Characterization of Peptidyl-Prolyl Cis-Trans Isomerase- and Calmodulin-Binding Activity of a Cytosolic Arabidopsis thaliana Cyclophilin AtCyp19-3

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.

The rolB plant oncogene affects multiple signaling protein modules related to hormone signaling and plant defense

“…ROC2 (CYP19-3): ROC2 physically interacts with calmodulins (CAMs) and thus affects a broad array of reactions controlled by CAMs 48 . These include the response to stress (mediated by the CBL-interacting serine/threonine-protein kinase 6 (SIP3), BTB and TAZ domain protein 4 (BT4) and basic leucine-zipper proteins; Fig.…”

The peptidyl-prolyl cis-trans isomerase activity of the wheat cyclophilin, TaCypA-1, is essential for inducing thermotolerance in Escherichia coli

“…Several studies have shown that besides cis-trans isomerization, members of cyclophilin family also perform additional functions in the cell [5] . We recently demonstrated that an Arabidopsis cyclophilin, AtCyp19-3, which is 72.67% identical to TaCypA-1, interacts with CaM in a Ca 2+ -dependent manner [19] . CaM is one of the most well characterized sensors of Ca 2+ , and it consists of four EF-hands that bind to Ca 2+ cooperatively [20] .…”

Plant Cyclophilins: Multifaceted Proteins With Versatile Roles

“…Contingent upon the presence of an extra loop of four or more amino acid residues present at residue 50 corresponding to hCYPA, the cyclophilins are classified as divergent or nondivergent (Dornan et al, 1999). The divergent loop cyclophilins such as TaCYPA-1 (Sekhon et al, 2013), CsCYP (Campos et al, 2013) and Cat r 1 (Ghosh et al, 2014) are similar to hCYPA in their active site composition and CsA binding characteristics except for the presence of a characteristic additional loop (consensus sequence XXGKXLH corresponding to amino acid residues 48-54 in TaCYPA-1), two conserved Cys residues (Cys40 and Cys168) and a conserved glutamate (Glu83) residue (Kaur et al, 2015;Vasudevan et al, 2015). On the contrary, the nondivergent cyclophilins such as hCYPA, SmCYPA and AtCYP20-3 or ROC4 (Rotamase Cyclophilin 4) lack the additional loop and are characterized by two conserved Cys residues at positions 122 and 126 (Gourlay et al, 2007;Laxa et al, 2007).…”

“…The PPIase activity of cyclophilins, in general, is regulated in a redox-dependent or independent manner. Contrary to the E. coli cyclophilin PPIB, that is regulated by redox-independent mechanisms (Hayano et al, 1991;Kaur et al, 2015), the PPIase activity of AtCYP19-3 (ROC2), AtCYP20-3, SmCYPA, CsCYP and TaCYPA-1 is subject to redox regulation (Motohashi et al, 2003;Gourlay et al, 2007;Laxa et al, 2007;Campos et al, 2013;Kaur et al, 2015Kaur et al, , 2017. Furthermore, the redoxregulatory mechanisms observed in different cyclophilins are also distinct.…”