to readily obtain pharmacophore information on the protein. Furthermore, if the 3D structure of the protein is known, this information also helps in orienting the ligand in the binding pocket.

Deriving Ligand Orientation in Weak Protein–Ligand Complexes by DEEP‐STD NMR Spectroscopy in the Absence of Protein Chemical‐Shift Assignment

“…These resonances, although lacking a specific assignment, are typical of aliphatic and aromatic amino acids, and we can exclude the presence of the NH resonances in the spectra as the protein was solvated in a D 2 O buffer. The identified resonances from the TEMPOL‐attenuated TOCSY spectra of Rg NanH‐GH33 were indeed in very good agreement with the predicted chemical shifts of key aliphatic and aromatic residues in the binding pocket of the enzyme (Ile258, Ile338, Val502, Thr557, Tyr525, Tyr677 and Trp698) …”

“…Recently, we developed the DiffErential EPitope mapping saturation transfer difference (DEEP‐STD) NMR methodology for weak protein–ligand interactions, as an extension of the general STD NMR method . The DEEP‐STD NMR technique allows the orientation of the ligand to be derived through differential selective saturation of different sets of key protein residues in the binding site.…”

“…For the development of our protocol, we combined this approach with DEEP‐STD NMR using the structurally characterized catalytic domain (belonging to glycoside hydrolase family 33, GH33) of the intramolecular trans ‐sialidase (IT‐sialidase) from the human gut symbiont Ruminococcus gnavus , Rg NanH‐GH33, in complex with 2,7‐anhydro‐Neu5Ac (PDB ID: 4X4A) as a benchmark . Rg NanH‐GH33 is a 489‐residue domain that can be considered out of the typical range for swift assignment and structure determination by NMR spectroscopy and was previously used to develop the DEEP‐STD NMR approach …”

“…As the absence of chemical‐shift assignment of the protein prevents the irradiation frequencies to specific protons in the binding pocket from being known, we propose here a novel approach: instead of using a single pair of frequencies to determine the differential epitope map of the ligand (DEEP‐STD map), an averaging approach should be followed. First, the DEEP‐STD factors for each experiment resulting from all the possible pairs of aliphatic and aromatic frequencies experimentally identified before are calculated (Figure S3).…”

“… Abstract Differential epitope mapping saturation transfer difference (DEEP‐STD) NMR spectroscopy is a recently developed powerful approach for elucidating the structure and pharmacophore of weak protein–ligand interactions, as it reports key information on the orientation of the ligand and the architecture of the binding pocket . The method relies on selective saturation of protein residues in the binding site and the generation of a differential epitope map by observing the ligand, which depicts the nature of the protein residues making contact with the ligand in the bound state.…”