Nuclear Magnetic Resonance Studies Demonstrate Differences in the Interaction of Retinoic Acid with Two Highly Homologous Cellular Retinoic Acid Binding Proteins

Cellular retinoic acid binding protein-I (CRABP-I) and cellular retinoic acid binding protein-II (CRABP-II) are highly homologous, 15 kDa proteins which bind all-trans-retinoic acid. In the adult, CRABP-II is expressed predominately in the epidermis, while CRAPB-I is expressed in a variety of tissues. To obtain structural information which could aid the design of more selective ligands, isotope-directed NMR methods were employed to observe the CRABP-bound conformation of 13C-labeled retinoic acid and to identify its contact points with neighboring amino acids. Analysis of HMQC, HMQC-TOCSY, and 13C-TOCSY-REVINEPT on CRABP-bound (2,3,6,7,8,9,10,11,19-13C)- and (1,4,5,8,9,16, 17,18,19-13C)-all trans-retinoic acid allowed the unambiguous assignment of all labeled protons and their attached 13C resonances. The volumes of 16 olefinic proton-methyl NOE cross-peaks measured from 30-ms 13C-(omega 2)-filtered 1H NOESY experiments were used to determine the conformations about the 6-, 8-, and 10-single bonds of the retinoic acid polyene chain. These spectra show qualitatively distinct NOE patterns for the two CRABPs. Measured cross-peak volumes for CRABP-II bound retinoic acid were well predicted by a single, static conformational having a 6-s torsion angle of -60 degrees skewed from a cis conformation. In contrast, for CRABP-I no single, static conformation was able to match the pattern of cross-peaks, suggesting motion about the 6-s bond. The measured 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 13C-labeled protons in the ring portion of retinoic acid and protein protons were different between CRABP-I and CRABP-II. These differences coincide well 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.