NMR study of the binding of all- trans-retinoic acid to type II human cellular retinoic acid binding protein

Abstract

Cellular RA binding proteins are thought to play important roles in the metabolism of retinoic acid (RA), a hormonally active metabolite of vitamin A that has profound effects on cell growth, differentiation and morphogenesis. Binding of RA to type II human cellular RA binding proteins (CRABPII) has been investigated by NMR spectroscopy. The sequential resonance assignments of CRABPII in the presence of RA were established by heteronuclear three-dimensional NMR at pH 7.3. The resonance assignments of the bound RA were achieved by homonuclear two-dimensional NMR. The secondary structures of holo-CRABPII determined by NMR were essentially the same as revealed by the crystal structure of holo-CRABPII. Most of the nuclear Overhauser effects (NOEs) between CRABPII and the bound RA were consistent with those predicted from the crystal structure of holo-CRABPII. The results suggested that the conformations of holo-CRABPII in solution and in the crystalline state are highly similar. Compared to apo-CRABPII, most part of the ligand binding pocket, especially the ligand entrance, was stabilized greatly by binding of RA. Ser12-Leu18, one of the structure elements that constitute the ligand binding pocket, became more mobile upon binding of RA. Intramolecular NOEs between protons of the bound RA indicated that the carboxylate end of the bound RA is well fixed but the β-ionone ring is mobile.