Abstract
It has been suggested that electrostatic interactions are critical for binding of retinoic acid by cellular retinoic acid-binding proteins (CRABP-I and CRABP-II). However, the roles of two conserved arginine residues (Arg-111 and Arg-131 in CRABP-I; Arg-111 and Arg-132 in CRABP-II) that interact with the carboxyl group of retinoic acid have not been evaluated. A novel competitive binding assay has been developed for measuring the relative dissociation constants of the site-directed mutants of CRABPs. Arg-111 and Arg-132 of CRABP-II were replaced with methionine by site-directed mutagenesis. The relative dissociation constants of R111M and R132M (Kd (R111M)/Kd (CRABP-II) and Kd (R132M)/Kd(CRABP-II)) were determined to be 40-45 and 6-8, respectively. The ring protons of the aromatic residues of the wild-type CRABP-II and the two mutants were sequentially assigned by two-dimensional homonuclear NMR in conjunction with three-dimensional heteronuclear NMR. Detailed analysis of the nuclear Overhauser effect spectroscopy spectra of the proteins indicated that the conformations of the two mutants are highly similar to that of the wild-type CRABP-II. These results taken together showed that Arg-111 and Arg-132 are important for binding retinoic acid but contribute to the binding energy only by approximately 2.2 and 1.2 kcal/mol, respectively. In addition, the relative dissociation constant of CRABP-II and CRABP-I (Kd (CRABP-II)/Kd (CRABP-I)) was determined to be 2-3, in close agreement with that calculated using the apparent Kd values determined under the same conditions by fluorometric titrations.
Highlights
Retinoic acid (RA),1 a hormonally active metabolite of vitamin A, has profound effects on cell growth, differentiation, and morphogenesis
Two isoforms (CRABP-I and cellular retinoic acid-binding proteins (CRABPs)-II) have been characterized. Both CRABP-I and CRABP-II bind to all-transretinoic acid, but they differ in several respects: (i) CRABP-I has higher affinity for RA than CRABP-II [3,4,5,6]; (ii) CRABP-I is expressed in many adult mouse and human tissues, but the expression of CRABP-II is limited to skin [7, 8]; and (iii) RA stimulates expression of CRABP-II but not that of CRABP-I [8]
Previous site-directed mutagenesis studies suggest that both Arg111 and Arg-131 are critical for CRABP-I to bind RA [13] but that only Arg-132 is important for CRABP-II to bind RA [14]
Summary
Materials—Nonradioactive RA was purchased from Sigma. [11,123H]RA was purchased from DuPont NEN. The equilibria in the two compartments that contained the wild-type CRABP-II and mutant proteins can be described by Equations 2 and 3 (similar equations can be written for CRABP-I and CRABP-II),. Where CWT and CMT are the measured radioactivities of the two compartments containing the wild-type CRABP-II and the mutant, respectively. Since RA is not stable even in the dark, the assay was redesigned to match the equilibrium conditions by varying the ratio of the protein concentrations of the wild-type and the mutant ([MT]total/ [WT]total). The spectral width was 7200 Hz. For wild-type CRABP-II, one phase-sensitive DQF-COSY spectrum [19], two MLEV-17 clean TOCSY spectra (20 –22) with mixing times of 20 and 40 ms and one NOESY spectrum [23, 24] with a mixing time of 150 ms were acquired. Chemical shifts were referenced to internal sodium 3-(trimethyl silyl)-propionate-2,2,3,3-d4
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