Mapping the ligand binding site at protein side-chains in protein-ligand complexes through NOE difference spectroscopy

This report describes a novel NMR approach for mapping the interaction surf ace between an unlabeled ligand and a C-13,N-15-labeled protein. The method relies on the spin inversion properties of the dipolar relaxation pathways and records the differential relaxation of two spin modes, where ligand an d protein H-1 magnetizations are aligned either in a parallel or anti-paral lel manner. Selective inversion of protein protons is achieved in a straigh tforward manner by exploiting the one-bond heteronuclear scalar couplings ( (1)J(CH), (1)J(NH)). Suppression of indirect relaxation pathways mediated b y bulk water or rapidly exchanging protons is achieved by selective inversi on of the water signal in the middle of the NOESY mixing period. The method does not require deuteration of the protein or well separated spectral reg ions for the protein and the ligand, respectively. Additionally, in contras t to previous methods, the new experiment identifies side-chain enzyme liga nd interactions along the intermolecular binding interface. The method is d emonstrated with an application to the B-12-binding subunit of glutamate mu tase from Clostridium tetanomorphum for which NMR chemical shift changes up on B-12-nucleotide loop binding and a high-resolution solution structure ar e available.