Engineering of monomeric FK506-binding protein 22 with peptidyl prolyl cis-trans isomerase Importance of a V-shaped dimeric structure for binding to protein substrate
Engineering of monomeric FK506-binding protein 22 with peptidyl prolyl cis-trans isomerase Importance of a V-shaped dimeric structure for binding to protein substrate
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DOI：
10.1111/j.1742-4658.2009.07116.x
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发表时间：
2009-08-01
期刊：
影响因子：
5.4
通讯作者：
Kanaya, Shigenori
中科院分区：
文献类型：
--
作者：
Budiman, Cahyo;Bando, Keisuke;Kanaya, Shigenori
fk506结合蛋白22 （FKBP22）来自精神营养细菌Shewanella sp. SIB1，是一种具有肽基丙氨酸顺式反式异构酶（PPIase）活性的二聚体蛋白。每个单体由205个氨基酸残基组成。根据三级模型，SIB1 FKBP22呈v形结构，其中两个单体在其n端相互作用。每个单体由一个带有二聚化核心的n端结构域和一个c端催化结构域组成，它们被一个40个残基长的α -螺旋分开。为了阐明这种v形结构的作用，我们构建了一个突变蛋白，其中n结构域通过一个柔性连接体串联重复。这种蛋白被命名为NNC-FKBP22，它被设计成两个重复的n结构域折叠成类似于Shewanella sp. SIB1 FKBP22野生型蛋白（WT）的结构。NNC-FKBP22以his标记的形式在大肠杆菌中过量产生，经过纯化并进行了生化表征。凝胶过滤层析和超离心分析表明，NNC-FKBP22以单体形式存在。用差示扫描量热法分析热变性表明，NNC-FKBP22与WT蛋白一样，具有两个转变。NNC-FKBP22对肽和蛋白底物均表现出PPIase活性。然而，与与WT蛋白相当的肽底物活性相比，它对蛋白质底物的活性降低了5到6倍。表面等离子体共振分析表明，NNC-FKBP22与α -乳白蛋白的还原形式结合的亲和力比WT弱6倍。这些结果表明，SIB1 FKBP22的v形结构对于有效结合蛋白质底物很重要。
FK506-binding protein 22 (FKBP22) from the psychrotrophic bacterium Shewanella sp. SIB1 is a homodimeric protein with peptidyl prolyl cis-trans isomerase (PPIase) (EC 5.2.1.8) activity. Each monomer consists of 205 amino acid residues. According to a tertiary model, SIB1 FKBP22 assumes a V-shaped structure, in which two monomers interact with each other at their N-termini. Each monomer consists of an N-terminal domain with a dimerization core and a C-terminal catalytic domain, which are separated by a 40-residue-long alpha-helix. To clarify the role of this V-shaped structure, we constructed a mutant protein, in which the N-domain is tandemly repeated through a flexible linker. This protein, termed NNC-FKBP22, is designed such that two repetitive N-domains are folded into a structure similar to that of the Shewanella sp. SIB1 FKBP22 wild-type protein (WT). NNC-FKBP22 was overproduced in Escherichia coli in a His-tagged form, purified and biochemically characterized. Gel-filtration chromatography and ultracentrifugation analyses indicate that NNC-FKBP22 exists as a monomer. Analysis of thermal denaturation using differential scanning calorimetry indicates that NNC-FKBP22 unfolds with two transitions, as does the WT protein. NNC-FKBP22 exhibited PPIase activity for both peptide and protein substrates. However, in contrast to its activity for peptide substrate, which was comparable to that of the WT protein, its activity for protein substrate was reduced by five- to six-fold, compared to that of the WT. Surface plasmon resonance analyses indicate that NNC-FKBP22 binds to a reduced form of alpha-lactalbumin with a six-fold weaker affinity than that of WT. These results suggest that a V-shaped structure of SIB1 FKBP22 is important for efficient binding to a protein substrate.