Structural and biological characterisation of the gut-associated cyclophilin B isoforms from Caenorhabditis elegans.
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.
Picken
Nichola C
NC
Structural Biochemistry Group, Institute of Cell and Molecular Biology, University of Edinburgh, Michael Swann Building, UK.
Eschenlauer
Sylvain
S
Taylor
Paul
P
Page
Antony P
AP
Walkinshaw
Malcolm D
MD
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017173:Caenorhabditis elegans / Q000737:chemistry* / Q000196:embryology / Q000235:genetics; D029742:Caenorhabditis elegans Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D021983:Cyclophilins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016572:Cyclosporine / Q000494:pharmacology; D004229:Dithiothreitol / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000378:metabolism; D020869:Gene Expression Profiling; D006801:Humans; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D019696:Peptidylprolyl Isomerase; D010641:Phenotype; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism; D018510:RNA, Helminth / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D013270:Stomach / Q000737:chemistry
D029742:Caenorhabditis elegans Proteins; D018832:Molecular Chaperones; D009842:Oligopeptides; D020033:Protein Isoforms; D012330:RNA, Double-Stranded; D018510:RNA, Helminth; C081786:cyclophilin B; C043307:succinyl-alanyl-alanyl-prolyl-phenylalanine-4-nitroanilide; D016572:Cyclosporine; D021983:Cyclophilins; D019696:Peptidylprolyl Isomerase; D004229:Dithiothreitol