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

Andrew W. Norris, Ding Rong, D. André d'Avignon, Michael Rosenberger, Kenzabu Tasaki, Ellen Li

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

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 CRABP-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 l3C-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- (ω)-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 conformation having a 6-s torsion angle of -60° 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° ± 30°, 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.

Original languageEnglish (US)
Pages (from-to)15564-15573
Number of pages10
JournalBiochemistry
Volume34
Issue number47
DOIs
StatePublished - Nov 1995
Externally publishedYes

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