Biochemical and structural characterization of a divergent loop cyclophilin from Caenorhabditis elegans

Cyclophilin 3 (CYP-3) is one of the most abundantly expressed cyclophilin i soforms in the free living nematode Caenorhabditis elegans, The detailed po st-embryonic expression pattern of the cyp-3 transcript is unusual, peaking during early larval development. The spatial expression pattern was examin ed via reporter gene analysis demonstrating that the cyp-3 transcript is ex clusively expressed in the single anterior excretory cell. Recombinant cycl ophilin 3 has been purified, crystallized and solved to a resolution of 1.8 Angstrom. The peptidyl-prolyl isomerase activity of CYP-3 has been charact erized 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 d rug cyclosporin A binds and inhibits CYP-3 with an IC50 value of 16 nM, com parable with the range of values found for human cyclophilin A The x-ray st ructure shows that the overall fold and active site geometry is similar to other cyclophilin structures. There are however a number of distinctive fea tures, 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 residu es 48 and 54 (KSGKPLH); two cysteine residues (Cys(40) and Cys(168)) that a re in close proximity but remain in the unoxidized form, and two Other cons erved residues, His(54) and Glu(83). We suggest that these features are fun ctionally important for the role played by this class of cyclophilins durin g cellular responses to stress caused by changes in the redox environment o r by upregulation of cellular activity. This study represents a detailed bi ological, biochemical, and structural characterization of a single cyclophi lin isoform in the model organism Caenorhabditis elegans.