Solid-state nuclear magnetic resonance analysis of the conformation of an inhibitor bound to thermolysin.

A number of structural experimental methods are available to determine the receptor-bound conformation of ligands as part of the process of rational drug design, including X-ray diffraction and solution-state NMR. Not all receptor/ligand systems are amenable to these types of analyses due to difficulties in sample preparation or inherent limitations of the methods. Rotational echo double-resonance (REDOR) NMR is a solid-state, magic angle-spinning technique that measures the dipolar coupling between specifically labeled nuclei and enables the determination of internuclear distance. In previous studies of helical peptides, we have verified the ability of REDOR NMR to measure distances accurately and precisely. In this study we use REDOR and double REDOR to measure distances between backbone atoms in a phosphonamidate transition-state inhibitor bound to thermolysin. The 31P-13C', 31P-15N, and 31P-13C alpha distances (3.61 +/- 0.10, 3.89 +/- 0.12, and 5.37 +/- 0.13 A, respectively) measured in a complex of Cbz-GlyP-[1-13C]Leu-[15N,2-13C]Ala and the enzyme are consistent with those observed by X-ray diffraction in other comparable thermolysin/inhibitor complexes (average values of 3.58 +/- 0.04, 3.91 +/- 0.13, and 5.17 +/- 0.18 A, respectively). These results demonstrate that REDOR NMR is a viable alternative to more traditional methods such as X-ray diffraction, transferred NOESY, and isotope-edited NOESY for characterizing the receptor-bound conformation of ligands.