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.


Selection of irradiation frequencies.
Identification of residues in the binding pocket being directly irradiated, for proteins of known structures.
General considerations for DEEP-STD NMR studies: For proteins of known 3D structure, if their chemical shift assignments are known, directly irradiated protons can be easily identified in the binding pocket. In many cases, however, the protein chemical shifts are not available. In those cases, statistical averages of chemical shifts of the residues present in the binding pocket (from existing NMR databases) can be used. Alternatively, the 3D structure of the protein can be used to predict the chemical shifts using existing software.
In the present work, the selection of irradiation frequencies was based on predictions of chemical shifts using the available 3D structures of the proteins by using ShiftX2 (http://www.shiftx2.ca). [2] Additionally, the chemical shifts of selected protons on the proteins were also checked against average chemical shift histograms from the Biological Magnetic Resonance Data Bank (http://www.bmrb.wisc.edu/). [3] All molecular graphics were generated with Schrodinger Maestro

Exchange rate of polar protons and Differential D 2 O/H 2 O Epitope Mapping
In H 2 O, the exchange rate of exchangeable polar protons on amino acid side chains of the protein in the ligand binding pocket has a strong influence on the ability of these protons (and their adjacent non-exchangeable ones) to transfer magnetization to a bound ligand. In H 2 O, slow exchanging protons will contribute to transfer the magnetization from the binding site to the ligand. In contrast, in the case of fast exchanging protons, their exchange with the bulk water will be too fast for them to take part efficiently in the protein-ligand saturation transfer process. In addition, the magnetization of non-exchangeable protein protons in the binding pocket close in space to fast exchanging protein protons will be lost in the bulk water due to an efficient exchange-mediated magnetization leakage ( Figure S1). [4] Based on these premises, the presence of slow exchanging protons in the binding pocket will enhance the STD of ligands protons in close contact with them, if the experiment is performed in H 2 O as opposed to D 2 O (a red "+" in Figure S1). It is worth noting that the enhancement of STD is "relative"; that is, after comparison of the "binding epitopes" (relative normalized STDs) under the two conditions (H 2 O and D 2 O), and not by comparing the absolute STD values (see Equation-1 below).
The presence of "isolated" fast exchanging protons will not have a significant effect on the STD of the ligand protons when increasing the percentage of protonated sites over deuterated ones (in light water they will exchange fast and they will be "invisible" in terms of magnetization transfer). However, as mentioned above, the presence of fast exchanging protons near to nonexchangeable protein protons leads to leakage of the magnetization of the latter. What is more, if the latter is/are in close contact with ligand protons (X'H-H in Figure S1), the result will be a relative reduction of the STD of the closest ligand protons (a purple "-" in Figure S1). This

DEEP-STD data processing protocol
The first step in the analysis of DEEP-STD NMR data consists in determining which of the two experimental conditions, experiment-1 (exp1) or experiment-2 (exp2), produced stronger STD intensities (comparing their total sum of STD values). The experiment giving rise to stronger STD intensities will be called "experiment-1". Next, the ratio of STD intensities is calculated for each proton of the ligand. These ratios report not only on differences in the epitopes but also on the different global level of protein saturation achieved in both experiments (e.g., saturation on the aromatics leads to a reduced global saturation of the protein, as the number of aromatic residues is normally much lower than the number of aliphatic ones). For that reason, to obtain the differential epitope ("DEEP-STD map"), the intrinsic differences in protein saturation must be removed. This assures that the data reveal only those differences purely arising from the different types of amino acids hit by the saturating radiofrequency. To that aim, the average ratio of STDs over all protons must be calculated For very large multimeric protein complexes spin diffusion will play a prominent role, making the differences between different irradiations frequencies very small. Nevertheless, it might be still possible to pick them up, due to the "differential" nature of the determined epitope in the DEEP-STD NMR method. It is not possible to give an upper limit of applicability of the method as it will depend not only on the molecular weight of the receptor but also on the internal dynamics of the protein (increased internal mobility will reduce the effect of spin diffusion).

5.
STD raw and processed data     Epitope: no DEEP-STD map of the ligand was obtained. c) Crystal structure of the complex (PDB ID: 1EEI). [5] The slow exchangeable protons in the binding pocket are enclosed in a green surface. The ligand polar protons have been omitted.

CORCEMA-ST validation of Differential Epitope Mapping for RgNanH-GH33
We have used theoretical full matrix relaxation calculations implemented in CORCEMA-ST [6] to validate the DEEP-STD NMR approach. CORCEMA-ST allows to predict STD intensities of a protein-ligand complex given the Cartesian co-ordinates of all the partners in the binding equilibrium (free state ligand and protein, as well as bound state complex). We tried to reproduce the differential epitope mapping at 0.5 s for the complex 2,7-anhydro-Neu5Ac with GH33, running the CORCEMA-ST calculations simulating the two approaches experimentally followed: (i) differential frequency STD (0.60 ppm/6.55ppm; Figure