3-DIMENSIONAL STRUCTURE OF BOVINE HEART FATTY-ACID-BINDING PROTEIN WITH BOUND PALMITIC ACID, DETERMINED BY MULTIDIMENSIONAL NMR-SPECTROSCOPY

The three-dimensional structure of the hole form of recombinant cellular bovine heart fatty-acid-binding protein (H-FABP(c)), a polypeptide of 133 amino acid residues with a molecular mass of 15 kDa, has been determined by multidimensional homonuclear and heteronuclear NMR spectroscopy applied to uniformly N-15-labeled and unlabeled protein. A nearly complete set of H-1 and N-15 chemical shift assignments was obtained. A total of 2329 intramolecular distance constraints and 42 side-chain chi(1) dihedral-angle constraints were derived from cross-relaxation and J coupling information. 3D nuclear Overhauser enhancement and exchange spectroscopy combined with heteronuclear multiple-quantum coherence (NOESY-HMQC) experiments, performed on a sample of uniformly C-13-labeled palmitic acid bound to unlabeled cellular heart fatty-acid-binding protein revealed 10 intermolecular contacts that determine the orientation of the bound fatty acid. An ensemble of protein conformations was calculated with the distance-geometry algorithm for NMR applications (DIANA) using the redundant dihedral-angle constraint (REDAC) strategy. After docking the fatty acid into the protein, the protein-ligand arrangement was subject to distance-restrained energy minimization. The overall conformation of the protein is a beta-barrel consisting of 10 antiparallel beta-strands which form two nearly orthogonal beta-sheets of five strands each. Two short helices form a helix-turn-helix motif in the N-terminal region of the polypeptide chain. The palmitic acid is bound within the protein in a U-shaped conformation close to the two helices. The obtained solution structure of the protein is consistent with a number of fatty-acid-binding-protein crystal structures.