Biochemical and structural characterization of a divergent loop cyclophilin from Caenorhabditis elegans.
Cyclophilin 3 (CYP-3) is one of the most abundantly expressed cyclophilin isoforms in the free living nematode Caenorhabditis elegans. The detailed post-embryonic expression pattern of the cyp-3 transcript is unusual, peaking during early larval development. The spatial expression pattern was examined via reporter gene analysis demonstrating that the cyp-3 transcript is exclusively expressed in the single anterior excretory cell. Recombinant cyclophilin 3 has been purified, crystallized and solved to a resolution of 1.8 A. The peptidyl-prolyl isomerase activity of CYP-3 has been characterized against the substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, and gives a k(cat)/K(m) value of 2.4 x 10(6) M(-1) s(-1). The immunosuppressive drug cyclosporin A binds and inhibits CYP-3 with an IC(50) value of 16 nM, comparable with the range of values found for human cyclophilin A. The x-ray structure shows that the overall fold and active site geometry is similar to other cyclophilin structures. There are however a number of distinctive features, and we use this structure and amino acid sequence alignment analysis to identify a subgroup of "divergent-loop cyclophilins". This subgroup has a number of uniquely conserved features: an additional loop between residues 48 and 54 (KSGKPLH); two cysteine residues (Cys(40) and Cys(168)) that are in close proximity but remain in the unoxidized form, and two other conserved residues, His(54) and Glu(83). We suggest that these features are functionally important for the role played by this class of cyclophilins during cellular responses to stress caused by changes in the redox environment or by up-regulation of cellular activity. This study represents a detailed biological, biochemical, and structural characterization of a single cyclophilin isoform in the model organism Caenorhabditis elegans.
Dornan
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Structural Biochemistry Group, Institute of Cell and Molecular Biology, The University of Edinburgh, Michael Swann Building, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, Scotland.
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D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology*; D018360:Crystallography, X-Ray; D016572:Cyclosporine / Q000493:pharmacokinetics; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D020033:Protein Isoforms; D011994:Recombinant Proteins; D016572:Cyclosporine; D019696:Peptidylprolyl Isomerase