Arginine 132 of cellular retinoic acid-binding protein (type II) is important for binding of retinoic acid.
Cellular retinoic acid-binding protein type II (CRABP-II) is one of two small molecular weight, cytosolic proteins which specifically bind retinoic acid (RA). Crystallographic and site-directed mutagenesis studies of several related proteins have indicated that either one or two conserved amino acid residues, homologous to positions Arg111 and Arg132 of CRABP-II, are important for the binding of the hydrophobic ligand. In this report we have prepared site-directed mutations of these two positions of CRABP-II, Arg111 and Arg132, as well as Lys82 to determine the role of these residues in the binding of RA. Recombinant wild type and mutant CRABP-II proteins were expressed and purified, and the affinity for retinoids was determined by fluorometric titration and binding of 3H-labeled compounds. K82A displayed an identical Kd for all-trans-RA as wild type CRABP-II and the Kd for all-trans-RA of R111A was only slightly higher. On the other hand, the two Arg132 mutants, R132A and R132Q, of CRABP-II demonstrated undetectable binding of all-trans-RA. Taken together these data demonstrate that Arg132 is a critical amino acid residue for the binding of RA by CRABP-II.
Chen
L X
LX
Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Zhang
Z P
ZP
Scafonas
A
A
Cavalli
R C
RC
Gabriel
J L
JL
Soprano
K J
KJ
Soprano
D R
DR
D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D006868:Hydrolysis; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D018168:Receptors, Retinoic Acid / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014212:Tretinoin / Q000378:metabolism*
D017931:DNA Primers; D018076:DNA, Complementary; D018168:Receptors, Retinoic Acid; D011993:Recombinant Fusion Proteins; C082928:retinoic acid binding protein II, cellular; D014212:Tretinoin; D001120:Arginine