Picken Nichola C, Eschenlauer Sylvain, Taylor Paul, Page Antony P, Walkinshaw Malcolm D
Structural Biochemistry Group, Institute of Cell and Molecular Biology, University of Edinburgh, Michael Swann Building, UK.
J Mol Biol. 2002 Sep 6;322(1):15-25. doi: 10.1016/s0022-2836(02)00712-x.
The free-living nematode Caenorhabditis elegans expresses 18 cyclophilin isoforms, eight of which are conserved single domain forms, comprising two closely related secreted or type B forms (CYP-5 and CYP-6). Recombinant CYP-5 has been purified, crystallised and the X-ray structure solved to a resolution of 1.75A. The detailed molecular architecture most strongly resembles the structure of human cyclophilin B with conserved changes in loop structure and N and C-terminal extensions. Interestingly, the active site pocket is occupied by a molecule of dithiothreitol though this has little effect on the geometry of the active site which is similar to other cyclophilin structures. The peptidyl-prolyl isomerase activity of CYP-5 has been characterised against the substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, and gives a k(cat)/K(m) value of 3.6x10(6)M(-1)s(-1) that compares with a value of 6.3x10(6)M(-1)s(-1) for human cyclophilin B. The immunosuppressive drug cyclosporin A binds and inhibits CYP-5 with an IC(50) value of 50nM, which is comparable to the value of 84nM found for human cyclophilin B. CYP-6 has 67% sequence identity with CYP-5 and a molecular model was built based on the CYP-5 crystal structure. The model shows that CYP-5 and CYP-6 are likely to have very similar structures, but with a markedly increased number of negative charges distributed around the surface of CYP-6. The spatial expression patterns of the cyclophilin B isoforms were examined using transgenic animals carrying a LacZ reporter fusion to these genes, and both cyp-5 and cyp-6 are found to be expressed in an overlapping fashion in the nematode gut. The temporal expression pattern of cyp-5 was further determined and revealed a constitutive expression pattern, with highest abundance levels being found in the embryo.
自由生活的线虫秀丽隐杆线虫表达18种亲环蛋白异构体，其中8种是保守的单结构域形式，包括两种密切相关的分泌型或B型形式（CYP-5和CYP-6）。重组CYP-5已被纯化、结晶，其X射线结构解析分辨率达到1.75埃。详细的分子结构与人类亲环蛋白B的结构最为相似，环结构以及N端和C端延伸部分有保守变化。有趣的是，活性位点口袋被二硫苏糖醇分子占据，不过这对活性位点的几何形状影响很小，该活性位点与其他亲环蛋白结构相似。已针对底物N-琥珀酰-Ala-Ala-Pro-Phe-对硝基苯胺对CYP-5的肽基脯氨酰异构酶活性进行了表征，其k(cat)/K(m)值为3.6×10(6)M(-1)s(-1)，而人类亲环蛋白B的值为6.3×10(6)M(-1)s(-1)。免疫抑制药物环孢素A以50nM的IC(50)值结合并抑制CYP-5，这与人类亲环蛋白B的84nM值相当。CYP-6与CYP-5有67%的序列同一性，并基于CYP-5晶体结构构建了分子模型。该模型表明，CYP-5和CYP-6可能具有非常相似的结构，但CYP-6表面分布的负电荷数量明显增加。使用携带与这些基因融合的LacZ报告基因的转基因动物研究了亲环蛋白B异构体的空间表达模式，发现cyp-5和cyp-6在线虫肠道中以重叠方式表达。进一步确定了cyp-5的时间表达模式，结果显示其为组成型表达模式，在胚胎中丰度水平最高。
