LIGAND PROTEIN ELECTROSTATIC INTERACTIONS GOVERN THE SPECIFICITY OF RETINOL-BINDING AND FATTY ACID-BINDING PROTEINS

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 (K(d) = 0.2 muM). 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 C-13 resonance. Furthermore, the binding of fatty acid to both proteins was accompanied by a selective perturbation of the guanidino C-13 resonance of a single arginine residue. In contrast, the binding of fatty acids to I-FABP(R106Q) was characterized by a 20-fold lower affinity (K(d) = 4 muM), a low enthalpic contribution to the free energy of binding (13%), and a solvent-accessible environment for the carboxyl group of the bound fatty acid, as indicated by the complete ionization shift of its carboxyl C-13 resonance. Unlike wild-type I-FABP, I-FABP(R106Q) bound one molecule of retinol or retinal with high affinity. In conclusion, the distinct binding properties of I-FABP and CRBP-II result largely from their differing abilities to form an ion-pair interaction between the ligand and one key residue at position 106/109. The ability or inability to form a ligand-protein electrostatic interaction may be a major determinant of the specificites of different members of the intracellular lipid-binding protein family.