inhibition of the calcineurin-nfat interaction by small organic
TRANSCRIPT
Inhibition of the Calcineurin-NFAT Interaction by Small Organic MoleculesReflects Binding at an Allosteric Site*
Sunghyun Kang†‡§#, Huiming Li†‡#, Anjana Rao†‡, and Patrick G Hogan†¶
From †The CBR Institute for Biomedical Research, 200 Longwood Avenue, Boston, Massachusetts02115, USA, and the ‡Department of Pathology, Harvard Medical School, 200 Longwood Avenue,Boston, Massachusetts 02115, USA
Running Title: INCA-binding site on calcineurin Aα
Address correspondence to Patrick G Hogan, The CBR Institute for Biomedical Research, 200 LongwoodAvenue, Boston, Massachusetts 02115, USA. Tel: 617-278-3057; Fax: 617-278-3280; E-mail:[email protected]
Transcriptional signalling from the Ca2+-calmodulin-activated phosphatase calcineurin to itssubstrate NFAT (also termed NFATc) is criticallydependent on a protein-protein docking interactionbetween calcineurin and the PxIxIT motif in NFAT.Several inhibitors of NFAT-calcineurin association(INCA compounds) prevent binding of NFAT or thepeptide ligand PVIVIT to calcineurin. Here weshow that the binding site on calcineurin for INCA1,INCA2, and INCA6 is centered on cysteine-266 ofcalcineurin Aα and does not coincide with the corePxIxIT-binding site. Although ample evidenceindicates that INCA1 and INCA2 react covalentlywith cysteine-266, covalent derivatization alone isnot sufficient for maximal inhibition of thecalcineurin-PVIVIT interaction, since the maleimideINCA12 reacts with the same site and produces onlyvery modest inhibition. Thus inhibition arisesthrough an allosteric change affecting the PxIxIT-docking site, which may be assisted by covalentbinding, but which depends on other specificfeatures of the ligand. The spatial arrangement ofthe binding sites for PVIVIT and INCA makes itprobable that the change in conformation involvesthe β11-β12 loop of calcineurin. The finding that anallosteric site controls NFAT binding opens newalternatives for inhibition of calcineurin-NFATsignalling.
Ca2+-calcineurin signalling serves vital purposes inmammalian cells, among them the provision of a directlink between mobilization of cytoplasmic Ca2+ and geneexpression (1,2). In the physiological activation of Tcells, calcineurin acts through NFAT-familytranscription factors and other transcriptional effectors(3-6). Levels of calcineurin in T cells are limiting, andupward or downward modulation of calcineurinenzymatic activity is directly reflected in increased or
decreased transcription from cytokine gene promoters(7-14). The immunosuppressive drugs cyclosporin A(CsA)1 and FK506 owe their clinical effectiveness tothe ability of CsA-cyclophilin or FK506-FKBP12complexes to inhibit calcineurin in cells of the immunesystem (15,16). However, the beneficial actions of CsAand FK506 are counterbalanced by serious toxicitiesattributed at least in part to their interference withcalcineurin signalling in other cells and tissues (17,18).
The centrality of calcineurin and the calcineurin-NFAT pathway to immune responses has suggested thatinterrupting signalling at any of several points couldprovide a therapeutic alternative to currentimmunosuppressive drugs. Promising target points arethe Ca2+ signal that activates calcineurin (19,20), theprotein-protein interaction of calcineurin and NFAT(21-25), and the cooperative binding of NFAT and AP1on DNA (26-30). Each strategy has the potential to bemore selective, and hence less toxic, than treatmentwith CsA or FK506, but none has yet advanced to thestage of small nonpeptide inhibitors that can be testedin vivo.
There is considerable evidence that targeting thecalcineurin-NFAT protein-protein interaction willproduce a selective inhibition. We have demonstratedthat the calcineurin-NFAT interaction is based onrecognition of a PxIxIT motif in NFAT, and that thisrecognition is essential for efficient signalling (23).Peptides that compete for binding at the PxIxIT-recognition site both inhibit the calcineurin-NFATinteraction in vitro and selectively inhibit calcineurin-NFAT signalling in cells (23,24,31). A recentlypublished study shows that a competitor peptidemodified to promote its uptake into cells can preventheterologous graft rejection in mice (32). Further, high-throughput screening of a library of organic compoundshas led to identification of nonpeptide inhibitors ofNFAT-calcineurin association (INCA compounds) (25).
JBC Papers in Press. Published on September 7, 2005 as Manuscript M502247200
Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
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These compounds interfere with calcineurin-NFATsignalling in cells, motivating a continued search forinhibitors having higher affinity and reducednonspecific toxicity, and suitable for in vivoadministration to animals.
The further development of inhibitors can be guidedby structural information about the sites of protein-protein interaction and inhibitor binding. To this end,we have determined the structure of the NFAT dockingsite on calcineurin, in which the PxIxIT recognitionpeptide of NFAT binds in an extended configurationand each conserved residue of the peptide directlycontacts calcineurin (33). Here we extend the structuralstudies by identifying a distinct binding site for INCAcompounds, at a cysteine residue adjacent to the PxIxITpeptide docking site. It is covalent binding or tightnoncovalent binding of the bulky INCA compounds atthis second site that allosterically inhibits recognition ofPxIxIT peptide and NFAT. Our findings serve toidentify the approaches that are most likely to befruitful in developing improved inhibitors. In additionthey suggest that calcineurin-substrate recognition, likecalcineurin catalytic activity (34-37), may be modulatedphysiologically by redox reactions.
EXPERIMENTAL PROCEDURES
cDNA constructs, protein expression, and syntheticpept ides— The expression construct for GST-calcineurin(2-347), encoding GST and the catalyticdomain of human calcineurin Aα with the substitutionsY341S, L343A, and M347D, has been described (24).Constructs encoding mutant proteins with individualC>A or C>V replacements, or with the combinedC>A//C266 and C>A//V266 replacements, were madeby PCR mutagenesis with appropriate primers andsubcloning. In each case the sequence of the cDNAinsert was verified.
GST-calcineurin fusion proteins were expressed inE. coli strain BL21-CodonPlus-RP (Stratagene) byovernight growth at 18°C after addition of 1 mM IPTG.Calcineurin was purified from the bacterial lysate byaffinity chromatography on Glutathione Sepharose(Amersham Biosciences) and cleavage in 50 mMTris.HCl pH 7.0, 150 mM NaCl, 1 mM EDTA, 1 mMDTT with PreScission Protease (AmershamBiosciences). Purified calcineurin(2-347) wasconcentrated by Centricon-30 filtration, aliquotted,flash frozen, and stored at –80°C. In most cases, theconcentration of DTT in the buffer was reduced to 0.1mM during concentration of the protein. This step andthe further substantial dilution into fluorescencepolarization assays minimized any interference ofresidual DTT with the INCA compounds. In somecases, protein was concentrated in buffer free of DTTimmediately before freezing.
PVIVIT 14-mer peptide (24) was labelled overnightat room temperature in a reaction containing 2 mgpeptide, 1.5 mg Oregon Green (Oregon Green 488carboxylic acid, succinimidyl ester *5-isomer*;Molecular Probes), and 5 µl diisopropylethylamine in190 µ l anhydrous N,N-dimethylformamide. Thelabelled peptide was purified by C18 reversed-phaseHPLC. The calcineurin(254-273)/C256S peptide,RGSSYFYSYPAVCEFLQHNN, was synthesized andHPLC purified at Tufts University Core Facility, andstored desiccated at –20°C.
Inhibitors— INCA1, INCA2, INCA6, and INCA12(Figure 1) were obtained from ChemBridge. Inhibitorstocks were prepared at 10 mM in anhydrous DMSOand stored desiccated at –20°C. Corresponding aliquotsof anhydrous DMSO were stored desiccated at –20°Cfor addition to control incubations. DMSO atconcentrations up to 1% had no effect on theparameters studied.
Fluorescence measurements— Fluorescencemeasurements were made on 10 µl samples in a black384-well plate (Molecular Devices) using thefluorescein filter set (excitation 485 nm, emission 530nm) in an Analyst plate reader (Molecular Devices).Each final reaction in 100 mM NaCl, 2 mM Mg acetate,20 mM HEPES pH 7.4, 0.1% (w/v) bovine IgGcontained calcineurin, 100 nM fluorescent PVIVIT, andother additions specified. Calcineurin was omitted formeasurements of the fluorescence emitted by unboundpeptide. In competitive binding assays, calcineurin wastypically present at 1 µM or 1.5 µM; in direct bindingtitrations, calcineurin was used at 0.15 µM to 10 µM.Except in time course experiments, adequate time wasallowed for the signal to reach a stable value, asverified by repeated readings of the same samples.Pretreatment with IAM, NEM, or INCA12 was for 30min to 2 hr.
Data from direct titrations with calcineurin havebeen fitted to a model for equilibrium binding to asingle class of sites (38). Competition and time coursedata for INCA compounds have been plotted withoutattempting to fit a theoretical curve, since the data arenot sufficient to specify parameters for covalentbinding, noncovalent binding, and side reactions of theINCA compounds.
The stability of calcineurin-INCA2 association wasexamined after equilibration of 6 µM calcineurin, 100nM labelled PVIVIT, and 20 µM INCA2 to produce anessentially complete blockade of PVIVIT binding. Toassess short-term stability of the complex, samples wereextracted by vortex mixing with 2 volumes of buffer-saturated ether for 2 min, and briefly centrifuged toseparate the phases. Extraction of samples containingonly calcineurin and labelled PVIVIT had no effect onpeptide binding and did not prevent subsequent
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displacement of peptide by INCA2. Extraction ofsamples containing only INCA compound, prior toincubation with calcineurin and PVIVIT, demonstratedthat removal of unbound INCA2 from the aqueousphase was complete within 10 sec. To assess long-termstability of the complex, samples containing PVIVITand blocked calcineurin were supplemented with 5 mMNEM or 5 mM DTT, and PVIVIT binding wasmonitored during the following 4 hr. Because thecalcineurin concentration in fluorescence polarizationassays is greater than the Kd of the calcineurin-PVIVITinteraction, each of these procedures is a sensitive testfor unmasking of blocked PVIVIT-binding sites.
Synthetic peptide-INCA reactions— Freshlydissolved RGSSYFYSYPAVCEFLQHNN peptide, 6µM in 200 mM NaCl, 4 mM Mg acetate, 20 mMHEPES pH 7.35, in 20 µl total volume, was incubatedfor 60 min at room temperature with 50 µM INCA1,INCA2, or INCA6. At the end of this incubation,samples were either immediately diluted into 240 µl0.1% (v/v) TFA and frozen; or further incubated for 60min after addition of NEM to 1 mM, then diluted into0.1% (v/v) TFA and frozen. Control samples werereacted first with 1 mM NEM for 60 min, then with 50µM INCA compound for 60 min.
Mass spectrometry— For MALDI-TOF massspectrometry, samples were desalted and concentratedby binding the peptides to C18 resin in a ZipTip(Millipore) and eluting with 70% (v/v) acetonitrile,0.1% (v/v) TFA. Eluted peptides were diluted 1:10with α-cyano-4-hydroxycinnamic acid matrix (10mg/ml in 50% (v/v) acetonitrile, 0.1% (v/v) TFA) anddried onto the target plate. Mass spectra were obtainedusing an Applied Biosystems Voyager-DE STRinstrument operated in reflector mode, with acceleratingvoltage 20 kV, grid voltage 72%, guide wire 0.1%, andextraction delay time 175 ns. Calibration wasperformed using the MSCAL2 mass standard set(Sigma).
A minor peak frequently observed in the peptidesample, having mass ~1776 Da, is apparently thepep t ide f ragment RGSSYFYSYPAVCEF.Corresponding peaks in samples that had beenincubated with INCA1, INCA2, or NEM— or insamples subjected to prolonged incubation with INCA6to allow the reaction to approach completion— wereshifted by the same mass as the main peak in thoseincubations. A peak of mass ~1397 Da, matching thecalculated mass of the fragment RGSSYFYSYPAV,was present occasionally both in untreated peptidesamples and in samples that had been incubated withNEM or INCA compounds. The reaction, or lack ofreaction, observed with these peptide fragmentsreinforces the conclusion that all the compoundsinvestigated reacted with the cysteine –SH group.
Cellular assays— Dephosphorylation of NFAT,nuclear import of NFAT, and induction of cytokinemRNAs in D5 T cells were assessed as described inprevious publications (23-25).
Calcineurin activity in cell lysates was measuredusing a standard assay (39) with phosphorylated RIIpeptide as substrate. D5 T cells were chilled on ice,collected by centrifugation at 4°C, and resuspended in50 mM Tris·HCl pH 7.5, 1 mM EDTA, 0.1 mM EGTA,0.2% (v/v) NP-40, 50 µ g/ml PMSF, 10 µg/mlleupeptin, 10 µg/ml aprotinin, and where specified 30mM sodium pyrophosphate. After 5 min on ice, lysateswere centrifuged, and supernatants were transferred tofresh tubes containing an equal volume of 50 mMTris·HCl pH 7.5, 100 mM NaCl, 2.5 mM CaCl2, 100µg/ml BSA, and 1.5 µM okadaic acid. Blank sampleswere prepared with resuspension buffer in place of celllysate. The reaction was initiated by addition of 32P-labelled RII peptide, each tube was incubated for 15min at 30°C, and the reaction was terminated by theaddition of 100 mM potassium phosphate buffercontaining 5% (w/v) trichloroacetic acid. Each samplewas applied to a Dowex AG 50W-X8 cation exchangecolumn (Bio-Rad), and the effluent was collected andits content of radiolabel determined by liquidscintillation counting.
Structural modelling— Structural modelling wasbased on the coordinates of calcineurin Aα from PDBentries 1AUI and 1TCO (40,41). The coordinates ofdocked PVIVIT peptide were from Li et al. (33).Figure 9 was prepared using RasMol.
RESULTS
The compounds INCA1, INCA2, INCA6, andINCA12 (Figure 1) have been shown to inhibitrecognition of NFAT by calcineurin, assessedquantitatively as binding of fluorescent PVIVIT peptideto calcineurin. Several observations raised thepossibility that a covalent INCA-protein complex isinvolved in inhibition of PVIVIT peptide binding.First, the most effective inhibitors were quinones orquinoneimines, compounds that are chemically reactiveand that are known to form protein adducts. Second,the kinetics of binding were slow at intermediateconcentrations of INCA compound, as illustrated forINCA1 (Figure 2A), with a plateau level of inhibitionreached only after tens of minutes at room temperature.This behavior is often indicative of formation of acovalent ligand-protein complex. Finally, INCA1,INCA2, and INCA6 were inactivated as inhibitors inthe competitive binding assay by preincubation withDTT (Figure 2B and not shown). Inactivation couldreflect either reduction of the compounds by DTT to aless reactive form or covalent reaction with DTT.
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Effect of sulfhydryl-modifying reagents— Proteinsulfhydryl groups are likely sites of covalentmodification by quinones. Consistent with thispossibility, preincubation of calcineurin with thesulfhydryl-modifying reagent iodoacetamide (IAM)impaired INCA2 competition, whereas preincubation ofcalcineurin by itself had no effect (Figure 3A). In moreextensive studies, preincubation of calcineurin with themaleimide INCA12 impaired competition by INCA1,INCA2, and INCA6 (Figure 3B and not shown).
Pretreatment of calcineurin with the sulfhydryl-modifying reagents IAM, N-ethylmaleimide (NEM),and INCA12 reduced the calcineurin-PVIVITpolarization signal even in the absence of INCAcompetitor (Figure 3 and not shown). Very lowconcentrations of INCA12 were sufficient to reduce thecalcineurin-PVIVIT polarization signal, but even highconcentrations did not fully eliminate the signal frombound PVIVIT (Figure 4A ). Similarly, for highconcentrations of IAM and NEM, the calcineurin-PVIVIT signal settled to a plateau that was well abovethe signal of free peptide (not shown). The explanationfor this behavior, documented below, is that pretreatedcalcineurin retains the ability to bind PVIVIT withreduced affinity.
In principle, the effect of the nonspecific alkylatingreagents IAM and NEM could be due either to trueimpairment of calcineurin-peptide binding or toimpaired immobilization of the fluorescent label whenPVIVIT peptide is bound. The second case would beevident in a reduction in the maximal polarizationsignal obtained by titrating fluorescent PVIVIT withIAM-pretreated or NEM-pretreated calcineurin, and acorresponding reduction in the slope of the centralportion of the binding curve. In fact the curves forpretreated calcineurin showed no change in slope, butinstead were shifted rightward to an extent compatiblewith a 1.5-2fold loss in affinity of the calcineurin-peptide interaction (Figure 4B). Thus IAM and NEMare themselves partial inhibitors of the calcineurin-PVIVIT interaction, and experiments described belowindicate that they act at the same site as the INCAcompounds.
Identification of a target cysteine residue— Theinhibitory activity common to IAM, maleimides, andquinones/quinoneimines strongly supported the notionthat these compounds derivatize a cysteine sulfhydrylof calcineurin. Pilot experiments examining the effectof several single C>A substitutions in calcineurin onthe inhibitory activity of INCA2 pointed to C266 as thelikely reactive residue, and more detailed studyconfirmed that the point mutant C266A was insensitiveto the inhibitory effect of INCA2 (Figure 5). Similarexperiments demonstrated that this substitution alsocompromised the effectiveness of INCA1, INCA6,IAM, and maleimides (not shown).
Conversely, the other surface-exposed cysteinesulfhydryl groups were not required for the action ofINCA compounds. The crystal structure of calcineurinshows six exposed cysteine residues in the catalyticdomain, C166, C184, C228, C256, C266, and C336.We compared calcineurin in which all exposed cysteineside chains except C266 were changed to alanine(C>A//C266) and calcineurin with the samesubstitutions plus a C266V substitution (C>A//V266).Binding of fluorescent PVIVIT to the two proteins wascomparable (Figure 6A). However, while replacementof the cysteines other than C266 did not compromisethe effectiveness of INCA1, INCA2, or INCA6, theC266V substitution in the context of these other C>Areplacements completely blocked the effects of theinhibitors (Figure 6B and not shown). The C266Vsubstitution in this context also blocked the effect ofNEM (not shown). These observations showed that thepresence of C266 is necessary and sufficient for acomplete block of PVIVIT binding by INCAcompounds.
Formation of a peptide-INCA adduct— We testedthe ability of the three INCA compounds to react witht h e s y n t h e t i c c a l c i n e u r i n p e p t i d eRGSSYFYSYPAVCEFLQHNN. The peptide iscalcineurin(254-273), with a C256S substitution toeliminate the possibility of covalent reaction at thatposition.
MALDI-TOF mass spectrometry demonstrated thatINCA1 and INCA2, at micromolar concentrations, reactcovalently with the synthetic calcineurin peptide(Figure 7A-C). Peptide-INCA adduct formation wasblocked in each case by prior incubation with excessNEM under conditions that derivatized the –SH group(Figure 7A and not shown). Conversely, NEM failed toreact with the peptide-INCA adducts present after a firstincubation with INCA1 and INCA2 (Figures 7B and7C), confirming that the INCA compounds block thesulfhydryl group. Since the peptide is unlikely to havea preferred conformation in solution, the experimentsshow that INCA1 and INCA2 are sufficiently reactiveto modify accessible cysteine sulfhydryl groups withoutnecessarily forming an initial noncovalent complex.
INCA6 reacted more slowly (Figure 7D), with fulllabelling of the peptide requiring several hours.Reaction was again blocked by pretreatment withexcess NEM. The sluggish reaction suggests thatcovalent reaction with C266 in calcineurin wouldrequire higher concentrations of INCA6, a localenvironment that increases the nucleophilicity of theC266 thiol, or a noncovalent interaction that assists intargeting INCA6 to the site.
Direct examination of tryptic digests of INCA6-treated calcineurin did not provide evidence of anINCA-protein adduct. Rather, the same tryptic peptidecontaining C266— identified by comparison with
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digests of C266V calcineurin— was detected in thedigests of untreated and treated calcineurin. However,it has been difficult to demonstrate adducts in trypticdigests of other proteins that are known to be covalentlymodified by quinones, probably because the S-quinonebond is labile (42,43). Lability of the adduct would beless likely to prevent its detection in the experimentswith synthetic peptide, where INCA compound ispresent in excess and the initial reaction mixture isdirectly examined by mass spectrometry.
Reversibility of modification by INCA2— Physicalassociation of INCA compounds with calcineurin hasbeen demonstrated by NMR spectroscopy and bycochromatography (25). Two further results indicatethat spontaneous dissociation of INCA2, if it occurs, isextremely slow. First, extraction with ether for 2 mindid not restore PVIVIT binding after blockade with theINCA compound was complete, even though thenonpolar INCA2, when not bound to calcineurin,rapidly partitions into ether. Second, incubation ofINCA2-blocked calcineurin with excess NEM, at anNEM concentration that rapidly blocks the inhibitorysite on unmodified calcineurin, did not lead to anyrecovery of PVIVIT binding during the subsequent 4hours. The modification by INCA2 is neverthelesschemically labile, since treatment with excess DTTlargely reversed the inhibitory effect.
Effects of INCA2 in cells— The chemical reactivityof INCA1 and INCA2 has raised the issue of theirsuitability for cellular studies. In previous work,INCA6 inhibited the dephosphorylation of NFAT andcalcineurin-NFAT signalling, without inhibitingcalcineurin enzymatic activity (25). INCA1 andINCA2 were not studied at that time because of theircytotoxicity, and even the less reactive INCA6exhibited toxicity in some types of cells.
It proved possible to examine the effects of INCA2in D5 T cells, for which INCA2 is not cytotoxic whenused at low micromolar concentrations. At first glance,INCA2 had effects similar to those of INCA6,preventing the dephosphorylation of NFAT, the nuclearimport of NFAT, and the induction of mRNAsencoding tumor necrosis factor-α (TNFα), interferon-γ(IFNγ), and macrophage inflammatory proteins MIP-1αand MIP-1β (Figure 8A-C, and not shown). However,closer examination revealed that the physiologicaleffects were associated with a general inhibition ofcalcineurin catalytic activity (Figure 8D). A likelymechanism is oxidation of calcineurin, throughreactions involving INCA2 itself or involving reactiveoxygen species derived from INCA2 metabolism,effects that are probably exacerbated by INCA2partitioning into lipids to produce elevated localconcentrations of the quinone in cells. These new datareinforce the point (25) that current INCA compoundsare most suited to probing the calcineurin-NFAT
interaction in vitro, and that their use to inhibit thecalcineurin-NFAT pathway in cells requires stringentcontrols.
DISCUSSION
The principal result of this study has been to locatethe binding site on calcineurin for the calcineurin-NFAT signalling inhibitors INCA1, INCA2, andINCA6. The position of the INCA-binding site, in thevicinity of C266 of calcineurin, and its spatialrelationship to the previously mapped NFAT dockingsite on calcineurin are illustrated in Figures 9A and 9B.The distance from Sγ of C266 to the nearest proline ringatom of the bound PVIVIT peptide is greater than 15Å.Thus, contrary to expectation, the most efficaciousINCA inhibitors do not displace PVIVIT peptide andNFAT by binding competitively in the core PxIxITrecognition site, but rather they act by inducing anallosteric change in the NFAT-binding site.
The probable locus of the allosteric change is theβ11-β12 loop of calcineurin, which positions residuesF299 and P300 to form the proline and isoleucinepockets of the peptide-binding site (33). C266 andother residues in or just preceding helix 10 are inintimate contact with the residues that anchor both endsof the β11-β12 loop (Figure 9C ): Y262 makesextensive contacts with the side chains of R292 andS301 and forms a template for the peptide backbonefrom residue 292 to residue 295; V265 and L269 are incontact with S301; and C266 itself is exposed in a canalon the surface of calcineurin, nestled against Y262 andin loose contact with the side chains of S294 and S301.Binding of a ligand in direct contact with the C266sulfhydryl, whether binding is covalent or noncovalent,is likely to require a local structural rearrangement andcould alter the conformation of the β11-β12 loop. Inaddition to any changes in the immediate neighborhoodof C266, movement of helix 10 could also repositionL275, in the short segment connecting helix 10 and βstrand 10, thereby altering its packing against F299 andits contribution to formation of the proline pocket.
A concise explanation of the inhibitory effect ofINCA compounds is that introduction of thesesubstituents at C266 induces a structural rearrangementthat alters the PVIVIT docking site, and that formationof a covalent —S-INCA bond provides part of theenergy for the rearrangement. In fact, all theefficacious INCA compounds have the potential forcovalent binding. INCA1 and INCA2, in particular, arehighly reactive with the cysteine sulfhydryl group.Their specific action on PVIVIT binding, viaderivatization of C266, could be simply a byproduct oftheir general reactivity, or there could be a noncovalentinteraction that targets the compounds to the vicinity of
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C266 prior to covalent binding. INCA6 is lessintrinsically reactive under our experimental conditions.Efficient formation of a calcineurin-INCA6 covalentadduct would require assistance, through targeting ofINCA6 to the site or through heightened reactivity ofthe C266 sulfhydryl. The possibility also remains thatINCA6 is not primarily a covalent inhibitor.
A more refined analysis of the reaction withcalcineurin will entail characterization of thecalcineurin-INCA adducts that arise underphysiological conditions. Steric and other constraints inthe protein might favor products that differ from thoseof the reaction with synthetic peptide discussed in thelegend to Figure 7. For example, the INCA2 linkage toprotein could involve a thioether bond, as observed inquinone cofactors of some amine dehydrogenases(44,45) and as inferred for many other protein-quinoneadducts (42,43,46-49); or an ipso adduct at the iminecarbon, as observed in the complex of N-acetyl-p-quinoneimine with papain (50). Cysteinyl-quinonethioether adducts are themselves highly reactive whenin the oxidized form (51), and could undergo furtherreactions following initial adduct formation. Analternative mechanism of quinone-initiatedmodification of proteins is the production of reactiveoxygen species (52), which in their turn react withcysteine side chains to give oxides of sulfur or sulfenylamides (52,53). However, NMR measurements supporta physical interaction between calcineurin and INCAcompounds (25), and the experiments with syntheticpeptide yielded no evidence that the INCA compoundsoxidize cysteine sulfhydryl groups under ourconditions.
A sidelight to our experiments is that the covalentinhibitors IAM, NEM, and INCA12 react with C266,and reduce the affinity of calcineurin for PVIVITmeasurably, but much less dramatically than INCA1,INCA2, and INCA6. This finding highlights the role ofan induced conformational change, rather than simplecovalent derivatization of C266, in causing inhibition.It further serves as an indicator that other partialinhibitors of calcineurin-PVIVIT binding identified byhigh-throughput screening (25) may bind covalently ornoncovalently at this allosteric site.
The current experiments were undertaken as a steptoward the identification of better inhibitors ofcalcineurin-NFAT signalling. Our earlier mapping ofthe PxIxIT-binding site (33) has provided a structuraltemplate for the design of inhibitors that competedirectly with the NFAT docking peptide. Inhibitorsbinding at the C266 site will present a less tractable taskfor structure-based design until the structure of thecalcineurin-INCA complexes has been determined.Two immediately workable approaches suggested byour experiments are further high-throughput screeningwith modifications of the screening assay— such as useof C266V calcineurin or preincubation of librarycompounds with DTT or another thiol— that will tipthe balance in the direction of noncovalent inhibitors;and examination of tethered ligands (54) focussed onthe site we have defined. Less conventionally, it mayprove possible to develop covalent ligands that aretargeted to the site by a noncovalent interaction and arerelatively unreactive with nonspecific sites (55,56).The pursuit of covalent inhibitors is an uncertainexercise in most cases, because of the difficulty ofdiscriminating between a desired increase in thestrength of the noncovalent interaction and an unwantedincrease in chemical reactivity. Here, though, thecombination of a single physiologically relevant site ofinteraction, judicious use of cysteine mutants, andcareful control experiments may provide an avenuearound this technical obstacle.
The sensitivity of protein-ligand affinity at thePxIxIT-binding site to minor modifications at C266,including carboxymethylation and the C266Asubstitution, raises an intriguing possibility forregulation of calcineurin signalling in cells. Previouswork has focussed attention on the possibility thatcalcineurin catalytic activity is regulated by thephysiological production of reactive oxygen species andoxidation of Fe2+ at the active site (34-37,57-59). Ifredox reactions or endogenous regulators in cells alsoact at the INCA-binding site, they could complementredox modulation of overall calcineurin catalyticactivity with a layer of regulation having selectiveeffects on different substrates.
Acknowledgment— We thank the Institute of Chemistry and Cell Biology, Harvard Medical School, for generouslyproviding access to the Analyst plate reader.
REFERENCES
1. Aramburu, J., Rao, A., and Klee, C.B. (2000) Curr. Top. Cell. Regul. 36, 237-295.
2. Rusnak, F., and Mertz, P. (2000) Physiol. Rev. 80, 1483-1521.
3. Rao, A., Luo, C., and Hogan, PG (1997) Annu. Rev. Immunol. 15, 707-747.
4. Serfling, E., Berberich-Siebelt, F., Chuvpilo, S., Jankevics, E., Klein-Hessling, S., Twardzik, T., and Avots, A. (2000) Biochim. Biophys. Acta 1498, 1-18.
by guest on February 4, 2018http://w
ww
.jbc.org/D
ownloaded from
5. Crabtree, G.R., and Olson, E.N. (2002) Cell 109 (supplement), S67-S79.
6. Hogan, PG, Chen, L., Nardone, J., and Rao, A. (2003) Genes Dev. 17, 2205-2232.
7. Fruman, D.A., Klee, C.B., Bierer, B.E., and Burakoff, S.J. (1992) Proc. Natl. Acad. Sci. USA 89, 3686-3690.
8. O'Keefe, S.J., Tamura, J., Kincaid, R.L., Tocci, M.J., and O'Neill, E.A. (1992) Nature 357, 692-694.
9. Clipstone, N.A., and Crabtree, G.R. (1992) Nature 357, 695-697.
10. Tsuboi, A., Masuda, E.S., Naito, Y., Tokumitsu, H., Arai, K., and Arai, N. (1994) Mol. Biol. Cell 5, 119-128.
11. Fruman, D.A., Pai, S.Y., Burakoff, S.J., and Bierer, B.E. (1995) Mol. Cell. Biol. 15, 3857-3863.
12. Batiuk, T.D., Pazderka, F., Enns, J., DeCastro, L., and Halloran, P.F. (1995) J. Clin. Invest. 96, 1254-1260.
13. Batiuk, T.D., Kung, L., and Halloran, P.F. (1997) J. Clin. Invest. 100, 1894-1901.
14. Bueno, O.F., Brandt, E.B., Rothenberg, M.E., and Molkentin, J.D. (2002) Proc. Natl. Acad. Sci. USA 99, 9398-9403.
15. Liu, J., Farmer, J.D. Jr., Lane, W.S., Friedman, J., Weissman, I., and Schreiber, S.L. (1991) Cell 66, 807-815.
16. Liu, J., Albers, M.W., Wandless, T.J., Luan, S., Alberg, D.G., Belshaw, P.J., Cohen, P., MacKintosh, C., Klee, C.B., and Schreiber, S.L. (1992) Biochemistry 31, 3896-3901.
17. Sigal, N.H., Dumont, F., Durette, P., Siekierka, J.J., Peterson, L., Rich, D.H., Dunlap, B.E., Staruch, M.J., Melino, M.R., Koprak, S.L., Williams, D., Witzel, B., and Pisano, J.M. (1991) J. Exp. Med. 173, 619-628.
18. Dumont, F.J., Staruch, M.J., Koprak, S.L., Siekierka, J.J., Lin, C.S., Harrison, R., Sewell, T., Kindt, V.M., Beattie, T.R., Wyvratt, M., and Sigal, N.H. (1992) J. Exp. Med. 176, 751-760.
19. Serafini, A.T., Lewis, R.S., Clipstone, N.A., Bram, R.J., Fanger, C., Fiering, S., Herzenberg, L.A., and Crabtree, G.R. (1995) Immunity 3, 239-250.
20. Venkatesh, N., Feng, Y., DeDecker, B., Yacono, P., Golan, D., Mitchison, T., and McKeon, F. (2004) Proc. Natl. Acad. Sci. USA 101, 8969-8974.
21. Loh, C., Shaw, K.T.-Y., Carew, J., Viola, J.P.B., Luo, C., Perrino, B.A., and Rao, A. (1996) J. Biol. Chem. 271, 10884-10891.
22. Wesselborg, S., Fruman, D.A., Sagoo, J.K., Bierer, B.E., and Burakoff, S.J. (1996) J. Biol. Chem. 271, 1274-1277.
23. Aramburu, J., García-Cozár, F., Raghavan, A., Okamura, H., Rao, A., and Hogan, PG (1998) Mol. Cell 1, 627-637.
24. Aramburu, J., Yaffe, M.B., López-Rodríguez, C., Cantley, L.C., Hogan, PG, and Rao, A. (1999) Science 285, 2129-2133.
25. Roehrl, M.H.A., Kang, S., Aramburu, J., Wagner, G., Rao, A., and Hogan, PG (2004) Proc. Natl. Acad. Sci. USA 101, 7554-7559.
26. Jain, J., McCaffrey, P.G., Valge-Archer, V.E., and Rao, A. (1992) Nature 356, 801-804.
by guest on February 4, 2018http://w
ww
.jbc.org/D
ownloaded from
27. Northrop, J.P., Ullman, K.S., and Crabtree, G.R. (1993) J. Biol. Chem. 268, 2917-2923.
28. Chen, L., Glover, J.N., Hogan, PG, Rao, A., and Harrison, S.C. (1998) Nature 392, 42-48.
29. Peterson, B.R., Sun, L.J., and Verdine, G.L. (1996) Proc. Natl. Acad. Sci. USA 93, 13671-13676.
30. Macián, F., García-Rodríguez, C., and Rao, A. (2000) EMBO J. 19, 4783-4795.
31. Liu, J., Arai, K., and Arai, N. (2001) J. Immunol. 167, 2677-2687.
32. Noguchi, H., Matsushita, M., Okitsu, T., Moriwaki, A., Tomizawa, K., Kang, S., Li, S.-T., Kobayashi, N., Matsumoto, S., Tanaka, K., Tanaka, N., and Matsui, H. (2004) Nat. Med. 10, 305- 309.
33. Li, H., Rao, A., and Hogan, PG (2004) J. Mol. Biol. 342, 1659-1674.
34. Wang, X., Culotta, V.C., and Klee, C.B. (1996) Nature 383, 434-437.
35. Rusnak, F., and Reiter, T. (2000b) Trends Biochem. Sci. 25, 527-529.
36. Namgaladze, D., Hofer, H.W., and Ullrich, V. (2002) J. Biol. Chem. 277, 5962-5969.
37. Sommer, D., Coleman, S., Swanson, S.A., and Stemmer, P.M. (2002) Arch. Biochem. Biophys. 404, 271-278.
38. Nardone, J., and Hogan, PG (1994) Proc. Natl. Acad. Sci. USA 91, 4417-4421.
39. Fruman, D.A., Pai, S.Y., Klee, C.B., Burakoff, S.J., and Bierer, B.E. (1996) Methods 9, 146-154.
40. Griffith, J.P., Kim, J.L., Kim, E.E., Sintchak, M.D., Thomson, J.A., Fitzgibbon, M.J., Fleming, M.A., Caron, P.R., Hsiao, K., and Navia, M.A. (1995) Cell 82, 507-522.
41. Kissinger, C.R., Parge, H.E., Knighton, D.R., Lewis, C.T., Pelletier, L.A., Tempczyk, A., Kalish, V.J., Tucker, K.D., Showalter, R.E,, Moomaw, E.W., Gastinel, L.N., Habuka, N., Chen, X, Maldonado, F., Barker, J.E., Bacquet, R., and Villafranca, J.E. (1995) Nature 378, 641-644.
42. Chang, M., Shin, Y.G., van Breemen, R,B,, Blond, S.Y., and Bolton, J.L. (2001) Biochemistry 40, 4811-4820.
43. van Zanden, J.J., Ben Hamman, O., van Iersel, M.L.P.S., Boeren, S., Cnubben, N.H.P., Lo Bello, M., Vervoort, J., van Bladeren, P.J., and Rietjens, I.M.C.M. (2003) Chem. Biol. Interact. 145, 139-148.
44. Datta, S., Mori, Y., Takagi, K., Kawaguchi, K., Chen, Z.W., Okajima, T., Kuroda, S., Ikeda, T., Kano, K., Tanizawa, K., and Mathews, F.S. (2001) Proc. Natl. Acad. Sci. USA 98, 14268-14273.
45. Satoh, A., Kim, J.K., Miyahara, I., Devreese, B., Vandenberghe, I., Hacisalihoglu, A., Okajima, T., Kuroda, S., Adachi, O., Duine, J.A., Van Beeumen, J., Tanizawa, K., and Hirotsu, K. (2002) J. Biol. Chem. 277, 2830-2834.
46. Hoffmann, K.J., Streeter, A.J., Axworthy, D.B., and Baillie, T.A. (1985) Mol. Pharmacol. 27, 566- 573.
47. Slaughter, D.E., Zheng, J., Harriman, S., and Hanzlik, R.P. (1993) Anal. Biochem. 208, 288-295.
48. Zheng, J., Cho, M., Jones, A.D., and Hammock, B.D. (1997) Chem. Res. Toxicol. 10, 1008-1014.
49. Boatman, R.J., English, J.C., Perry, L.G., and Fiorica, L.A. (2000) Chem. Res. Toxicol. 13, 853-860.
by guest on February 4, 2018http://w
ww
.jbc.org/D
ownloaded from
50. Chen, W., Shockcor, J.P., Tonge, R., Hunter, A., Gartner, C., and Nelson, S.D. (1999) Biochemistry 38, 8159-8166.
51. Hanzlik, R.P., Harriman, S.P., and Frauenhoff, M.M. (1994) Chem. Res. Toxicol. 7, 177-184.
52. Wang, Q., Dube, D., Friesen, R.W., LeRiche, T.G., Bateman, K.P., Trimble, L., Sanghara, J., Pollex, R., Ramachandran, C., Gresser, M.J., and Huang, Z. (2004) Biochemistry 43, 4294-4303.
53. Salmeen, A., Andersen, J.N., Myers, M.P., Meng, T.C., Hinks, J.A., Tonks, N.K., and Barford, D. (2003) Nature 423, 769-773.
54. Erlanson, D.A., Wells, J.A., and Braisted, A.C. (2004) Annu. Rev. Biophys. Biomol. Struct. 33, 199- 223.
55. Way, J.C. (2000) Curr. Opin. Chem. Biol. 4, 40-46.
56. Chen, G., Heim, A., Riether, D., Yee, D., Milgrom, Y., Gawinowicz, M.A., and Sames, D. (2003) J. Am. Chem. Soc. 125, 8130-8133.
57. Furuke, K., Shiraishi, M., Mostowski, H.S., and Bloom, E.T. (1999) J. Immunol. 162, 1988-1993.
58. Reiter, T.A., and Rusnak, F. (2002) J. Biol. Inorg. Chem. 7, 823-834.
59. Fujii, H., and Hirano, T. (2002) Eur. J. Neurosci. 16, 1777-1788.
FOOTNOTES
* This work was supported by grants R01 AI40127 and R21 AI059940 from the National Institutes of Health.
§ Present address: Proteomics System Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB),52 Eoeun-dong, Yuseong-gu, Daejon, 305-333, Republic of Korea
# These authors contributed equally.
¶ To whom correspondence should be addressed. Tel: 617-278-3057; Fax: 617-278-3280; E-mail:[email protected]
1 The abbreviations used are CsA, cyclosporin A; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; GST, glutathioneS-transferase; IAM, iodoacetamide; IgG, immunoglobulin G; INCA, inhibitor of NFAT-calcineurin association;IPTG, isopropyl-β-D-1-thiogalactopyranoside; NEM, N-ethylmaleimide; PVIVIT, 14mer peptideMAGPHPVIVITGPHEE-amide; TFA, trifluoroacetic acid.
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FIGURE LEGENDS
Figure 1. Structural formulae of the INCA compounds studied here. INCA1, INCA2, and INCA6 atmicromolar concentrations completely block the calcineurin-PVIVIT interaction. INCA12 produces onlypartial inhibition of the calcineurin-peptide interaction.
Figure 2. A, Time course of inhibition of fluorescent PVIVIT binding by INCA1. INCA1 (13 µM) wasincubated with calcineurin at room temperature or 0˚C for the time indicated, the reaction was quenchedby addition of 5 mM DTT and fluorescent PVIVIT, and the polarization of fluorescence was measured.The polarization signals from the calcineurin-PVIVIT mixture in the absence of INCA1 (bound) and fromfluorescent PVIVIT alone (free) are indicated. A similar slow onset of inhibition was seen withintermediate concentrations of INCA2 and INCA6. B, Concentration dependence of PVIVITdisplacement by INCA1 in the absence of DTT (filled symbols) and in the presence of 5 mM DTT (opensymbols). Polarization signals from the calcineurin-peptide mixture in the absence of inhibitor and fromfree peptide are also shown. DTT had a comparable effect on displacement by INCA2 and by INCA6.
Figure 3. Effect of alkylating reagents on competition by INCA inhibitors. A, Concentration dependenceof PVIVIT displacement by INCA2 from untreated calcineurin (filled symbols) and from calcineurinpretreated with 10 mM IAM (open symbols). B, Concentration dependence of PVIVIT displacement byINCA2 from untreated calcineurin (filled symbols) and from calcineurin pretreated with 30 µM INCA12(open symbols). INCA12 likewise prevented displacement of PVIVIT by INCA1 and INCA6.
Figure 4. Effect of alkylating reagents on PVIVIT binding. A, Concentration dependence of PVIVITdisplacement by INCA12. A prominent feature is that 1 µM INCA12, equimolar with calcineurin in theassay, substantially inhibits calcineurin-PVIVIT binding. Previous work (25) has shown that the plateauobserved at high concentrations of INCA12 continues up to 1 mM INCA12. B, Titration of fluorescentPVIVIT with increasing concentrations of untreated calcineurin (black squares and left fitted curve),calcineurin pretreated with 10 mM IAM (grey squares and middle fitted curve), and calcineurin pretreatedwith 10 mM NEM (white squares and right fitted curve).
Figure 5. Competition of INCA2 with PVIVIT for binding to wildtype calcineurin (filled symbols) andthe C266A mutant (open symbols). Note that binding to the mutant calcineurin in the absence of INCAcompounds is lower than binding to wildtype calcineurin.
Figure 6. Competition by INCA compounds is strictly dependent on the presence of C266. A, Titrationof fluorescent PVIVIT with increasing concentrations of C>A//C266 calcineurin (filled symbols) andC>A//V266 calcineurin (open symbols). B, INCA2 fails to inhibit PVIVIT binding to C>A//V266calcineurin. The same result was obtained for INCA1 and INCA6. The difference in binding of PVIVITto the two mutant proteins in the absence of competitor reflects the slightly lower affinity of C>A//V266calcineurin for PVIVIT and possibly also a small mismatch in the protein concentrations in thisexperiment.
Figure 7. MALDI-TOF MS analysis of the reactions of NEM and INCA compounds with the syntheticcalcineurin peptide. Data in each spectrum are plotted as intensity relative to the principal peak, and theabscissae have been labelled as mass (rather than as m/z) since all the species detected are singly charged.Values given are for the monoisotopic mass. A, The unmodified peptide (upper spectrum) is detected at2382.02 Da (calculated mass, 2382.06 Da). There is neither disulfide formation nor oxidation of cysteineto a sulfenic acid, sulfenyl-amide, sulfinic acid, or sulfonic acid. A minor peak at 1775.62 Da isprovisionally identified as the N-terminal fragment RGSSYFYSYPAVCEF (calculated mass, 1775.77Da), as discussed in Experimental Procedures. After reaction with 1 mM NEM for 60 min (lowerspectrum, displaced vertically for clarity) both the peptide and its N-terminal fragment have been
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derivatized at a single site, which can be confidently identified as the cysteine sulfhydryl given thereaction pH, 7.35, and the presence of a free sulfhydryl group in the peptide sample. B-D, Reactions withINCA1, INCA2, and INCA6. The INCA compounds were present at 50 µM initially, and reaction wasallowed to proceed for 60 min. The reaction step was followed in B and C by a further 60-min incubationwith NEM; however, omission of the NEM incubation did not alter the products observed. The peptide-INCA adducts were not further characterized, but chemically reasonable product structures, based on themasses, are shown as insets. B, The INCA1 reaction yields a principal peak at ~2732 Da. Examination ofthis peak at higher resolution reveals a minor overlapping peak at ~2730 Da that we interpret as theoxidized form of the adduct (Supplementary Figure 1). The peak at ~2125 Da is the product of reactionwith the ~1775 Da peptide fragment. C, The INCA2 reaction gives a peak at ~2536 Da, consistent withhydrolysis of the labile quinoneimine and loss of the –Cl substituents. An overlapping peak at ~2538 Dais resolvable when the data are plotted on an expanded m/z axis (not shown). D, In the INCA6 reaction,at 60 min, the main peak is unreacted peptide and the minor peak at ~2666 Da is the adduct.
Figure 8. Effects of INCA2 on calcineurin-NFAT signalling in cells. A, D5 murine helper T cells wereuntreated, treated with 1 µM ionomycin alone, or treated with ionomycin in the presence of 4 µM INCA2or 1 µM CsA and 100 nM FK506. The cytoplasmic or nuclear localization of NFAT1 was visualized byimmunocytochemical staining. B, D5 cells were left untreated or pretreated with 4 µM INCA2 or with 1µM CsA and 100 nM FK506 as indicated, then further incubated for 45 min without stimulation, with 10nM PMA, or with 10 nM PMA and 300 nM ionomycin. Induction of TNFα and IFNγ mRNAs wasanalyzed by an RNase protection assay. Lymphotoxin β (LTβ), L32, and GAPDH mRNAs, which are notinduced, serve as loading controls. C, D5 cells were treated as in Figure 8B. Induction of MIP-1α andMIP-1β mRNAs was analyzed by RNase protection assay, with RANTES, L32, and GAPDH mRNAsserving as loading controls. The order of lanes is the same as in Figure 8B. D, Dephosphorylation of RIIphosphopeptide in lysates from untreated cells (bar 2) or from cells treated with 4 µM INCA2 (bar 3).Total phosphatase activity was assessed by adding the nonspecific inhibitor sodium pyrophosphate (PPi)to the lysate (bar 4). Under the conditions of the assay, essentially all the phosphatase activity measuredis due to calcineurin (25, and not shown). No calcineurin activity was detected in lysates from cellsincubated with INCA2 for 30 min or 10 min at 37C (not shown) or, as here, in lysates preparedimmediately after addition of INCA2. Data plotted are the means of duplicate samples from arepresentative experiment.
Figure 9. Structural context of C266 in calcineurin Aα. A, Location of C266 relative to the PxIxIT-binding site, in a model of the calcineurin A (grey)-calcineurin B (black) heterodimer based oncoordinates from PDB entry 1AUI. B, Stereo view of the calcineurin-PVIVIT complex (33), indicatingthe minimum distance from C266 to bound PVIVIT peptide, 15.3 Å. For clarity only calcineurin Aresidues 260-278, 288-305, and 318-335 are depicted in ribbon representation. C, Stereo view ofcalcineurin A backbone and sidechain packing in the vicinity of C266. Residues in helix 10 and itsimmediate flanking segments are shown in white; the bulk of the β11-β12 loop and the initial residues ofβ strand 12, in lavender; and F299 and P300, in red. The side chains of Y262, V265, C266, and L269 areapposed to the base of the loop that positions residues F299 and P300 to form the PxI-binding subsite, andL275 is packed against F299.
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FIGURE 9, A and B
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SUPPLEMENTAL DATA
Supplementary Figure 1. MALDI-TOF analysis of peptide and peptide-INCA1 adduct peaks displayed athigher resolution. A, Peptide at ~2382 Da is resolved into a series of peaks reflecting the naturalabundance of the heavy isotopes 2H, 13C, 15N, 18O, and 34S. B, The adduct formed by brief reaction withINCA1 in 0.1% TFA, followed immediately by freezing, is resolved into a similar series of peaks,consistent with a single product of monoisotopic mass ~2732 Da. C, The INCA1 reaction of Figure 7Byielded a more complex pattern indicating an additional product of monoisotopic mass ~2730 Da.
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SUPPLEMENTARY FIGURE 1
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Sunghyun Kang, Huiming Li, Anjana Rao and Patrick G. Hoganbinding at an allosteric site
Inhibition of the calcineurin-NFAT interaction by small organic molecules reflects
published online September 7, 2005J. Biol. Chem.
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