Wang L, Yan H
Department of Biochemistry, Michigan State University, East Lansing 48824, USA.
Biochemistry. 1998 Sep 15;37(37):13021-32. doi: 10.1021/bi981021x.
The solution structure of a site-directed mutant of type-II human cellular retinoic acid binding protein (CRABPII) with Arg111 replaced by methionine (R111M) has been determined by NMR spectroscopy. The sequential assignments of the 1H and 15N resonances of apo-R111M were established by multinuclear multidimensional NMR. The solution structure was calculated from 2302 distance restraints and 77 phi dihedral restraints derived from the NMR data. The root-mean-square deviation of the ensemble of 28 refined conformers that represent the structure from the mean coordinate set derived from them was 0.54 +/- 0.26 and 0.98 +/- 0.23 A for the backbone atoms and all heavy atoms, respectively. The solution structure of apo-R111M is similar to that of wild-type apo-CRABPII. However, there are significant conformational differences between the two proteins, localized mainly to three segments (Leu19-Ala36, Glu73-Cys81, and Leu99-Pro105) clustered around the ligand entrance more than 17 A away from the point mutation. In apo-R111M, all the three segments move toward the center of the ligand entrance so that the opening of the ligand-binding pocket in apo-R111M is much smaller than that in wild-type apo-CRABPII. Furthermore, the ligand-binding pocket of apo-R111M, especially the ligand entrance, is much less flexible than that of apo-CRABPII. Surprisingly, apo-R111M is more similar to holo-CRABPII than to apo-CRABPII in both structure and dynamical properties. The conformational and dynamical changes caused by the mutation are similar to those induced by binding of RA, although the magnitudes of the changes caused by the mutation are smaller than those induced by binding of RA. The results suggest that Arg111 plays a critical role in determining the structure and dynamical properties of CRABPII.
已通过核磁共振光谱法确定了II型人细胞视黄酸结合蛋白（CRABPII）定点突变体（精氨酸111被甲硫氨酸取代，即R111M）的溶液结构。通过多核多维核磁共振确定了脱辅基R111M的1H和15N共振的序列归属。根据核磁共振数据得出的2302个距离约束和77个phi二面角约束计算出溶液结构。代表该结构的28个精制构象体的集合与从中导出的平均坐标集相比，主链原子和所有重原子的均方根偏差分别为0.54±0.26 Å和0.98±0.23 Å。脱辅基R111M的溶液结构与野生型脱辅基CRABPII的结构相似。然而，这两种蛋白质之间存在显著的构象差异，主要集中在三个片段（Leu19 - Ala36、Glu73 - Cys81和Leu99 - Pro105），这些片段聚集在距离点突变超过17 Å的配体入口周围。在脱辅基R111M中，所有这三个片段都向配体入口中心移动，因此脱辅基R111M中配体结合口袋的开口比野生型脱辅基CRABPII中的小得多。此外，脱辅基R111M的配体结合口袋，尤其是配体入口，比脱辅基CRABPII的灵活性小得多。令人惊讶的是，脱辅基R111M在结构和动力学性质上与全酶CRABPII比与脱辅基CRABPII更相似。尽管突变引起的变化幅度小于视黄酸（RA）结合诱导的变化幅度，但突变引起的构象和动力学变化与RA结合诱导的变化相似。结果表明，精氨酸111在决定CRABPII的结构和动力学性质方面起着关键作用。
