title: Ligand-protein electrostatic interactions govern the specificity of retinol- and fatty acid-binding proteins.
abstract: Cellular retinol-binding protein II (CRBP-II) and intestinal fatty acid-binding protein (I-FABP) are both expressed in small intestinal enterocytes and exhibit 31% sequence identity. I-FABP binds a single molecule of long-chain fatty acid and forms an ion-pair electrostatic interaction between the cationic side chain of arginine-106 and the anionic fatty acid carboxyl group. In contrast, CRBP-II binds all-trans-retinol or -retinal and contains a glutamine residue in the corresponding position, residue 109. We have characterized and compared the interactions of fatty acids and retinoids with I-FABP, CRBP-II, and two reciprocal mutant proteins. The mutants were designated CRBP-II(Q109R), where glutamine-109 was replaced by arginine, and I-FABP(R106Q), where arginine-106 was replaced by glutamine. As monitored by titration calorimetry and carbon-13 NMR spectroscopy, the fatty acid-binding properties of CRBP-II(Q109R) were found to be essentially identical to those of wild-type I-FABP. Both proteins bound 1 molecule of fatty acid with identical affinities (Kd = 0.2 microM). The enthalpic contribution to the total free energy of binding was large for both proteins: 66% and 87%, respectively. In addition, the carboxyl groups of fatty acids bound to both proteins were solvent-inaccessible. There was little or no change in the ionization state of the bound fatty acid over a wide pH range, as monitored by the chemical shift of the fatty acid carboxyl 13C resonance. Furthermore, the binding of fatty acid to both proteins was accompanied by a selective perturbation of the guanidino 13C resonance of a single arginine residue.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: M G
authors.initials: MG
authors.lastname: Jakoby
authors.forename: K R
authors.initials: KR
authors.lastname: Miller
authors.forename: J J
authors.initials: JJ
authors.lastname: Toner
authors.forename: A
authors.initials: A
authors.lastname: Bauman
authors.forename: L
authors.initials: L
authors.lastname: Cheng
authors.forename: E
authors.initials: E
authors.lastname: Li
authors.forename: D P
authors.initials: DP
authors.lastname: Cistola
mesh_terms: D000818:Animals; D001120:Arginine; D002151:Calorimetry; D002247:Carbon Isotopes; D002352:Carrier Proteins / Q000378:metabolism*; D004560:Electricity; D004926:Escherichia coli / Q000235:genetics; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism*; D005973:Glutamine; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012172:Retinaldehyde / Q000378:metabolism; D012177:Retinol-Binding Proteins / Q000378:metabolism*; D054840:Retinol-Binding Proteins, Cellular; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014801:Vitamin A / Q000378:metabolism
chemical_list: D002247:Carbon Isotopes; D002352:Carrier Proteins; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D008024:Ligands; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; C518461:Rbp2 protein, rat; D011994:Recombinant Proteins; D012177:Retinol-Binding Proteins; D054840:Retinol-Binding Proteins, Cellular; D005973:Glutamine; D014801:Vitamin A; D001120:Arginine; D012172:Retinaldehyde