Intracellular localization of AtCyp19-3.

<p>The upper panel <b>(A)</b> shows the accumulation pattern of AtCyp19-3:YFP chimeric protein. The lower panel <b>(B)</b> shows the sub-cellular interaction of AtCyp19-3-1:YFPn and OsCaM1:YFPc chimeric proteins by bi-molecular fluorescence complementation. The chimeric polypeptide containing AtCyp19-3_(71–176), (which did not show binding to CaM by gel overlay assay) with YFPn served as a negative control for <i>in vivo</i> studies. BF: bright field; Fl: fluorescence image; M: merged image, N: nucleus; C:cytoplasm.</p

Redox control mechanisms in AtCyp19-3 and TaCypA-1 (A)

<p>Potential metal binding site (Site1) in TaCypA-1 comprising of Cys-122, His-99 and Cys-126 amino acids (yellow) as predicted by TEMSP. Cys-122-Cys-126 pair (3.905 <i>Å</i>) in TaCypA-1 which may be involved in Redox 2-Cys mechanism similar to SmCypA, is also shown. <b>(B)</b> Second potential metal binding site (Site 2) consisting of Cys-40, His-54 and Cys-168 (red). Disulfide bond between Cys-40 and Cys-168 (5.489 <i>Å</i>), and hydrogen bond network linking side chains of Glu-83(red) with divergent loop residues Lys-48 and Ser-49 (magenta) in TaCypA-1 are implicated in allosteric control similar to Redox 2-Cys Mechanism in CsCypA. <b>(C)</b> Cartoon showing His-54 and disulfide bridge forming pair Cys-40-Cys-168 (5.4 <i>Å</i>) in AtCyp19-3. This triad (yellow) may form a metal binding site in AtCyp19-3. <b>(D)</b> Hydrogen bond connecting side chain carboxyl group of Glu-83(red) with main chain amide group of Lys-48 (green) from the divergent loop in AtCyp19-3. Formation of Cys-40-Cys-168 disulfide bond disrupts this interaction and closes the active site. Distances have been shown as black dashed lines while hydrogen bonds as black solid lines.</p

SDS-PAGE analysis of purified recombinant fusion proteins and their confirmation by immunoblotting with anti-His antibodies.

<p><b>(A)</b> SDS-PAGE analysis of purified full length recombinant AtCyp19-3, and deleted versions AtCyp19-3 _(35–176) and AtCyp19-3 _(71–176). Total proteins were isolated from recombinant <i>E</i>.<i>coli</i> BL21(DE3) before (UI) and after induction (I) with 0.5 mM IPTG, and purified by Ni-NTA affinity column. Arrows indicate the purified recombinant proteins. <b>(B)</b> Confirmation of the purified recombinant proteins (arrows) by immunoblotting with the anti-His antibodies. M: markers.</p

Comparative analysis of biochemical parameters of various cyclophilins.

<p>Comparative analysis of biochemical parameters of various cyclophilins.</p

Calmodulin (CaM) gel-overlay assay of the purified recombinant AtCyp19-3 and its truncated versions.

<p>The purified proteins were resolved by using 15% SDS-PAGE followed by transfer on to Hybond C nitrocellulose membrane. Proteins transferred on to membrane, after Ponceau S staining <b>(A)</b>, were allowed to renature in the presence <b>(B)</b> and absence of Ca²⁺<b>(C)</b> for 16 h at 4°C. The membrane was incubated with biotinylated-CaM in the presence (+Ca²⁺) and absence of Ca²⁺(-Ca²⁺), followed by probing with streptavidin-alkaline phosphate conjugate. CaM-binding proteins were visualized using nitro-blue tetrazolium/5-bromo-4-chloro-3-indolyphosphate as substrate. <b>(D)</b> Helical wheel projection (<a href="http://rzlab.ucr.edu/scripts/wheel/wheel.cgi" target="_blank">http://rzlab.ucr.edu/scripts/wheel/wheel.cgi</a>) of the predicted CaM-binding domain (CaMBD) of AtCyp19-3. The dashed line separate the hydrophilic and the hydrophobic faces of the amphiphilic CaMBD. Hydrophilic residues: circles; hydrophobic residues: diamonds; potentially negatively charged: triangles; potentially positively charged: pentagons. Hydrophobic residues are depicted in green and the amount of green decreases proportionally to the hydrophobicity, with zero hydrophobicity coded as yellow. Hydrophilic residues are shown as red, with the amount of red being proportional to the hydrophilicity. Light blue represents the potentially charged residues. M-protein markers (kDa).</p

Effect of copper on the peptidyl prolyl <i>cis-trans</i> isomerase (PPIase) activity of purified recombinants AtCyp19-3 and TaCypA-1 (A)

<p>Effect of Cu²⁺ on PPIase activity of AtCyp19-3 and TaCypA-1. First order rate constants were analysed using GraFit 4.0 software. The residual PPIase activity (%) is relative to uninhibited control. <b>(B)</b> Inhibition constants (k_i) of Cu²⁺ for AtCyp19-3 and TaCypA-1 were determined as gradient of the line of the best fit from a plot of [Cu²⁺]/(1-k/k_o) against k_o/k, where k is the rate constant at any given Cu²⁺ concentration and k_o is the rate constant in the absence of heavy metal. The slope of the line represents the ki. Data represent the mean ± SD of triplicates.</p

Multiple sequence alignment of cyclophilin proteins from <i>Triticum aestivum</i> (TaCypA-1), <i>Cajanus cajan</i> (CcCyp), <i>Camellia sinensis</i> (CsCyp), <i>Arabidopsis thaliana</i> (AtCyp18-3, AtCyp19-2, AtCyp19-1, AtCyp18-4, AtCyp19-3, AtCyp20-3), <i>Thellungiela halophila</i> (ThCyp1), <i>Oryza sativa</i> (OsCyp2), <i>Schistosoma mansoni</i> (SmCypA) and <i>Homo-sapiens</i> (hCypA) was performed using clustalw2 server (http://www.ebi.ac.uk/Tools/msa/clustalw2/).

<p>The ESPript 3.0 multiple-alignment editor (<a href="http://espript.ibcp.fr/ESPript/ESPript/" target="_blank">http://espript.ibcp.fr/ESPript/ESPript/</a>) was used for the final presentation. The positions of the various α-helices and β-sheets in reference to the TaCypA-1 crystal structure (4HY7) are indicated at the top of the figure in red. Names of displayed cyclophilins sequences under investigation (AtCyp19-3 and TaCypA-1) have been colored red while for SmCypA and CsCyp are colored blue. Active site residues are marked by blue asterisks while the Cys residues corresponding to TaCypA-1 are marked with red triangles below the figure. The plant-specific divergent loop residues are shaded in green colour. Relative accessibility of amino acid is displayed below the alignment block (Blue: accessible, Cyan: intermediate and white: buried). The active site residues in all the sequences are very well conserved except for AtCyp19-1 for which a portion of protein sequence containing the first three active site residues and part of the divergent loop is deleted. Four Cys residues (C-40, C-69, C-122, C-168) have been conserved in all sequences except for Cys-40 in hCypA (mutated to S) and Cys 69 in AtCyp20-3 (mutated to I). TaCypA-1 has two additional Cys residues with the one corresponding to position 126 being also observed in OsCyp2 and SmCypA.</p

PPIase activity of purified recombinant fusion protein AtCyp19-3

<p><b>(A)</b> Hydrolysis of N-succinyl-ala-ala-pro-phe-p-nitroanilidine (peptidyl prolyl <i>cis-trans</i> isomerase or PPIase activity) in the presence of 4.34 nM of recombinant AtCyp19-3 and 52.4 nM and 65.87 nM each of AtCyp19-3_(35–176) and AtCyp19-3_(71–176) proteins. The rate of reaction is expressed as first order rate constant (k) Bovine serum albumin (BSA) was used as a negative control. <b>(B)</b> Effect of purified AtCyp19-3 on rate constant. <b>(C)</b> Effect of cyclophilin inhibitor, cyclosporin A (CsA), and FKBP inhibitor, FK506, on the PPIase activity of AtCyp19-3 <b>(D)</b> Determination of inhibition constant (k_i) of AtCyp19-3 for CsA. Inhibition constant (k_i) for CsA was determined as gradient of the line of the best fit from a plot of [CsA]/(1-k/k_o) against k_o/k, where k is the rate constant at any given CsA concentration and k_o is the rate constant in the absence of CsA. The slope of the line represents the k_i.</p

Effect of N-ethylmaleimide (500 μM) on peptidyl prolyl <i>cis-trans</i> isomerase (PPIase) activity in the presence of 22 nM each of AtCyp19-3 and TaCypA-1.

<p>The residual PPIase activity was calculated as a percentage of the initial activity before the addition of N-ethylmaleimide and expressed as a function of incubation time.</p

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

<div><p>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 <i>cis-trans</i> 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 <i>Arabidopsis</i> 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 <i>Arabidopsis</i> cyclophilin, AtCyp19-3, which demonstrated that this protein is enzymatically active and possesses peptidyl-prolyl <i>cis-trans</i> isomerase (PPIase) activity that is specifically inhibited by CsA with an inhibition constant (K_i) 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 <i>in vitro</i> 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 <i>in vivo</i> also, thus, validating the <i>in vitro</i> observations. However, the PPIase activity of the <i>Arabidopsis</i> cyclophilin was not affected by CaM. The implications of these findings are discussed in the context of Ca²⁺ signaling and cyclophilin activity in <i>Arabidopsis</i>.</p></div