NUCLEAR-MAGNETIC-RESONANCE STUDIES DEMONSTRATE DIFFERENCES IN THE INTERACTION OF RETINOIC ACID WITH 2 HIGHLY HOMOLOGOUS CELLULAR RETINOIC ACID-BINDING PROTEINS

Cellular retinoic acid binding protein-I (CRABP-I) and cellular retino ic acid binding protein-II (CRABP-II) are highly homologous, 15 kDa pr oteins which bind all-trans-retinoic acid. In the adult, CRABP-II is e xpressed predominately in the epidermis, while CRABP-I is expressed in a variety of tissues. To obtain structural information which could ai d the design of more selective ligands, isotope-directed NMR methods w ere employed to observe the CRABP-bound conformation of C-13-labeled r etinoic acid and to identify its contact points with neighboring amino acids. Analysis of HMQC, HMQC-TOCSY, and C-13-TOCSY-REVINEPT on CRABP -bound (2,3,6,7,8,9,10,11,19(-)(13)C)- and (1,4,5,8,9,16,17,18,19-C-13 )-all-trans-retinoic acid allowed the unambiguous assignment of all la beled protons and their attached C-13 resonances. The volumes of 16 ol efinic proton-methyl NOE cross-peaks measured from 30-ms C-13-((omega) 2)-filtered H-1 NOESY experiments were used to determine the conformat ions about the 6-, 8-, and 10-single bonds of the retinoic acid polyen e chain. These spectra show qualitatively distinct NOE patterns for th e two CRABPs. Measured cross-peak volumes for CRABP-II bound retinoic acid were well predicted by a single, static conformation having a 6-s torsion angle of -60 degrees skewed from a cis conformation. In contr ast, for CRABP-I no single, static conformation was able to match the pattern of cross-peaks, suggesting motion about the 6-s bond. The meas ured cross-peaks were best described by 8-s and 10-s torsion angles of 180 degrees +/- 30 degrees, a trans configuration, for both proteins. The pattern of intermolecular NOESY cross-peaks between C-13-labeled protons in the ring portion of retinoic acid and protein protons were different between CRABP-I and CRABP-II. These differences coincide wel l with nearby amino acid substitutions in the recently reported X-ray structures of crystalline CRABP-I and CRABP-II and may assist rational design of selective ligands.