Rouvière-Fourmy N, Craescu C T, Mispelter J, Lebeau M C, Baulieu E E
Institut National de la Santé et de la Recherche Médicale U350, Institut Curie, Orsay, France.
Eur J Biochem. 1995 Aug 1;231(3):761-72.
FKBP59, a 59-kDa FK506 binding protein, was discovered in heterooligomeric complexes containing nontransformed, non-DNA binding, steroid receptors. Sequence similarity search and secondary structure prediction suggested that the protein has a multi-domain organization, the N-terminal domain having a great similarity to human FKBP12 (12-kDa FK506-binding protein). FKBP59 binds immunosuppressant FK506 and has peptidylprolyl cis-trans-isomerase activity, both properties being localized in the N-terminal domain (FKBP59-I). In order to characterize its conformational features and to better understand its biological significance, we overexpressed and 15N-labeled this domain (149 amino acids) in Escherichia coli and initiated an NMR structural study in solution. Almost complete sequence-specific assignment of the 1H and 15N resonances was achieved using two-dimensional and three-dimensional homonuclear and heteronuclear experiments. Localization of the secondary structure elements was derived essentially from C alpha H chemical shift distribution along the sequence, the short-range and medium-range NOE connectivities and exchange kinetics of amide protons. The domain has a structured part comprising six beta-strands and a three-turn alpha-helix between K87 and M96. The first 17 residues are highly flexible and show no regular secondary structure. The beta-sheet structure, derived from long-range connectivities between backbone protons, consists of six beta-strands defined as follows: B1, V22-I24; B2, V32-K37; B3, D50-L61; B4, T64-S68 and F76-L80; B5, E100-K107; B6, L127-F137. They are organized in an antiparallel beta-sheet with the connecting topology +1, +3, +1, -3, +1. The alpha-helix connects strand B4 to strand B5. Globally, the structure of FKBP59-I, derived from the present work, is similar to the NMR-derived structures of uncomplexed FKBP12. However, several conformational differences were noted at this level of structural analysis. The beta-sheet of the FKBP59 domain has an additional strand at the N-terminal and the alpha-helix is longer by about one helical turn. In addition, strand B4 has two components, separated by a large bulge (seven residues); the first component was observed in the X-ray or NMR structures of complexed FKBP12 but not in the NMR-derived, uncomplexed structure.
FKBP59是一种59千道尔顿的FK506结合蛋白，在含有未转化、非DNA结合的类固醇受体的异源寡聚复合物中被发现。序列相似性搜索和二级结构预测表明，该蛋白具有多结构域组织，其N端结构域与人类FKBP12（12千道尔顿的FK506结合蛋白）具有高度相似性。FKBP59能结合免疫抑制剂FK506并具有肽基脯氨酰顺反异构酶活性，这两种特性都位于N端结构域（FKBP59-I）。为了表征其构象特征并更好地理解其生物学意义，我们在大肠杆菌中对该结构域（149个氨基酸）进行了过表达和15N标记，并启动了溶液中的核磁共振结构研究。使用二维和三维同核及异核实验几乎完成了1H和15N共振的全序列特异性归属。二级结构元件的定位主要源自沿序列的CαH化学位移分布、短程和中程NOE连接性以及酰胺质子的交换动力学。该结构域有一个结构化部分，由六条β链和K87与M96之间的一个三圈α螺旋组成。前17个残基高度灵活，没有规则的二级结构。由主链质子之间的长程连接性得出的β折叠结构由六条β链组成，定义如下：B1，V22-I24；B2，V32-K37；B3，D50-L61；B4，T64-S68和F76-L8 O；B5，E100-K107；B6，L127-F137。它们以反平行β折叠的形式组织，连接拓扑为+1、+3、+1、-3、+1。α螺旋将链B4连接到链B O。总体而言，本研究得出的FKBP59-I的结构与未结合的FKBP12的核磁共振衍生结构相似。然而，在这个结构分析水平上注意到了几个构象差异。FKBP59结构域的β折叠在N端有一条额外的链，α螺旋长约一圈。此外，链B4有两个部分，由一个大的凸起（七个残基）隔开；第一个部分在结合的FKBP12的X射线或核磁共振结构中观察到，但在核磁共振衍生的未结合结构中未观察到。
