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J. Biol. Chem., Vol. 280, Issue 45, 37698-37706, November 11, 2005

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Inhibition of the Calcineurin-NFAT Interaction by Small Organic Molecules Reflects Binding at an Allosteric Site^*

Sunghyun Kang12, Huiming Li2, Anjana Rao, and Patrick G. Hogan3

From the CBR Institute for Biomedical Research, Boston, Massachusetts 02115 and the Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115

Transcriptional signaling from the Ca²⁺-calmodulin-activated phosphatase calcineurin to its substrate NFAT (nuclear factor of activated T cells, also termed NFATc) is critically dependent on a protein-protein docking interaction between calcineurin and the PXIXIT motif in NFAT. Several inhibitors of NFAT-calcineurin association (INCA compounds) prevent binding of NFAT or the peptide ligand PVIVIT to calcineurin. Here we show that the binding site on calcineurin for INCA1, INCA2, and INCA6 is centered on cysteine 266 of calcineurin A and does not coincide with the core PXIXIT-binding site. Although ample evidence indicates that INCA1 and INCA2 react covalently with cysteine 266, covalent derivatization alone is not sufficient for maximal inhibition of the calcineurin-PVIVIT interaction, because the maleimide INCA12 reacts with the same site and produces only very modest inhibition. Thus, inhibition arises through an allosteric change affecting the PXIXIT docking site, which may be assisted by covalent binding but depends on other specific features of the ligand. The spatial arrangement of the binding sites for PVIVIT and INCA makes it probable that the change in conformation involves the 11-12 loop of calcineurin. The finding that an allosteric site controls NFAT binding opens new alternatives for inhibition of calcineurin-NFAT signaling.

Received for publication, February 28, 2005 , and in revised form, September 7, 2005.

^* This work was supported by National Institutes of Health Grants R01 AI40127 and R21 AI059940. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at www.jbc.org) contains supplemental Fig. 1, a MALDI-TOF analysis of peptide and peptide-INCA1 adduct peaks displayed at higher resolution.

¹ Present address: Proteomics System Center, Korea Research Inst. of Bioscience & Biotechnology (KRIBB), 52 Eoeun-dong, Yuseong-gu, Daejon, 305-333, Republic of Korea.

² These authors contributed equally to this work.

³ To whom correspondence should be addressed: CBR Inst. for Biomedical Research, 200 Longwood Ave., Boston, MA 02115. Tel.: 617-278-3057; Fax: 617-278-3280; E-mail: hogan{at}cbr.med.harvard.edu.

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