Abstract
Cyclophilins, which bind to immunosuppressant cyclosporin A (CsA), are ubiquitous proteins and constitute a multigene family in higher organisms. Several members of this family are reported to catalyze cis-trans isomerisation of the peptidyl-prolyl bond, which is a rate limiting step in protein folding. The physiological role of these proteins in plants, with few exceptions, is still a matter of speculation. Although Arabidopsis genome is predicted to contain 35 cyclophilin genes, biochemical characterization, imperative for understanding their cellular function(s), has been carried only for few of the members. The present study reports the biochemical characterization of an Arabidopsis cyclophilin, AtCyp19-3, which demonstrated that this protein is enzymatically active and possesses peptidyl-prolyl cis-trans isomerase (PPIase) activity that is specifically inhibited by CsA with an inhibition constant (Ki) of 18.75 nM. The PPIase activity of AtCyp19-3 was also sensitive to Cu2+, which covalently reacts with the sulfhydryl groups, implying redox regulation. Further, using calmodulin (CaM) gel overlay assays it was demonstrated that in vitro interaction of AtCyp19-3 with CaM is Ca2+-dependent, and CaM-binding domain is localized to 35–70 amino acid residues in the N-terminus. Bimolecular fluorescence complementation assays showed that AtCyp19-3 interacts with CaM in vivo also, thus, validating the in vitro observations. However, the PPIase activity of the Arabidopsis cyclophilin was not affected by CaM. The implications of these findings are discussed in the context of Ca2+ signaling and cyclophilin activity in Arabidopsis.
Highlights
A peptide bond in a folded protein can attain either a trans or cis conformation
As part of our ongoing studies to understand the functional significance of different cyclophilins, in the present study we examined the biochemical properties of AtCyp19-3 (ROC2), which shares 82.4% similarity with AtCyp18-3 (ROC1), 73.3% with AtCyp19-1(ROC3), 50.6% with AtCyp20-3 (ROC4), 81.8% with AtCyp18-4 (ROC5) and 86.4% with AtCyp19-2 (ROC6) (S2 Table) [7]
The peptidyl prolyl cis-trans isomerase (PPIase) activity of the purified AtCyp19-3 was determined by the proteolytic cleavage assay using chymotrypsin
Summary
A peptide bond in a folded protein can attain either a trans or cis conformation. The trans form is sterically favoured and proteins are synthesized with peptide bonds in this form [1]. The functional role of thiol groups in regulating the PPIase activity of purified AtCyp19-3, TaCypA-1 and PpiA was studied by incubating the proteins (22 nM) in assay buffer containing 500 μM N-ethylmaleimide (NEM) (diluted from 50 mg/ml stock prepared in 100% EtOH) for different time intervals and different concentrations of CuSO4 [47], followed by estimation of PPIase activity.
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