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.
Three-dimensional modeling of the complex between retinoic acid-binding protein (CRABP) and retinoic acid suggests that binding of the ligand is mediated by interaction between the carboxyl group of 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 beta-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.
Zhang
J
J
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
Liu
Z P
ZP
Jones
T A
TA
Gierasch
L M
LM
Sambrook
J F
JF
D000595:Amino Acid Sequence; D001120:Arginine; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D050556:Fatty Acid-Binding Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D004676:Myelin Basic Protein / Q000235:genetics; D018996:Myelin P2 Protein; D009363:Neoplasm Proteins; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D018168:Receptors, Retinoic Acid; D012689:Sequence Homology, Nucleic Acid; D014212:Tretinoin / Q000378:metabolism*
D002352:Carrier Proteins; D050556:Fatty Acid-Binding Proteins; D004676:Myelin Basic Protein; D018996:Myelin P2 Protein; D009363:Neoplasm Proteins; D018168:Receptors, Retinoic Acid; D014212:Tretinoin; D001120:Arginine