Protein-bound conformation of a specific inhibitor against Candida albicans myristoyl-CoA : protein N-myristoyltransferase in the ternary complex with CaNmt and myristoyl-CoA by transferred NOE measurements

Transferred nuclear Overhauser enhancement (trNOE) experiments have been pe rformed to study the bioactive conformation(s) of Ro09-3472/000 derivatives in the ternary complex with Candida albicans myristoyl-CoA: protein N-myri stoyltransferase (CaNmt) and myristoylCoA (MyrCoA). A critical step in the trNOE study is to identify 'true' trNOEs in the spectra. Nonspecific bindin g of ligands to target proteins and/or spin diffusion effects can give rise to 'false' trNOEs, which may lead to an incorrect conclusion if used to de rive bound conformations. In this study for all ligands the observed trNOEs arose from specific binding interactions with the active site of CaNmt. Th is was shown by displacing the ligand with the known tightly binding active -site inhibitor 1 [Devadas, B., Zupec, M.E., Freeman, S.K., Brown, D.L., Na garajan, S., Sikorski, J.A., McWherter, C.A., Getman, D. P. & Gordon, J.I. (1995) J. Med. Chem. 38, 1837-1840] and measuring the resonance linewidths in the NMR spectrum before and after addition of the competitive inhibitor. The compounds were also tested for nonspecific protein binding with bovine serum albumin (BSA) using the same method. Of the six compounds tested, Ro 09-3700/001 (racemate) and its optically pure enantiomers, Ro09-4764/001(S) and Ro09-4765/001(R), showed both specific binding to CaNmt and no interac tion with BSA. The NMR data of these molecules in the ternary complex with CaNmt/MyrCoA could thus be used for a detailed structural analysis. Thereby , the conformation of the bound ligand was obtained from a conformational s earch using the observed trNOEs as a selection filter. The NMR-determined c onformations are in good agreement with the recently solved CaNmt-bound X-r ay structures of two similar Ro09-3472/000 derivatives.