Norris A W, Rong D, d'Avignon D A, Rosenberger M, Tasaki K, Li E
Department of Medicine, Washington University, St. Louis, Missouri 63110, USA.
Biochemistry. 1995 Nov 28;34(47):15564-73. doi: 10.1021/bi00047a023.
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.
细胞视黄酸结合蛋白-I（CRABP-I）和细胞视黄酸结合蛋白-II（CRABP-II）是高度同源的15 kDa蛋白，可结合全反式视黄酸。在成体中，CRABP-II主要在表皮中表达，而CRABP-I在多种组织中表达。为了获得有助于设计更具选择性配体的结构信息，采用了同位素定向核磁共振方法来观察13C标记视黄酸与CRABP结合的构象，并确定其与相邻氨基酸的接触点。对与CRABP结合的（2,3,6,7,8,9,10,11,19-13C）-和（1,4,5,8,9,16,17,18,19-13C）-全反式视黄酸进行HMQC、HMQC-TOCSY和13C-TOCSY-REVINEPT分析，可明确归属所有标记质子及其相连的13C共振峰。从30毫秒的13C-（ω2）-滤波1H NOESY实验测得的16个烯丙基质子-甲基NOE交叉峰的体积，用于确定视黄酸多烯链6-、8-和10-单键周围的构象。这些光谱显示两种CRABP的NOE模式在质量上有明显差异。对于与CRABP-II结合的视黄酸，通过单一的静态构象可以很好地预测测得的交叉峰体积，该构象的6-s扭转角为-60度，偏离顺式构象。相比之下，对于CRABP-I，没有单一的静态构象能够匹配交叉峰模式，这表明6-s键存在运动。两种蛋白测得的交叉峰最好用8-s和10-s扭转角为180度±30度（反式构型）来描述。视黄酸环部分的13C标记质子与蛋白质质子之间的分子间NOESY交叉峰模式在CRABP-I和CRABP-II之间有所不同。这些差异与最近报道的结晶CRABP-I和CRABP-II的X射线结构中附近的氨基酸取代很好地吻合，可能有助于合理设计选择性配体。
