1H and 15N resonance assignments and secondary structure of cellular retinoic acid-binding protein with and without bound ligand.
Sequence-specific assignments for the 1H and 15N backbone resonances of cellular retinoic acid-binding protein (CRABP), with and without the bound ligand, have been obtained. Most of the side-chain resonances of both apo- and holo-CRABP have also been assigned. The assignments have been obtained using two-dimensional homonuclear and heteronuclear NMR data, and three-dimensional 1H-15N TOCSY-HMQC and NOESY-HMQC experiments. The secondary structure, deduced from nuclear Overhauser effects, amide H/D exchange rates and H alpha chemical shifts, is analogous in both forms of the protein and is completely consistent with a model of CRABP that had been constructed by homology with the crystal structure of myelin P2 protein [Zhang et al. (1992) Protein Struct. Funct. Genet., 13, 87-99]. This model comprises two five-stranded beta-sheets that form a sandwich or beta-clam structure, and a short N-terminal helix-turn-helix motif that closes the binding cavity between the two sheets. Comparison of the data obtained for apo- and holo-CRABP indicates that a region around the C-terminus of the second helix is much more flexible in the apo-protein. Our data provide experimental evidence for the hypothesis that the ligand-binding mechanism of CRABP, and of other homologous proteins that bind hydrophobic ligands in the cytoplasm, involves opening of a portal to allow entry of the ligand into the cavity.
Rizo
J
J
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235-9041.
Liu
Z P
ZP
Gierasch
L M
LM
D000595:Amino Acid Sequence; D006859:Hydrogen / Q000737:chemistry; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009590:Nitrogen Radioisotopes / Q000737:chemistry; D017433:Protein Structure, Secondary; D018168:Receptors, Retinoic Acid / Q000737:chemistry*
D008024:Ligands; D009590:Nitrogen Radioisotopes; D018168:Receptors, Retinoic Acid; D006859:Hydrogen