Targets of immunophilin-immunosuppressant complexes are distinct highly conserved regions of calcineurin A.

Cárdenas, María E.; Muir, R S; Breuder, T.; Heitman, Joseph

doi:10.1002/j.1460-2075.1995.tb07277.x

Cited by 107 publications

(111 citation statements)

References 63 publications

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“…Trp-352 is another residue whose mutant phenotype is not obvious from the structure. The W352C equivalent mutant of yeast CMP-1 exhibited FK506 resistance, but no change in sensitivity to CsA (25). As Trp-352 has hydrophobic contacts and forms a hydrogen bond with both CsA and FK506 (Fig.…”

Section: Resultsmentioning

confidence: 94%

“…The unique hydrogen bond between Tyr-341 of CNA and CsA can account for the observation that mutation of Tyr-341 to Phe rendered T lymphocytes and yeast resistant to CsA, but not to FK506 (24,25). Similarly, mutation of Thr-350 of yeast CN CMP-1 to either Lys or Arg, which corresponds to Val-314 in human CNA, leads to selective resistance to CsA.…”

Section: Resultsmentioning

confidence: 99%

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Crystal structure of calcineurin–cyclophilin–cyclosporin shows common but distinct recognition of immunophilin–drug complexes

Huai

Kim

Liu

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

253

191

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Calcineurin, a Ca 2؉ ͞calmodulin-dependent protein phosphatase, is the common target for two immunophilin-immunosuppressant complexes, cyclophilin A-cyclosporin A (CyPA-CsA) and FKBP-FK506. How the two structurally distinct immunophilin-drug complexes bind the same target has remained unknown. We report the crystal structure of calcineurin (CN) in complex with CyPA-CsA at 2.8-Å resolution. The CyPA-CsA complex binds to a composite surface formed by the catalytic and regulatory subunits of CN, where the complex of FK506 and its binding protein FKBP also binds. While the majority of the CN residues involved in the binding are common for both immunophilin-immunosuppressant complexes, a significant number of the residues are distinct. Unlike immunosuppressants ͉ FK506 ͉ FK506-binding protein ͉ protein phosphatase ͉ T cell C alcineurin (CN) is a unique serine͞threonine protein phosphatase (PP3 or PP2B) in its regulation by the second messenger Ca 2ϩ and calmodulin. CN is involved in many biological processes, including immune responses (1-5), the second messenger cAMP pathway (3, 6), Na ϩ ͞K ϩ ion transportation in the nephron (7), cell cycle progression in lower eukaryotes (8), cardiac hypertrophy (9), and memory formation (10, 11). The most extensively studied function of CN is its role in the signal transduction pathway during T cell activation. CN has been shown to be a common receptor for two immunophilinimmunosuppressant complexes, cyclophilin A-cyclosporin A (CyPA-CsA) and FK506-binding protein (FKBP)-FK506 (12, 13). The binding of CyPA-CsA or FKBP-FK506 inhibits the calcium-dependent dephosphorylation of the transcription factor nuclear factor of activated T cell (NFAT) by CN, thus blocking T cell receptor-mediated cytokine transcription and T-cell activation (14,15).Since the discovery of CN as a common target for both CyPA-CsA and FKBP-FK506 complexes, how two structurally distinct immunophilin-immunosuppressant complexes recognize a common target has remained a mystery. While the crystal structures of CN and its complex with FKBP-FK506 have shed light on the molecular recognition of CN by 17), the CyPA-CsA-CN structure has remained elusive. We report here the crystal structure of CyPA-CsA-CN at 2.8-Å resolution. The structure revealed that CyPA-CsA and FKBP-FK506 are bound to the same surface of CN, thus providing a molecular basis for understanding the regulation of CN activity by the immunophilin-immunosuppressant. MethodsProtein Purification. The catalytic subunit (CNA, 61 kDa) and regulatory subunit (CNB, 19 kDa) of the ␣ isoform of human CN were coexpressed in Escherichia coli and purified by using a previously described procedure with slight modifications (18). The recombinant human CyPA was expressed as a glutathione S-transferase (GST) fusion protein in E. coli BL21 and purified by glutathione-agarose beads (Sigma). The CyPACsA-CN complex was prepared by a four-step procedure: (i) purification of recombinant human CN by Ni NTA (Qiagen, Valencia, CA) and Q-Sepharose (Pharmacia) columns, (ii) purific...

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Section: Resultsmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

Crystal structure of calcineurin–cyclophilin–cyclosporin shows common but distinct recognition of immunophilin–drug complexes

Huai

Kim

Liu

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

253

191

View full text Add to dashboard Cite

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“…To test this possibility, we took advantage of the interaction between Fpr1p and Cna1p that is elicited by a small membrane-permeable molecule FK506, a macrolide isolated from Streptomyces tsukubaensis (23)(24)(25)(26). FK506 was used to trigger the interaction between a Gal80-Fpr1p fusion and plasma membrane-localized Myr-Cna1p.…”

Section: Fig 2 Lowering the Gal3p Expression Level Causes Reductionmentioning

confidence: 99%

Gene activation by interaction of an inhibitor with a cytoplasmic signaling protein

Peng

Hopper

2002

Proc. Natl. Acad. Sci. U.S.A.

105

127

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Galactose-inducible genes (GAL genes) in yeast Saccharomyces cerevisiae are efficiently transcribed only when the sequencespecific transcription activator Gal4p is activated. Activation of Gal4p requires the interaction between the Gal4p inhibitory protein Gal80p and the galactokinase paralog, Gal3p. It has been proposed that Gal3p binds to a Gal80p-Gal4p complex in the nucleus to activate Gal4p. Here, we present evidence that the Gal3p-Gal80p interaction occurs in the cytoplasm, and concurrently, Gal80p is removed from Gal4p at the GAL gene promoter. We also show that GAL gene expression can be activated by heterologous protein-protein interaction in the cytoplasm that is independent of galactose and Gal3p function. These results indicate that galactose-triggered Gal3p-Gal80p association in the cytoplasm activates Gal4p in the nucleus.

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“…First, the N⑀1 nitrogen of the conserved Trp 352 of the calcineurin A-subunit forms a bifurcated H-bond with both the 13-and 15-methoxy groups of FK506. Mutation of this residue in yeast calcineurin prevents the FKBP12-FK506 complex from binding while retaining calcineurin phosphatase activity (Cardenas et al, 1995;Hemenway and Heitman, 1999). Second, the conserved Leu 343 of the A-subunit is in proximity (4.2 Å) to the methyl moiety of the 15-methoxy group of FK506.…”

mentioning

confidence: 99%

Genetically Engineered Analogs of Ascomycin for Nerve Regeneration

Revill¹,

Voda²,

Reeves³

et al. 2002

J Pharmacol Exp Ther

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The polyketides FK506 (tacrolimus) and FK520 (ascomycin) are potent immunosuppressants that function by inhibiting calcineurin phosphatase through formation of an FKBP12-FK506/ 520-calcineurin ternary complex. They also have calcineurinindependent neuroregenerative properties in cell culture and animal models of nervous system disorders. Based on the crystal structure of the FKBP12-FK506-calcineurin complex, we deduced that the 13-and 15-methoxy groups of FK506 or FK520 are important for inhibition of calcineurin phosphatase but not for binding to FKBP12. By genetic modification of the FK520 gene cluster, we generated 13-and 15-desmethoxy analogs of FK520 that contain hydrogen, methyl, or ethyl instead of methoxy at one or both of these positions. These analogs bind FKBP12 tightly, have decreased calcineurin phosphatase inhibition and immunosuppressive properties, and enhance neurite outgrowth in cell cultures. A representative compound was also shown to accelerate nerve regeneration and functional recovery in the rat sciatic nerve crush model.

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Targets of immunophilin-immunosuppressant complexes are distinct highly conserved regions of calcineurin A.

Cited by 107 publications

References 63 publications

Crystal structure of calcineurin–cyclophilin–cyclosporin shows common but distinct recognition of immunophilin–drug complexes

Crystal structure of calcineurin–cyclophilin–cyclosporin shows common but distinct recognition of immunophilin–drug complexes

Gene activation by interaction of an inhibitor with a cytoplasmic signaling protein

Genetically Engineered Analogs of Ascomycin for Nerve Regeneration

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