PDBsum entry 2p6b

Hydrolase/hydrolase regulator

PDB id: 2p6b

Contents

Protein chains

14 a.a.

357 a.a.

146 a.a.

Ligands

PO4 ×2

Metals

_CA ×8

_ZN ×2

_FE ×2

Waters ×564

References listed in PDB file

Key reference

Title

Structure of calcineurin in complex with pvivit peptide: portrait of a low-Affinity signalling interaction.

Authors

H.Li, L.Zhang, A.Rao, S.C.Harrison, P.G.Hogan.

Ref.

J Mol Biol, 2007, 369, 1296-1306. [DOI no: 10.1016/j.jmb.2007.04.032]

PubMed id

17498738

Abstract

The protein phosphatase calcineurin recognizes a wide assortment of substrates and controls diverse developmental and physiological pathways in eukaryotic cells. Dephosphorylation of the transcription factor NFAT and certain other calcineurin substrates depends on docking of calcineurin at a PxIxIT consensus site. We describe here the structural basis for recognition of the PxIxIT sequence by calcineurin. We demonstrate that the high-affinity peptide ligand PVIVIT adds as a beta-strand to the edge of a beta-sheet of calcineurin; that short peptide segments containing the PxIxIT consensus sequence suffice for calcineurin-substrate docking; and that sequence variations within the PxIxIT core modulate the K(d) of the interaction within the physiological range 1 muM to 1 mM. Calcineurin can adapt to a wide variety of substrates, because recognition requires only a PxIxIT sequence and because variation within the core PxIxIT sequence can fine-tune the affinity to match the physiological signalling requirements of individual substrates.

Figure 2.
Figure 2. Structure of human calcineurin in complex with 14mer PVIVIT peptide. (a) Ribbon diagram of PVIVIT (red) sandwiched between two calcineurin heterodimers in the asymmetric unit. CNA molecule A is colored light blue, and its associated CNB, light green. CNA molecule C is colored dark blue, and its associated CNB, purple. (b) Backbone hydrogen bonds connecting PVIVIT peptide to β-sheets of the two CNA molecules. (c) Schematic diagram of (b) depicting the position and register of the peptide with respect to β-strands 14 of the two CNA molecules. Residues numbered in boldface project toward the viewer.

Figure 3.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2007, 369, 1296-1306) copyright 2007.

Secondary reference #1

Title

Crystal structures of human calcineurin and the human fkbp12-Fk506-Calcineurin complex.

Authors

C.R.Kissinger, H.E.Parge, D.R.Knighton, C.T.Lewis, L.A.Pelletier, A.Tempczyk, V.J.Kalish, K.D.Tucker, R.E.Showalter, E.W.Moomaw.

Ref.

Nature, 1995, 378, 641-644.

PubMed id

8524402

Secondary reference #2

Title

X-Ray structure of calcineurin inhibited by the immunophilin-Immunosuppressant fkbp12-Fk506 complex.

Authors

J.P.Griffith, J.L.Kim, E.E.Kim, M.D.Sintchak, J.A.Thomson, M.J.Fitzgibbon, M.A.Fleming, P.R.Caron, K.Hsiao, M.A.Navia.

Ref.

Cell, 1995, 82, 507-522. [DOI no: 10.1016/0092-8674(95)90439-5]

PubMed id

7543369

Secondary reference #3

Title

Affinity-Driven peptide selection of an nfat inhibitor more selective than cyclosporin a.

Authors

J.Aramburu, M.B.Yaffe, C.López-Rodríguez, L.C.Cantley, P.G.Hogan, A.Rao.

Ref.

Science, 1999, 285, 2129-2133. [DOI no: 10.1126/science.285.5436.2129]

PubMed id

10497131

Figure 2.
Fig. 2. The VIVIT peptide is a potent inhibitor of the NFAT-calcineurin interaction, and its substitution into the calcineurin docking site enhances the calcineurin responsiveness of NFAT1. (A) Inhibition of the NFAT-calcineurin interaction (9). Calcineurin (Cn) was activated with calmodulin (CaM) and CaCl[2] (Ca^2+), and its binding to GST (lane 1) and GST-NFAT1 (residues 1 through 415) (lanes 2 through 11) was evaluated by protein immunoblotting. Cn A, calcineurin A chain. (B and C) Inhibition of the calcineurin-mediated dephosphorylation of NFAT proteins (9). Lysates of HeLa cells expressing HA-NFAT1 (B) or lysates from HEK 293T cells expressing HA-NFAT1, HA-NFAT2, or HA-NFAT4 (C) were incubated with the phosphatase inhibitor sodium pyrophosphate (NaPPi, lane 1) or with activated calcineurin (Cn+CaM+Ca^2+) in the absence or presence of peptides at the indicated micromolar concentrations. The phosphorylation status of NFAT proteins was evaluated by protein immunoblotting with anti-HA. The positions of phospho- and dephospho-NFAT are indicated by arrows in (B). (D) Inhibition of NFAT-dependent gene expression. (Left panel) Jurkat cells were cotransfected with a 3xNFAT-Luc reporter plasmid and with expression plasmids encoding murine NFAT1, GFP, GFP-SPRIEIT, or GFP-VIVIT as indicated (13). (Right panel) Jurkat cells were cotransfected with a 3xNFAT-Luc reporter plasmid and with expression plasmids encoding GFP, GFP-VIVIT, and murine NFAT1, human NFAT2, and human NFAT4 as indicated (11). Twenty-four hours after transfection, luciferase activity induced by endogenous NFAT (Endog.) or by overexpressed NFAT proteins was measured in unstimulated cells and in cells stimulated for 6 hours with PMA and ionomycin. (E) Substitution of the VIVIT sequence into NFAT1 (12). Cl.7W2 murine T cells were transfected with wild-type HA-NFAT1-GFP or with the mutant HA-NFAT1[VIVIT]-GFP, in which HPVIVITGP replaces SPRIEITPS. Cells were stimulated with ionomycin (Iono) at the concentrations indicated in the absence or presence of 1 µM CsA, and the phosphorylation status of NFAT1 was assessed by protein immunoblotting with anti-HA. (F) Localization of NFAT1 and NFAT1[VIVIT] in cells (12). HeLa cells transiently expressing wild-type HA-NFAT1-GFP or HA-NFAT1[VIVIT]-GFP were left untreated or were treated with 10 µM CsA (for 16 hours). NFAT1 proteins were visualized in fixed cells by GFP fluorescence.

Figure 3.
Fig. 3. The VIVIT peptide selectively inhibits NFAT activation but not calcineurin activity. (A) The VIVIT peptide did not inhibit calcineurin phosphatase activity, assayed as radiolabel released (counts per minute × 10^ 3) from ^32P-phospho-RII peptide (9). The numbers next to each peptide label indicate peptide concentrations (micromolar). CsA/CypA complexes were used at 10 µM. (B) Selective inhibition of NFAT reporter activity (11). Jurkat cells were cotransfected with 3xNFAT-Luc (left panel) or 2xNF- B-Luc (right panel) reporter plasmid, and with GFP and GFP-VIVIT expression plasmids as indicated (measured in micrograms of plasmid per 10^6 cells). Twenty-four hours after transfection, cells were left untreated (open bars) or were stimulated for 6 hours with PMA and ionomycin (solid bars). (C) Calcineurin dependence of NFAT and NF- B reporter activity in T cells (11). Jurkat cells were transfected with 3xNFAT-Luc (left panel) or 2xNF- B-Luc (right panel) reporter plasmid. Twenty-four hours after transfection, cells were left unstimulated or were stimulated for 6 hours with PMA and ionomycin (P+I) in the absence or presence of CsA. (D) Inhibition of NFAT-dependent activation of the IL-2 and TNF- promoters (11). Jurkat cells were cotransfected with GFP or GFP-VIVIT expression plasmids and with luciferase reporter plasmids driven either by the human IL-2 promoter (left panel) or by the human TNF- promoter (right panel). Twenty-four hours after transfection, cells were left unstimulated (open bars) or were stimulated for 6 hours with PMA and ionomycin (solid bars) or with anti-CD3 and anti-CD28 (hatched bars).

The above figures are reproduced from the cited reference with permission from the AAAs

Secondary reference #4

Title

Structural delineation of the calcineurin-Nfat interaction and its parallels to pp1 targeting interactions.

Authors

H.Li, A.Rao, P.G.Hogan.

Ref.

J Mol Biol, 2004, 342, 1659-1674. [DOI no: 10.1016/j.jmb.2004.07.068]

PubMed id

15364589

Figure 6.
Figure 6. Model of the CnAα–VIVIT complex obtained from docking simulations with 5mer VIVIT peptide. a, The peptide is red in a ball-and-stick representation, and CnAα is grey in a ribbon diagram, with β strands 11–14 highlighted in yellow. b, In the model, the side-chains of Ile8 and Ile10 project toward M290 of CnAα. c, The predicted hydrogen bond between Thr11 of VIVIT and N330 of CnAα is shown. d, van der Waals packing interaction between Ile10 of the VIVIT peptide and I331 and Y288 of CnAα.

Figure 9.
Figure 9. Topography of the docking sites on calcineurin and PP1. a, Views from the end of each site that would be occupied by the N-terminal residues of the corresponding peptide, illustrating the rather different surface topographies. F299 and other labelled residues in the foreground surround the proline pocket. b, Views from the end of each site that would be occupied by the C-terminal residues of the corresponding peptide, illustrating the similar topographies of the hydrophobic troughs in the two proteins. The trough in calcineurin, however, is closed off by the side-chains of F299 and P300, thereby forming the Ile8 recess.

The above figures are reproduced from the cited reference with permission from Elsevier