An improved, time-efficient approach to extract accurate distance restraints for NMR 2 structure calculation

Abstract. Exact Nuclear Overhauser Enhancement (eNOE) yields highly accurate, ensemble averaged 1H-1H distance restraints with an accuracy of up to 0.1 Å for the multi-state structure determination of proteins as well as for Nuclear Magnetic Resonance Molecular Replacement (NMR2) to determine the structure of the protein-ligand interaction site in a time-efficient manner. However, in the latter application, the acquired eNOEs lack the obtainable precision of 0.1 Å because of the asymmetrical nature of the filtered NOESY experiment used in NMR2. This error is further propagated to the eNOE equations used to fit for and extract the distance restraints. In this work, a new analysis method is proposed to obtain inter-molecular distance restraints from the filtered NOESY spectrum more accurately and intuitively by dividing the NOE cross-peak by the corresponding diagonal peak of the ligand. The method termed diagonal-normalized eNOEs was tested on the data acquired by Torres et al. (Torres et al., 2020) on the complex of PIN1 and a small, weak-binding phenylimidazole fragment. The diagonal-normalised eNOE derived distance restraints NMR2 yielded the right orientation of the fragment in the binding pocket, and produced a structure that more closely resembles the benchmark X-ray structure (2XP6) (Potter et al., 2010) with an average heavy atom RMSD of 1.681 Å than the one produced with traditional NMR2 with an average heavy atom RMSD of 3.628 Å, attributed to the higher precision of the evaluated distance restraints.