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Results: 7

1.
Figure 7

Figure 7. Predicted binding mode of LxVP based on docking and molecular dynamic simulations. From: A conserved docking surface on calcineurin mediates interaction with substrates and immunosuppressants.

The LxVP peptide (ball and sticks) is positioned parallel to the B-subunit-binding helix of CnA (orange), and forms additional contacts with residues in CnB (green). In the zoom view (top), hydrogen bonds and salt bridges are shown as dashed lines. CN residues predicted by MD simulation to be involved in ligand binding are labeled and numbered. The interactions are detailed in 2D (right) and summarized in a table (bottom): vdW; van der Waals.

Antonio Rodríguez, et al. Mol Cell. ;33(5):616-626.
2.
Figure 3

Figure 3. Calcineurin binding by LxVPc1 requires CnA and CnB subunits. From: A conserved docking surface on calcineurin mediates interaction with substrates and immunosuppressants.

(A) Flag-tagged full-length (521) and deletion-mutant versions of human CnA-α. Functional regions are underlined and conserved domains indicated: B = CnB binding domain; C = calmodulin binding domain; AI = auto-inhibitory domain.
(B) HEK cells were co-transfected with CnB and the indicated flag-tagged CnA construct. CN was pulled down from lysates with GST-LxVPc1 in the presence of 200 μM LxVPc1 or control peptide. Bound and unbound CN was detected with anti-Flag.
(C–E) Pull-down experiments with (C,E) GST-LxVPc1 or GST or (D) GST-PxIxITc2. (C,D) CnB and CnA deletion mutants were expressed in bacteria, purified and used in pull-down experiments with (C) GST-LxVPc1 or GST or (D) GST-PxIxITc2. (E) Binding of bovine CN to GST-LxVPc1 in the presence of Ca2+ (1.5mM) and calmodulin (600nM) or EGTA (5mM). Bound and unbound CN subunits were detected by western blot.

Antonio Rodríguez, et al. Mol Cell. ;33(5):616-626.
3.
Figure 4

Figure 4. LxVPc1 peptide and immunosuppressant drugs dock at a common region in calcineurin. From: A conserved docking surface on calcineurin mediates interaction with substrates and immunosuppressants.

(A–B) CN was pulled-down with immunosuppressant-immunophilin complexes formed between (A) CsA and GST-CypA and (B) FK506 and GST-FKBP12. Binding was competed with LxVPc1 synthetic peptide. Scrambled (C) and mutant LxVP (LxVPc1mut) peptides were used as controls. Bound CN was detected by western blot. CN did not bind GST-immunophilins in the absence of the corresponding immunosuppressant (first lanes).
(C) Identification of CN residues putatively involved in interaction with LxVPc1 peptide by in silico docking (AutoDock). A surface representation of CnA (red) bound to CnB (green) is shown. The in silico modeling locates the LxVPc1 peptide (sticks) in a hydrophobic groove comprising amino acids from both subunits. The positions of the identified amino acids are shown in the primary structures of CnA (blue) and CnB (yellow).
(D–F) CN subunits carrying individual alanine substitutions of the residues identified by in silico analysis were used in pull-down experiments with (D) GST-LxVPc1 (right) or GST-PxIxITc2 (left), (E) GST-immunophilins, or (F) GST-NFATc1. Bound CN was eluted and detected by western blot with anti-CnA or anti-CnB mAb. A representative experiment is shown.

Antonio Rodríguez, et al. Mol Cell. ;33(5):616-626.
4.
Figure 2

Figure 2. Inhibition of calcineurin phosphatase activity by LxVPc1 peptide and contribution of LxVP-motif residues to calcineurin binding. From: A conserved docking surface on calcineurin mediates interaction with substrates and immunosuppressants.

(A) LxVPc1 inhibits CN phosphatase activity on the 19mer phosphopeptide RIIp. Inorganic phosphate release in the presence of the indicated synthetic peptides (AID=auto-inhibitory domain of CN) was determined spectrophotometrically. Maximal activity was defined as phosphate release in the absence of synthetic peptide. Data are from a representative experiment performed in triplicate (means ± sd).
(B) LxVPc1 increases CN phosphatase activity on the small compound pNPP. Each LxVPc1 concentration was assayed three times and the corresponding values (means ± sd) plotted as percentages of phosphate release in the presence of mutant LxVPc1 peptide (LxVPc1mut: LxVP>AxAA).
(C) LxVPc1 mutant peptides were generated in which each residue of the 15mer wt sequence was replaced in turn by alanine or glycine. The impact of each mutation was tested on the inhibition of CN phosphatase activity toward RIIp. The graph shows plots for wt LxVPc1 (dashed line) and mutant peptides that strongly reduced inhibitory activity; n≥3 for each peptide concentration.
(D) CN pull-down with GST-fusions of the LxVPc2 sequence (NFATc2) bearing individual or grouped substitutions based on LxVPc1. Peptide sequences are shown with LxVPc1-directed mutations shaded. c2 and c1 are the wt LxVPc2 and LxVPc1 sequences. The blot shows a representative pull-down experiment of four.

Antonio Rodríguez, et al. Mol Cell. ;33(5):616-626.
5.
Figure 6

Figure 6. Rcn1 interacts with Cna1 in vivo via an LxVP motif. From: A conserved docking surface on calcineurin mediates interaction with substrates and immunosuppressants.

(A) GST or GST-Rcn1 was expressed in yeast strain Y258 and purified. Co-purification of Cna1 with GST-tagged proteins was assessed by western blot. Where indicated, Rcn1–Cna1 interaction was competed in vitro with LxVPc1 or LxVPc1mut peptides.
(B) Left. The Rcn1 YLKVP motif inhibits mammalian CN phosphatase activity toward RIIp. Inorganic phosphate release in the presence of YLKVP, YaKaa or LxVPc1 synthetic peptides was determined spectrophotometrically. 100% activity was defined as phosphate release in the absence of peptide. Data are from a representative experiment performed in triplicate (means ± sd). Right. Binding of CN to GST fusions of the Rcn1 YLKVP motif, its mutated form YaKaa or LxVPc1. Pull down experiments were conducted in the presence of Ca2+ (1.5mM) and calmodulin (600nM), and bound CN detected by western blot.
(C) Binding of mammalian CN to GST-YLKVP or GST-LxVPc1 was competed by adding either LxVPc1 peptide or increasing concentrations of CsA-CypA complex (10 and 100μM). CsA alone was used as negative control.
(D,E) The Rcn1-LxVP sequence is required for interaction with CN in vivo. (D) GST-Rcn1 or GST-Rcn1-YaKaa was expressed in yeast strain Y258 and purified. Co-purification of Cna1 was analyzed as in (A). (E) Two-Hybrid interaction between the Cna1-Gal4-DNA binding domain fusion and Gal4 activation domain fusions with Rcn1 or Rcn1-YaKaa was assayed in strain PJ69-4A (GAL1 HIS3). Cells were analyzed by dilution plating on medium lacking tryptophan and leucine for selection of plasmids and, where indicated, lacking histidine to assay reporter construct activation. Growth was scored after 4 days at 30°C.

Antonio Rodríguez, et al. Mol Cell. ;33(5):616-626.
6.
Figure 5

Figure 5. Mutations of key calcineurin residues involved in interaction with LxVPc1 impair signaling in yeast. From: A conserved docking surface on calcineurin mediates interaction with substrates and immunosuppressants.

(A) Alignment of human calcineurin Aα (hCnA), Saccharomyces cerevisiae calcineurin (yCnA) and Neurospora crassa calcineurin A (nCnA). The conserved yCnA residues W388 and F392 (equivalent to W352 and F356 in human CnA) are indicated.
(B) Two-hybrid assay assessing interaction between wt or mutant Cna1 proteins and the substrates Hph1, Slm1 and Slm2, the regulator Rcn1 (homolog of mammalian RCAN1), and the CN B subunit (Cnb1). Growth was assayed by spotting serial dilutions of yeast strains on medium lacking tryptophan and leucine to select for both Gal4-fusion plasmids, and where indicated on medium lacking histidine to assay reporter activation. Interactions with Hph1, Slm1 and Slm2 were assessed using histidine deficient medium supplemented with 3mM 3-AT (see Experimental Procedures). DBD: Gal4–DNA-binding-domain fusion of wt or mutant CNA1. AD: Gal4–activation-domain fusions containing the indicated open reading frames. All plasmids were transformed into strain pJ69-4A (GAL1-HIS3). Cell growth was scored after 4–5 days at 30°C.
(C) Crz1 transcriptional activity. β-galactosidase activity was measured in strain BY1001 (Δcna1 Δcna2 4X-CDRE-lacZ) transformed with vector pRS314 encoding wt or mutant CNA1. Cells were grown in the presence of FK506 (FK) or vehicle (ET), and where indicated 200 mM CaCl2 was added 2 h before harvest.
(D) Growth of cells expressing wt or mutant CNA alleles under environmental stress. Cells of strain cna1Δ, cna2Δ were transformed with empty vector, CNA1, cna1W388A or cna1F392A and scored by spotting serial dilutions on rich YPD medium or YPD containing 4mM MnCl2, 800 mM NaCl + ET solvent, or NaCl + 2 μg/ml FK506. Cell growth was scored after 3 days at 30°C.

Antonio Rodríguez, et al. Mol Cell. ;33(5):616-626.
7.
Figure 1

Figure 1. LxVPc1 blocks NFAT-dependent signaling. From: A conserved docking surface on calcineurin mediates interaction with substrates and immunosuppressants.

(A) Schematic representation of the N-terminal regulatory and CN-interacting domain of NFAT proteins, based on the structure of human NFATc1. Conserved regions among NFAT members (c1–c4) are indicated: AD = activation domain; DBD = DNA binding domain. The PxIxIT and LxVP sequences from NFAT members are shown. For LxVP motifs, sequences are aligned to show conserved residues (black); identical residues are shaded. The consensus reference LxVP motif is shown below. Φ, aromatic residue.
(B) Jurkat T cells were infected with lentivirus encoding a GFP fusion of either wt LxVPc1 or LxVPc1mut (control) peptide. Infection efficiency was monitored by flow cytometry. Plots show cell number versus GFP fluorescence. Non-infected cells were used as negative control (dotted line). Viruses encoding LxVPc1 (dotdashed line) or LxVPc1mut (solid line) infected the whole population, and CsA treatment (200 ng/ml) did not alter GFP expression (dashed line).
(C) Western blot showing the effect of LxVPc1 on NFAT activation. Infected cells were stimulated (30 min) with PMA (P, 10 ng/ml) plus Ca2+ ionophore (Io, 1μM). As a positive control of NFAT-pathway inhibition, cells infected with LxVPc1mut were treated with CsA (200 ng/ml). The blot shows expression of NFATc2 in total cell lysates, and the positions of phosphorylated (P) and dephosphorylated (deP) proteins are indicated. Φ, non-infected cells.
(D and F) Expression of NFAT-regulated genes. Total mRNA was extracted after P+Io stimulation (4h), and mRNA levels of RCAN1.4 and IL-2 (D), and IL-3, IL-8 and IL-13 (F) were quantified by Q-PCR. Rq values were calculated by comparing the amount of mRNA post stimulation with that obtained with corresponding non-stimulated cells. 100% corresponds to the amount of mRNA induced in cells infected with control virus (LxVPc1mut). Data are means ± sd of three independent experiments. ***, p<0.0001 vs. LxVPc1 mut.
(E) NFAT-dependent transcription. Infected cells were transiently transfected with plasmids encoding luciferase under control of the RCAN1.4- or the IL-2 promoter. After 24h, cells were pretreated (30min) with 200 ng/ml CsA or vehicle and stimulated with P+Io (4h). Luciferase activity is expressed as fold induction over baseline levels in transfected unstimulated cells (means ± sd; n=3). *, p<0.05 vs. LxVPc1 mut; **, p<0.001 vs. LxVPc1 mut.

Antonio Rodríguez, et al. Mol Cell. ;33(5):616-626.

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