Mutating the charged residues in the binding pocket of cellular retinoic acid‐binding protein simultaneously reduces its binding affinity to retinoic acid and increases its thermostability

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Abstract
Three‐dimensional modeling of the complex between retinoic acid‐binding protein (CRABP) and retinoic acid suggests thatbinding of the ligand is mediated by interaction between the carboxyl groupof retinoic acid and two charged amino acids (Arg‐111 and Arg‐131) whose side chains project into the barrel of the protein. To assess the contribution of these amino acids to protein–ligand interaction, amino acid substitutions were made by oligonucleotide‐directed, site‐specific mutagenesis. The wild‐type and mutant proteins were expressed in E. coli and subsequently purified. Like wild‐type CRABP, the mutant proteins are composed mainly of β‐strands as determined by circular dichroism in the presence and absence of ligand, and thus presumably are folded into the same compact barrel structure as the wild‐type protein. Mutants in which Arg‐111 and Arg‐131 are replaced by glutamine bind retinoic acid with significantly lower affinity than the wild‐type protein, arguing that these two residues indeed interact with the ligand. The mutant proteins are more resistant to thermal denaturation than wild‐type CRABP in the absence of retinoic acid, but they are not as thermostable as the CRABP–retinoic acid complex. These data suggest a model for CRABP–retinoic acid interaction in which the repulsive forces between the positively‐charged arginine residues provide conformational flexibility to the native protein for retinoic acid to enter the binding pocket. Elimination of the positively‐charged pair of amino acids produces a protein that is more thermostable than wild‐type CRABP but less effective at ligand‐binding.