structure of the calcineurin-nfat complex: defining a t cell activation switch using solution nmr...

Structure

Article

Structure of the Calcineurin-NFAT Complex:Defining a T Cell Activation Switch UsingSolution NMR and Crystal CoordinatesKoh Takeuchi,1 Michael H.A. Roehrl,1,2 Zhen-Yu J. Sun,1 and Gerhard Wagner1,*1 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston,

MA 02115, USA2 Department of Pathology and Laboratory Medicine, Massachusetts General Hospital, Harvard Medical School, 55 Fruit Street,Boston, MA 02114, USA

*Correspondence: [email protected]

DOI 10.1016/j.str.2007.03.015

SUMMARY

Calcineurin (Cn) is a serine/threonine proteinphosphatase that plays pivotal roles in manyphysiological processes, including cell prolifer-ation, development, and apoptosis. Most prom-inently, Cn targets the nuclear factors of acti-vated T cell (NFATs), transcription factors thatactivate cytokine genes. Calcium-activated Cndephosphorylates multiple residues within theregulatory domain of NFAT, triggering joint nu-clear translocation. This relies crucially on theinteraction between the catalytic domain of Cn(CnCat) and the conserved PxIxIT motif locatedin a region distinct from the dephosphorylationsites of NFAT. Here, we present the structure ofthe complex between the 39 kDa CnCat anda 14 residue peptide containing a PVIVIT seg-ment that was derived from affinity-driven pep-tide selection based on the conserved PxIxITmotif of NFATs. The structure of the complexwas determined by using NMR assignmentsand structural constraints and the coordinatesof the CnCat crystal structure. The NMR analy-sis relied on recently developed labeling andspectroscopic techniques. The VIVIT peptideis accommodated in a hydrophobic cleftformed by b strands 11 and 14, and the loopbetween b strands 11 and 12, forming a shortparallel b sheet with the exposed b strand 14in Cn. The side chains of conserved residuesin the PxIxIT sequences make extensive inter-actions with conserved residues in Cn, whilethose of nonconserved residues are solventexposed. The architecture of the interfaceexplains the diversity of recognition sequencescompatible with NFAT function and uncovers apotential targeting site for immune-suppressiveagents. The structure reveals that the orienta-tion of the bound PxIxIT directs the phosphory-

Structure 15,

lation sites in NFAT’s regulatory domain towardthe Cn catalytic site.

INTRODUCTION

Calcineurin (Cn) is a heterodimeric phosphoprotein serine/

threonine phosphatase that is activated by Ca2+/calmod-

ulin (Figure 1A). The active site of calcineurin is located in

the CnA subunit, which is 57–59 kDa in mammals, and

is regulated by interaction with the 19kDa CnB subunit

(Rusnak and Mertz, 2000). CnA has one Zn and one Fe

ion in the catalytic core, and both metals are indispens-

able for catalytic activity (Mondragon et al., 1997). Cn

was originally identified as a major calmodulin-binding

protein in the brain (Klee et al., 1979; Stewart et al.,

1982) but has since been shown to play pivotal roles in

a wide variety of other biological contexts, such as regula-

tion and development of the immune, nervous, cardiovas-

cular, and musculoskeletal systems, and to be involved in

apoptosis and necrosis (Aramburu et al., 2000, 2004;

Clipstone and Crabtree, 1992; Hemenway and Heitman,

1999; Hogan and Li, 2005; Rusnak and Mertz, 2000).

The most studied role of Cn is its role in T cell signaling.

Increased intracellular calcium levels activate Cn, which

causes it to dephosphorylate multiple residues in nuclear

factors of activated T cells (NFATs) (Jain et al., 1993).

Phosphorylation sites are located in NFAT’s regulatory

domain in three different serine-rich motifs, termed SRR1,

SP2, and SP3 (Figure 1B). Dephosphorylation of these

serine residues is thought to cause exposure of nuclear

localization signal sequences triggering translocation of

the dephosphorylated NFAT-Cn complex to the nucleus,

where NFAT activates target gene expression (e.g., IL2)

(Rao et al., 1997). The DNA-binding domain of NFAT is

located in the REL-homology region (RHR), which also

contains the binding sites for activator protein 1 (AP1) pro-

teins, such as FOS and JUN. The structures of RHR alone,

and the RHR-DNA and RHR-DNA-FOS-JUN complexes,

which have been determined with NMR and/or X-ray crys-

tallography, revealed the cooperativity aspects of tran-

scriptional activation by NFAT (Chen et al., 1998; Wolfe

et al., 1997; Zhou et al., 1998). Due to its role in T cell

activation, Cn has been targeted for the development of

587–597, May 2007 ª2007 Elsevier Ltd All rights reserved 587

mailto:[email protected]

Structure

Structure of the Calcineurin-NFAT Complex

Figure 1. Schematic Representation of the Domain Structure of Human Calcineurin and Human NFAT2

(A) For CnA, the locations of the binding sites for CnB, calmodulin (CaM), and the autoinhibitory domain (AI) are indicated with gray boxes. For CnB,

four EF hand motifs are indicated.

(B) The location of the calcineurin binding site, the serine-rich region 1 (SRR1), the SPxx-repeat motifs 2 and 3 (SP2 and SP3), and nuclear localization

and export signals of hNFAT2 are indicated.

immune-suppressant drugs. The two most successful Cn

inhibitors discovered so far, cyclosporin A (CsA) and

tacrolimus (FK506), block Cn’s phosphatase activity by

obscuring its catalytic site. They are presented as stable

trimeric complexes with the intracellular proteins cyclo-

philin and FKBP12, respectively (Griffith et al., 1995;

Huai et al., 2002; Kissinger et al., 1995). Although highly

successful, CsA and FK506 have severe potential side

effects, which may be due to intrinsic toxicity; more likely,

however, these effects result from the general inhibition of

the enzymatic activity of Cn, which plays other biologically

important roles besides NFAT activation (Kiani et al.,

2000).

It is known that Cn-NFAT signaling depends on an inter-

action between the Cn catalytic domain (CnCat) and the

conserved PxIxIT motif, which is located N-terminal to

the phosphorylation sites in NFAT’s regulatory domain

(Aramburu et al., 1998) (Figure 1B). Interfering with the

Cn-NFAT interaction by mutating the conserved sequence

impairs NFAT’s activation capabilities (Aramburu et al.,

1998). While the interaction between CnCat and the PxIxIT

motif of NFAT is indispensable for NFAT activation, it does

not depend on the polypeptide segment containing the

phosphorylation sites (Aramburu et al., 1998; Garcia-

Cozar et al., 1998). Based on the topologies of this inter-

action, an alternative strategy for more specific inhibition

of Cn-NFAT signaling has been proposed by targeting

the NFAT interaction site rather than the enzyme’s cata-

lytic site, in order to achieve selectivity in suppressing

T cell activation (Roehrl et al., 2004a, 2004b). Affinity

selection from a consensus PxIxIT sequence-based com-

588 Structure 15, 587–597, May 2007 ª2007 Elsevier Ltd All ri

binatorial peptide library showed that Cn is able to accom-

modate a variety of sequences without significantly losing

their affinity and some of the selected peptides had

a higher Cn binding affinity than the original NFAT se-

quences (Aramburu et al., 1999). While the native targeting

sequences have relatively low affinity for Cn (Kd �20 mM

for SPRIEIT sequence in NFAT1), the best selected pep-

tide with the sequence VIVIT at the center exhibits a 50-

fold higher affinity for Cn (Aramburu et al., 1999). Efforts

to identify the VIVIT peptide binding site with X-ray crystal-

lography have failed so far because crystals cracked after

the soaking with the peptide in various Cn crystal forms

(Roehrl et al., 2004a, 2004b). This observation suggested

that the VIVIT peptide might bind to a conserved crystal

contact site, which maps to a conserved hydrophobic

surface around b strand 13 (319–325) and b strand 14

(328–334), and the idea was recently supported by a com-

bination study of photoaffinity crosslinking, mutation, and

in silico docking simulations (Li et al., 2004).

To obtain a more detailed picture of the Cn-NFAT inter-

action, we have determined the structure of Cn-VIVIT pep-

tide complex by using solution NMR techniques. The VIVIT

peptide binds to the exposed b strand 14 while forming

a short parallel b sheet. The conserved hydrophobic side

chains of the PxIxIT motif are located in a hydrophobic

cleft formed by b strands 11 and 14 and the b11-b12

loop (i.e., the loop between b strand 11 and 12). On the

other hand, nonconserved residues in the PxIxIT motif

are largely exposed to solvent. These results provide a

detailed view of NFAT recognition by Cn and reveal the

mechanism by which Cn specifically recognizes the

ghts reserved

Structure


Figure 2. 1H-15N TROSY HSQC Spectra of 2H15N-Labeled CnCat

The spectra without and with the 14-mer VIVIT peptide are shown in red and black, respectively. For the spectrum with the VIVIT peptide, a 1.2-fold

excess of peptide was added to 2H15N-labeled CnCat.

conserved PxIxIT sequence. The information is potentially

useful for developing new bioactive compounds that

target Cn-NFAT interaction.

RESULTS AND DISCUSSION

Identification of the VIVIT Binding Site on Calcineurin

The 39 kDa catalytic domain of calcineurin, which includes

residues 2–347, was prepared based on the method

developed by Aramburu et al. with minor modifications

(Aramburu et al., 1999). As shown in Figure 2, the TROSY1H-15N HSQC spectrum of CnCat is well dispersed, re-

flecting the a-b mixed structure of the CnCat domain

(Griffith et al., 1995; Huai et al., 2002; Jin and Harrison,

2002; Kissinger et al., 1995). About 210 resonances out

of 326 expected resonances are identified in the TROSY1H-15N HSQC spectrum of perdeuterated CnCat. One

explanation for the absence of some of the resonances

is insufficient D/H exchange after expression of the protein

in D2O. The other possibility is the paramagnetic effect,

since CnCat possesses a paramagnetic iron (Fe3+ or

Fe2+) in its catalytic center. To ascertain the main cause

for the missing resonances, we utilized a partial deutera-

tion strategy where the protein was cultured on deuter-

ated media but in H2O to avoid the back exchange prob-

lem (Lohr et al., 2003). This strategy, however, rescued

only 20 additional resonances out of the �110 missing

signals, indicating that paramagnetic effect, rather than

insufficient back exchange, is the primary reason for the

missing resonances. Indeed, the results of our main-chain

Structure 15, 5

assignments of the protein revealed that resonances orig-

inating from any hydrogens within a 10 A distance from the

iron in the catalytic center of CnCat could not be observed.

Upon the addition of VIVIT peptide, CnCat resonances

showed chemical shift perturbations indicative of the

slow-exchange kinetics, confirming that the VIVIT peptide

forms a stable complex with CnCat. The chemical shift

changes were saturated at a 1:1 stoichiometric amount

of peptide to protein indicating a 1:1 complex between

CnCat and the VIVIT peptide. Main-chain resonances of

CnCat in complex with VIVIT peptide were assigned with

the data from standard TROSY triple-resonance experi-

ments and information from selective labeling. Comparing

the spectra of free and bound CnCat, we identified the res-

onances shifted upon binding of the VIVIT peptide (peaks

shifted more than half peak width). Figures 3B and 3C

show the distribution of the residues affected by VIVIT

binding. Chemical shift changes were observed for >30

resonances, which reside in the C terminus of helix 3 (res-

idue number 72–74), b strand 1 (79), the C-terminal side of

helix 4 (103–106), the H4-b3 loop (i.e., the loop between

helix 4 and b strand 3) (111), b strand 3 (114), the b5-b6

turn (192–194), the b7-b8 loop (240 and 242), the C termi-

nus of helix 12 (271 and 272), b strand 10 (277), the b11-

b12 loop (293, 294, 296, 298, and 299), and b strands 13

and 14 (324–335). Most of these residues are mapped

into a contiguous surface, which includes two (lower and

upper) clefts on the CnCat structure (colored in magenta

in Figure 3C). The lower cleft includes the C terminus of

helix 3 and b strand 13, while the upper cleft includes


Structure


Figure 3. Summary of VIVIT Peptide-

Binding Experiments for CnCat

(A) Intensity reduction ratio for each resonance

in the cross-saturation experiment. A satura-

tion time of 300 ms was applied to the methyl

proton resonances of the VIVIT peptide. The

residues that experienced a significant inten-

sity reduction are colored in red. The error

bars are estimated from the signal-to-noise

ratios of the resonances analyzed.

(B and C) Sites perturbed by the binding of

VIVIT are mapped on the ribbon (B), and sur-

face (C), of the CnCat structure. Red spheres

or surfaces indicate the sites that experienced

both cross-saturation and chemical-shift per-

turbations. Yellow indicates the sites that

showed chemical-shift changes upon VIVIT

binding. Blue residues were not affected in

both experiments. White surfaces were not

assigned. Magenta shadows in (C) indicate

the positions of the two clefts on the CnCat

structure.

the b strands 11 and 14 and the b11-b12 loop. The types of

amino acids that line these two clefts are significantly dif-

ferent. The upper cleft consists of hydrophobic or aro-

matic residues, while the lower cleft is lined primarily by

hydrophilic and charged residues. The total solvent-cces-

sible surface area for the shifted residues is 1400 A, which

is much larger than what a single 14-mer peptide can

cover. Given the fact that the peptide and the protein

form a 1:1 complex, it seems that CnCat has a secondary

site that allosterically changes its structure upon the bind-

ing of the NFAT peptide to its cognate site. The overall

structure of CnCat, however, doesn’t seem to change

significantly since the global features of the spectra do

not alter dramatically upon VIVIT binding. To identify the

direct VIVIT binding site, a cross-saturation experiment

was performed for 2H15N CnCat in complex with unlabeled

VIVIT peptide. Selective irradiation to the methyl reso-

nances of VIVIT caused a significant signal intensity loss

for the resonances of Met329 and Ile331 on b strand 14

(Figure 3A). The data strongly indicate that the upper cleft

rather than lower cleft is the direct binding site of VIVIT

(Figure 3C). Moreover, since the upper cleft consists

mostly of hydrophobic residues, the nature of the cleft is

also favorable for accommodating the hydrophobic VIVIT

peptide.

590 Structure 15, 587–597, May 2007 ª2007 Elsevier Ltd All rig

Identification of the Cn Binding Site

on the VIVIT Peptide

For precise identification of the CnCat-binding sequence

on the VIVIT peptide, the main-chain resonance assign-

ment of the free VIVIT peptide and in complex with CnCat

was accomplished with triple-resonance experiments

(Figure 4). The VIVIT peptide binds with slow-exchange

kinetics, which is consistent with the features observed

on the CnCat side. As shown in Figure 4A, the region

from Ile6 to Gly10 experiences a significant downfield shift

upon binding, indicating that this segment forms an

extended secondary structure in the complex. This obser-

vation was also supported by the upfield shift of Ca reso-

nances upon binding to CnCat (data not shown). Val5

exhibited a significant downfield chemical-shift change.

Figure 4B displays the normalized chemicals shift for

amide resonances ([DdH2 + (DdN / 5)2]1/2) of VIVIT peptide

upon binding to CnCat (upper). The lower panel reports

the result of cross-saturation for the same complex. The

region from Val5 to Gly10 showed >0.2 ppm chemical-

shift changes and was also strongly saturated in the

cross-saturation experiment, indicating the region binds

tightly to the CnCat. His12 also showed a modest chemi-

cal shift perturbation, while the regions before His3 or after

Glu13 do not seem to interact with the protein.

hts reserved

Structure


Structure of the CnCat-VIVIT Complex

To obtain a detailed view of the Cn-NFAT interaction, we

then set out to determine the structure of the Cn-VIVIT

peptide complex by simulated annealing of the VIVIT pep-

tide in the presence of the X-ray structure of free CnCat

(PDB code: 1AIU) using structural information derived

from NMR spectroscopy. In total, we used 22 intermole-

cular distance restraints, 52 intramolecular distance re-

straints within the VIVIT peptide, and 9 main-chain angular

Figure 4. Summary of CnCat-Binding Experiments for VIVIT

Peptide(A) 1H-15N TROSY HSQC spectra of 2H15N-labled VIVIT peptide with-

out (red) and with (blue) CnCat. The free and bound resonances are

connected by a dotted line with the assignments.

(B) Upper panel: normalized chemical-shift perturbation upon binding

to CnCat. Lower panel: intensity reduction ratio for each resonance

in the cross-saturation experiment. A saturation time of 300 ms was

applied for the methyl proton resonances of CnCat.

Structure 15,

constraints for I6-G10 in the VIVIT peptide. Taking advan-

tage of the knowledge of the VIVIT binding site on CnCat,

the side chains of the residues forming the VIVIT peptide

binding site in CnCat were allowed to be flexible during

the simulation. Other parts of CnCat were tightly re-

strained by distance and angular constraints calculated

from the X-ray structure of free CnCat.

Figure 5A shows a set of converged structures of the

Cn-VIVIT complex based on the structural restraints

derived from NMR experiments. The VIVIT binding site is

located on the side of the CnCat molecule. There is no

VIVIT peptide atom within 18 A distance from the catalytic

core iron, which may explains how the peptide inhibits

NFAT binding without affecting the catalytic activity of

CnCat (Aramburu et al., 1998). The total buried surface

area of the peptide is 698 ± 70 A2, which corresponds to

32% ± 3% of the total exposed surface of the peptide.

The conserved PxIxIT sequence of the VIVIT peptide is

particularly well ordered in the structure. The root-mean-

square deviation values are 0.68 ± 0.32 A and 1.15 ±

0.39 A for main-chain and all heavy atoms, respectively,

in this region, due to the extensive number of experimental

constraints applied (see Figure S1 available with this arti-

cle online). The VIVIT peptide binds on the exposed b

strand 14 of CnCat forming a short four-residue parallel

b sheet (Ile6 to Thr9) (Figures 5B and 5C). The parallel

b sheet structure is supported by interstrand NOEs shown

in Figure 5C. In the calculated structures, intermolecular

main-chain hydrogen bonds are observed for M329

(CO)-V7 (HN) (7/10), I331 (HN)-V7 (CO) (10/10), and I331

(CO)-T9 (HN) (10/10). The VIVIT peptide forms a bend

at the N-terminus of the conserved sequence between

Pro4 and Val5. This bend allows for contact between

the N-terminus of the peptide and the b11-b12 loop.

Figure 6A shows the surface of CnCat with the color rep-

resentation derived from NMR-binding experiments. The

results of the cross-saturation experiment reliably identify

the region corresponding to the core VIVIT binding site,

where a tight interaction between the peptide and the

protein occurs. In contrast, chemical-shift perturbation

experiments also report weak interacting regions such

as the b11-b12 loop, where the N terminus of the peptide

interacts, or the lower cleft shown in Figure 3C, which

apparently senses allosteric effects. It is also known that

mutations to the linker region joining the catalytic domain

and the regulatory domain of Cn (S337P, H339L, or L343S)

disrupt the binding of Cn to NFAT1 (Rodriguez et al.,

2005). Since there is no atom from the bound VIVIT pep-

tide within 6 A from these mutated residues, there appears

to be no direct interaction between the linker region and

the VIVIT peptide. However, because the mutated resi-

dues are C-terminal to the VIVIT binding b strand 14 of

CnCat, these mutations might allosterically affect the

structure of the VIVIT binding site in CnCat and disrupt

the binding between Cn and NFAT.

Architecture of the PxIxIT/Cn Interface

Figures 6B and 6C show enlarged views of the binding site

of the conserved PxIxIT motif with a color representation


www.ncbi.nlm.nih.gov

Structure


Figure 5. NMR Model of the CnCat-VIVIT

Peptide Complex

(A) Stereo view of an ensemble of ten final NMR

structures of the CnCat-VIVIT peptide com-

plex. Blue lines show the backbone trace of

CnCat. Red lines show the backbone trace of

the VIVIT peptide with the side-chain heavy

atoms for the PxIxIT motif (i.e., Pro4-Thr9).

(B) Ribbon representation of the CnCat-VIVIT

peptide complex with the lowest energy func-

tion.CnCat is shown in light blue,and VIVIT pep-

tide is shown in red with side chains for the

PxIxIT motif. In (A) and (B), the two metal ions

are shown as spheres (yellow, Fe; purple, Zn).

(C) A schematic representation of the second-

ary structure elements in the CnCat-VIVIT pep-

tide complex. The hydrogen bonds observed in

the structure are marked by black dotted lines.

Blue dotted arrows indicate the observed inter-

strand NOEs.

based on the property of residues. The VIVIT binding site

on Cn is very hydrophobic and forms several hydrophobic

pockets in CnCat structures. As shown in Figure 6B and

the cartoon of Figure 6D, Phe195 in b strand 6, Tyr324 in

b strand 13, and M329 and Ile331 in b strand 14 of CnCat

form the base of the hydrophobic pockets. Leu275 in the

H12-b11 loop, and Pro300 in the b11-b12 loop define

the boundary of the hydrophobic regions. Tyr288 and

Met 290 in b strand 11 and Phe299 in the b11-b12 loop

separate this section into three hydrophobic pockets. All

residues that form the hydrophobic pockets are con-

served among different species. Each of the conserved

hydrophobic pockets accommodates one of the residues

from the conserved motif in the VIVIT peptide. Pro4 in the

VIVIT peptide fits into a hydrophobic cleft between Leu275

and Phe299 of CnCat. Ile6 is the least exposed residue

(10% of the total surface is exposed) in the conserved mo-

tif, and a hydrophobic pocket formed by Met290, Phe299,

Pro300, and Met328 in CnCat accommodates the side

chain of Ile6. Ile8 is sandwiched between Tyr288 and

Met290 and forms an extensive interaction with Ile331 in

a nested rearrangement (Wouters and Curmi, 1995). Mu-

tation of Ile331 to Ala is known to reduce the affinity of

the peptide to protein by more than 20-fold, indicating

that the side-chain hydrophobic interaction between

these residues is energetically important (Li et al., 2004).


Although, Ile6 and Ile8 are conserved within NFATs, these

interaction sites can potentially accommodate a different

type of amino acid residue with hydrophobic properties.

Indeed, it has been shown that even the least exposed

Ile6 can be mutated to Phe, Val, or Leu without signifi-

cantly loosening the affinity to CnCat (Aramburu et al.,

1999). Two valine residues (Val5 and Val7) in between

these conserved residues are oriented toward the solvent,

allowing extensive substitution of these residues. The po-

sitions corresponding to Val5/Val7 is Arg/Glu in hNFAT1

and hNFAT2, Ser/Arg in hNFAT3, and Ser/Gln in hNFAT4.

In a similar recognition motif in the calcineurin inhibitor

Cabin1, the corresponding residues are Glu/Thr. How-

ever, since Val328 in b strand 14 of CnCat makes hydro-

phobic contacts with Val5 and Val7, hydrophobic residues

may be more preferable compared to the native hydro-

philic sequences. This might be one of the reasons why

the VIVIT sequence has a higher affinity relative to native

sequences. Thr9 of the VIVIT peptide is exposed despite

being conserved within NFATs. As shown in Figure 6C,

Thr9 is in close proximity to Asn330 and Arg332, both of

which are conserved in CnCat. Indeed, the energetic inter-

action between the side chains of Thr9 and Asn330 is

shown by mutational analysis (Li et al., 2004). Since the

g-hydroxyl group of Thr9 is observed in some of our struc-

tures to form hydrogen bond with the side-chain amide of

hts reserved

Structure


Figure 6. Interface of the CnCat-VIVIT Peptide Interaction

(A) Sites that are perturbed by the binding of the VIVIT peptide are mapped on the CnCat surface in the CnCat-VIVIT peptide complex. Red surfaces

indicate sites that experienced both cross-saturation and chemical-shift perturbation. Yellow indicates the sites that showed chemical-shift changes

upon binding of the VIVIT peptide. Blue residues were not affected in both experiments. White surfaces were not assigned. The VIVIT peptide is shown

as a ribbon representation with the side chains for the PxIxIT motif.

(B) and (C) show the interface of CnCat and the conserved PxIxIT motif. In (B), the conserved residues in CnCat are shown with a colored surface

representation based on the properties of each amino acid. Hydrophobic, acidic, basic, and hydrophilic residues are shown in yellow, red, blue,

and white, respectively. The VIVIT peptide is shown as a ribbon representation with stick representation for the side-chain heavy atoms of the PxIxIT

motif. The conserved Pro4, Ile6, Ile8, Thr9 are shown in dark red, while nonconserved Val6 and Val8 are colored in pink. In (C), the conserved residues

in CnCat are shown as a stick model with color representation based on the properties of each amino acid. Hydrophobic, acidic, basic, and hydro-

philic residues are shown in yellow, red, blue, and light blue, respectively. VIVIT is shown in the same representation as in (B).

(D) Schematic representation for the interaction between the PxIxI sequence and hydrophobic pockets of CnCat. The VIVIT peptide binds on the

hydrophobic floor formed by Phe195, Tyr324, Val328, Met329, and Ile331. The conserved PxIxI sequence is accommodated in the hydrophobic

pockets separated by Leu275, Tyr288, Met290, and Phe299. The peptide backbone is represented by the red arrow, and conserved and non-

conserved side chains are shown in dark and light red, respectively.

Asn330 (30% of structures) and the guanidine group of

Arg332 (40% of structures), hydrogen-bonding interaction

appears to be the source of the energetic interaction.

To confirm our complex model and rule out the possibil-

ity that the hydrophobic binding pockets for Ile6 and Ile8

may instead accommodate Val5 and Val7 in a different

flipped orientation, selective NOE difference experiments

were performed with a complex of [ul-2H, FIMY-1H] Cn

and [ul-2H15N, Id1LdVg -1H13C] VIVIT (Reibarkh et al.,

2006; Takahashi et al., 2006). Figure 7A shows the1H-13C HSQC spectra of [ul-2H, ILV-Me-1H] VIVIT with

and without [ul-2H15N, Id1LdVg -1H13C] Cn. Chemical-shift

Structure 15,

changes upon CnCat binding were more significant for

Ile6 and Ile8 compared to Val5 and Val7, and the reso-

nances of the isoleucines were less intense than the valine

resonances. These results indicate a stronger preference

of Cn for Ile6 and Ile8 over Val5 and Val7, which is consis-

tent with our structural model. Selective irradiation of the

aromatic proton resonances of either Tyr288 or Phe299

selectively saturated the Ile6 and Ile8 methyl resonances

without affecting Val5 and Val7 resonances (Figure 7B).

The quantitative reduction in intensity also showed

a good correlation with the distance between irradiated

and saturated residues. The irradiation of Tyr288 caused


Structure


stronger saturation for Ile8 than Ile6, while that of Phe299

decreased the intensity of the Ile6 resonance more than

the Ile8 resonance. These results confirmed the orienta-

tion observed in the determined complex between CnCat

and the VIVIT peptide. A conserved interaction involving

the Thr9 side chain as well as the relative preference of

the hydrophobic pockets for the Ile residues may be the

determining factors for the unique orientation between

CnCat and the VIVIT peptide. In fact, the Ile-Ile pair (e.g.,

Ile8-Ile331) is one of the most preferred amino pairings

in a parallel b sheet (Fooks et al., 2006). In contrast, the

IleHB-ArgnHB pair (i.e., Ile8-Arg332), which would occur

in an alternative flipped interaction, is one of the most

unfavorable pairing of residues in a parallel b sheet.

Figure 7. Chemical-Shift Perturbation and Selective Cross-

Saturation Experiments Using Methyl Selectively Labeled

VIVIT Peptide

(A) HSQC spectra of [ul-2H15N, Id1LdVg -1H13C] VIVIT peptide with (red)

and without (blue) CnCat. Assignment for each resonance is also

shown.

(B) Selective cross-saturation experiment using [ul-2H15N, Id1LdVg-1H13C] VIVIT peptide in complex with [U-2H, FIMY-1H] CnCat. Intensity

reduction ratios for each methyl resonance with 250 ms of continuous

wave irradiation to aromatic resonances of Tyr288 and Phe299 are

shown. Average intensity reduction ratios of two g-methyl resonances

are shown for Val resonances. The error bars are estimated from the

signal-to-noise ratios of the resonances analyzed.


ThePxIxIT Interaction Orients NFAT’sPhosphorylated

Residues toward the Cn Catalytic Site

The topology of the PxIxIT interaction with CnCat raises

the question of how this relates to dephosphorylation

of the regulatory region (Figure 1). All of NFAT’s phosphory-

lation sites are C-terminal to the Cn-binding sequence.

The Cn binding sequence of hNFAT2, for example, is

118-PRIEIT-123 and the dephosphorylation regions are

172-SPASSLSSRSCNSE-185 (serine-rich region 1 [SRR1],

dephosphorylation residues are underlined and bolded),

233-SPRHSPSTSPRA-244 (SPXX-repeat motif 2 [SP2]),

and 278-SPHHSPTPSPHGSP-291 (SP3) (Macian, 2005).

The orientation of the C terminus of the bound VIVIT

peptide (Figure 5) directs the phosphorylated residues to-

ward the catalytic site of Cn. NFAT dephosphorylation is

thought to trigger exposing of the nuclear localization

sequence (NLS), 265-KRK-267, which is N-terminal to

SP3. Although close to the dephosphorylation sites, the

mechanism by which the NLS is activated is not clear with-

out additional structural information. A second potential

second NLS, 682-KRK-684, is located in the C-terminal

domain of NFAT2, but it is unclear how it would be

activated by dephosphorylation of the regulatory domain.

Conclusion

In summary, the structure of CnCat-VIVIT complex was

determined by using NMR-derived constraints and the

known crystal structure of CnCat. The VIVIT peptide forms

a parallel b sheet with the exposed b strand 14 in Cn. The

structure reveals that the bound peptide is oriented to

direct the phosphorylation sites toward the Cn catalytic

site. The conserved PxIxIT sequence is either recognized

by hydrophobic pockets or by forming a side-chain-side-

chain interaction with conserved residues in Cn. These

results provide a detailed view of NFAT recognition by Cn,

providing insight on how Cn specifically recognizes the

conserved PxIxIT motif. The information obtained here

may be useful for developing small compounds that spe-

cifically inhibit the Cn-NFAT interaction. The hydrophobic

pockets of the PVIVIT binding site we characterized in this

study define a target site that could accommodate inhibi-

tors of the CnCat/NFAT interaction. The NMR assignments

established here will provide the means for future charac-

terization of complexes between CnCat and inhibitors,

such as described in Roehrl et al. (2004a).

EXPERIMENTAL PROCEDURES

All chemicals were purchased from Sigma unless otherwise noted.

1-13C amino acids were purchased from Cambridge Isotope Labora-

tories. Celtone base media were purchased from Spectra Stable

Isotopes.

Expression and Purification of the Catalytic Domain

of Human Calcineurin

The catalytic domain of human calcineurin Aa (CnCat), comprising

residues 2–347 with substitutions Y341S, L343A, and M347D (Ara-

mburu et al., 1999), was produced as a cleavable GST fusion protein

in the E. coli strain Rosetta (DE3). Cleavage of the fusion protein with

PreScission protease produces CnCat (2–347) with an additional

GPLG sequence at its N terminus. The CnCat domain obtained is

hts reserved

Structure


enzymatically active as described in our previous publication (Roehrl

et al., 2004b). A crystal structure of the protein prepared in this way

was solved and revealed a structure nearly identical to that of the cat-

alytic domain in full-length calcineurin A including the Fe-Zn cluster

(Michael, 2004). The CnCat protein, uniformly labeled with 2H, 13C,

and 15N, was obtained by growing the E. coli in modified M9 Celtone

medium, which consists of 1 kg/l 99.8% D2O, 8.5 g/l Na2HPO4, 3 g/l

KH2PO4, 0.5 g/l NaCl, 40 mg/l carbenicillin, 15 mg/l chloramphenicol,

1 g/l 15NH4Cl (99.9% enriched), 2 g/l 2H613C-glucose (97% enriched),

1 g/l 2H (>97% enriched), 13C (>97% enriched), 15N (>98% enriched)

Celtone Base Powder, 2 mM MgSO4, 0.1 mM CaCl2, 10 mg/l ZnSO4,

and 10 mg/l FeCl3. To obtain the partially 2H- and 15N-labeled CnCat

protein, modified M9 Celtone medium consisting of 1 liter H2O,

8.5 g/l Na2HPO4, 3 g/l KH2PO4, 0.5 g/l NaCl, 40 mg/l carbenicillin,

15 mg/l chloramphenicol, 1 g/l 15NH4Cl (99.9% enriched), 2 g/l 2H6-

glucose (97% enriched), 1g/l 2H (>97% enriched), 15N (>98% enriched)

Celtone Base Powder, 2 mM MgSO4, 0.1 mM CaCl2, 10 mg/l ZnSO4,

and 10 mg/l FeCl3 was used. To obtain selectively labeled CnCat,

E. coli was grown in modified M9 Celtone medium, and targeted

amino acids are added after induction of protein expression at seven

to ten times the amount present in the Celtone media (Spectra Stable

Isotopes) (Takeuchi et al., 2007).

All expression experiments were performed at 37�C; after reaching

an OD600 = 0.6, protein expression was induced by the addition of

1 mM IPTG. The cells were harvested after 36–48 hr of induction for

uniformly labeled samples, and after 24 hr for selectively labeled sam-

ples. The harvested cells were resuspended in 40 ml of PBS with 2 mM

dithiothreitol (DTT) and 0.2 mg ml�1 phenylmethylsufonylfuruolide

(PMSF) at 4�C. The suspended cells were then disrupted by sonication

and the insoluble fraction was removed by centrifugation for 20 min at

15,000 3 g. The supernatant was applied to a 5 ml column of Glutathi-

one Sepharose 4 Fast Flow. After washing the resin with 40 ml of PBS

with 2 mM DTT, the CnCat protein was eluted with 40 ml of PBS

containing 2 mM DTT and 25 mM of reduced glutathione. The elution

fraction was concentrated to 20 ml by using 10,000-MWC membrane

ultrafiltration. PreScission protease (0.5 units/20 mg CnCat) was added

to the concentrated elution fraction, and the solution was dialyzed

against 1 l of PBS with 2 mM DTT for 15 hr. The digested solution

was concentrated to 2 ml and subjected to PD-10 desalting column

to remove remaining glutathione. The fractions containing CnCat

were then applied to a 1.5 ml bed volume of Glutathione Sepharose

4 Fast Flow to remove PreScission protease and GST. The elution

protein was further purified with Superedex75 column. Traces of

GST in the Superedex75 elution fractions were then removed by pass-

ing the eluate through 1.5 ml bed volume of Glutathione Sepharose 4

Fast Flow. A typical final yield of CnCat was 2 mg/l culture.

Preparation of the VIVIT Peptide

A synthetic VIVIT 14-mer peptide (H4N-GPHPVIVITGPHEE-COOH,

Tufts-New England Medical Center peptide synthesis facility, Boston,

MA) was used as the nonlabeled form of peptide. The 2H15N13C-

labeled peptide was produced as a GB1 fusion protein in an E. coli

strain, BL21 (DE3). The GB1 fusion protein has the PreScission prote-

ase recognition site between GB1 and VIVIT peptide, which produces

VIVIT (14-mer) with an additional LEHHHHHH purification tag at its

C terminus. For the expression of uniformly 2H15N13C labeled VIVIT

peptide, E. coli harboring the GB1-VIVIT fusion vector was grown in

M9 Celtone medium, which consists of 1 kg/l 99.8% D2O, 8.5 g/l

Na2HPO4, 3 g/l KH2PO4, 0.5 g/l NaCl, 50 mg/l kanamycin, 1 g/l15NH4Cl (99.9% enriched), 2 g/l 2H6

13C-glucose (97% enriched),

1 g/l 2H (>97% enriched), 13C (>97% enriched), 15N (>98% enriched)

Celtone Base Powder, 2 mM MgSO4, and 0.1 mM CaCl2. The uniformly2H15N, ILV-methyl 1H13C-labeled ([ul-2H15N, ILV-1H13C-Methyl]) VIVIT

peptide was expressed in M9 minimum medium following the literature

by using [4-1H13C] a-ketobutyrate and [4,4-1H13C] a-ketoisovalerate

(Goto et al., 1999).

The cells were incubated at 37�C, and after reaching an OD600 = 0.6,

protein expression was induced by the addition of 1 mM of IPTG. The

Structure 15,

cells were harvested after 4–6 hr of induction and were resuspended in

40 ml of buffer A (50 mM Tris-HCl [pH 8.0], 300 mM NaCl, and 20 mM

imidazole) with 0.2 mg/ml PMSF at 4�C. The suspended cells were

disrupted by sonication, and the insoluble fraction was removed by

centrifugation at 15,000 3 g for 20 min. The supernatant was applied

to a 5 ml column of Ni-NTA agarose. After washing the resin with 40 ml

of buffer A, the GB1-VIVIT protein was eluted with 40 ml of buffer

consisting of 50 mM Tris-HCl (pH 7.5), 300 mM NaCl, and 300 mM

imidazole. The elution fraction was concentrated to 2 ml by 5000-

MWC-membrane ultrafiltration. PreScission protease and 1 mM DTT

were added to the concentrated elution fraction. The digested solution

was purified on a Superedex75 column. The final yield of the VIVIT

peptide was 20 mg/l culture.

NMR Spectroscopy

All experiments were performed on a Bruker Avance 750 spectrometer

equipped with a cryogenic probe. All spectra were collected with 0.4–

0.6 mM protein in 10 mM sodium phosphate buffer (pH 6.8) containing

150 mM NaCl, 2 mM DTT, and 90% H2O/10% D2O at 298 K. Spectra

were processed by using XWINNMR and analyzed with Sparky

(Goddard and Kneller, 2006). The backbone assignment of CnCat in

complex with the VIVIT peptide was accomplished by using standard

TROSY triple-resonance experiments (Ferentz and Wagner, 2000). A

500 mM sample of uniformly 2H-, 13C-, and 15N-labeled CnCat mixed

with 1.2 molar excess of the nonlabeled VIVIT peptide was prepared.

To confirm the assignments, four samples of perdeuterated 15N-

labeled CnCat amino acid specifically labeled with unlabeled Arg,

Lys, or 1-13C Val and 1-13C Leu were prepared. The backbone assign-

ment of the VIVIT peptide in free form and in complexed with CnCat

was accomplished by using standard triple-resonance experiment

without and with TROSY pulse scheme, respectively (Ferentz and

Wagner, 2000). For the observation of free VIVIT spectra, a 500 mM

concentration of 2H-, 13C-, and 15N-labeled VIVIT was prepared. To

record spectra of VIVIT in complex with CnCat, 2H-, 13C-, and 15N-

labeled peptide was complexed with 1.2 molar excess of nonlabeled

CnCat. For the assignment of the proton resonances of the VIVIT

peptide, 1H-TOCSY and 1H-NOESY (100 ms mixing time) spectra

were recorded for the nonlabeled VIVIT peptide in complex with

[U-2H15N13C] CnCat. To obtain the main-chain-main-chain and

main-chain-side-chain intermolecular NOE between VIVIT and CnCat,15N-NOESY-HSQC spectra with 200 ms mixing time were recorded for2H15N13C labeled VIVIT peptide or CnCat in the presence of a slight

excess amount of nonlabeled binding partner (Gross et al., 2003).

Side-chain-side-chain NOEs were obtained from recording a 13C-

NOESY-HSQC spectrum with 200 ms mixing time of a complex of

[ul-2H15N, Id1LdVg -1H13C] VIVIT peptide and [2H, FIMY-1H] CnCat.

Cross-saturation experiments were performed in accordance with

previous literature (Takahashi et al., 2006; Takahashi et al., 2000).

Structure Determination of CnCat-VIVIT Complex

The structure of the CnCat-VIVIT complex was determined by simu-

lated annealing of the VIVIT peptide in the presence of the X-ray struc-

ture of free CnCat (Kissinger et al., 1995) (PDB code: 1AIU) by using

structural information derived from NMR spectroscopy. The experi-

mental restraints include 22 intermolecular distance restraints be-

tween CnCat and VIVIT peptide, 52 intramolecular distance restraints

within VIVIT peptide, and 9 main-chain angular constraints for I6-G10

in VIVIT peptide. While calculating the structure, CnCat was modeled

by main-chain and side-chain angular restraints as well as amide-

amide, amide-alpha, and alpha-alpha intramolecular proton-proton

distance restraints and hydrogen-bonding restraints calculated from

X-ray structure of free CnCat (21–347). The side chains of the residues

near the VIVIT peptide binding sites in CnCat were left flexible by not

applying the angular restraints. The final structures, with statistical

results shown in Table 1, were calculated by using CYANA. We ran

100 sets of the final structure calculations with all of the determined

structural constraints and chose the ten best structures based on

lowest energy function, no NOE violations >0.5 A, and no angular



Structure


violations >5�. The structures were analyzed with the PROCHECK-

NMR (Laskowski et al., 1996) and MOLMOL (Koradi et al., 1996)

programs. The solvent-accessible surface area for amino acid residues

was calculated with a solvent radius of 1.4 A with the MOLMOL pro-

gram. Structural figures were generated with the MOLMOL program.

Supplemental Data

Supplemental Data describing experimental restraints applied for the

structure calculations are available at http://www.structure.org/cgi/

content/full/15/5/587/DC1/.

ACKNOWLEDGMENTS

This work was supported by the National Institutes of Health (grants

AI37581, GM47467, and EB 002026). K.T. is supported by Japan

Society of Promotion of Science.

Received: February 1, 2007

Revised: March 10, 2007

Accepted: March 27, 2007

Published: May 15, 2007

REFERENCES

Aramburu, J., Garcia-Cozar, F., Raghavan, A., Okamura, H., Rao, A.,

and Hogan, P.G. (1998). Selective inhibition of NFAT activation by

a peptide spanning the calcineurin targeting site of NFAT. Mol. Cell

1, 627–637.

Aramburu, J., Yaffe, M.B., Lopez-Rodriguez, C., Cantley, L.C., Hogan,

P.G., and Rao, A. (1999). Affinity-driven peptide selection of an NFAT

inhibitor more selective than cyclosporin A. Science 285, 2129–2133.

Table 1. Statistics for the Final Ten NMR Structures ofVIVIT Peptide in Complex with CnCat

Experimental NOE distance restraints 74

Intramolecule (VIVIT peptide) 52

Intermolecule 22

Backbone angular restraints 9

Ramachandran plot (Pro4-Thr9)a

Most favored region (%) 85.0

Additionally allowed region (%) 15.0

Generously allowed region (%) 0.0

Disallowed region (%) 0.0

Average rms differences to mean structure (A)

Backbone (N, Ca, C) (Pro4-Thr9) 0.68 ± 0.32

All heavy atoms (Pro4-Thr9) 1.15 ± 0.39

None of these ten structures exhibited distance violations>0.5 A or dihedral angle violations >5�. There is no bad con-

tact in the VIVIT peptide as well as the interface of Cn-VIVIT

interaction. The rmsd for covalent bonds and angles relative

to the standard dictionary is 0.001 A and 0.2�, respectively,with all covalent bonds and angles within 6.0 3 rmsd for VIVIT

peptide as well as the interface of Cn-VIVIT. Artificial con-

straints are applied to CnCat (see text for more detail).a The program PROCHECK-NMR (Laskowski et al., 1996) was

used to assess the stereochemical quality of the structures.

596 Structure 15, 587–597, May 2007 ª2007 Elsevier Ltd All righ

Aramburu, J., Rao, A., and Klee, C.B. (2000). Calcineurin: from struc-

ture to function. Curr. Top. Cell. Regul. 36, 237–295.

Aramburu, J., Heitman, J., and Crabtree, G.R. (2004). Calcineurin:

a central controller of signalling in eukaryotes. EMBO Rep. 5, 343–348.

Chen, L., Glover, J.N., Hogan, P.G., Rao, A., and Harrison, S.C. (1998).

Structure of the DNA-binding domains from NFAT, Fos and Jun bound

specifically to DNA. Nature 392, 42–48.

Clipstone, N.A., and Crabtree, G.R. (1992). Identification of calcineurin

as a key signalling enzyme in T-lymphocyte activation. Nature 357,

695–697.

Ferentz, A.E., and Wagner, G. (2000). NMR spectroscopy: a multi-

faceted approach to macromolecular structure. Q. Rev. Biophys. 33,

29–65.

Fooks, H.M., Martin, A.C.R., Woolfson, D.N., Sessions, R.B., and

Hutchinson, E.G. (2006). Amino acid pairing preferences in parallel

[beta]-sheets in proteins. J. Mol. Biol. 356, 32–44.

Garcia-Cozar, F.J., Okamura, H., Aramburu, J.F., Shaw, K.T.Y.,

Pelletier, L., Showalter, R., Villafranca, E., and Rao, A. (1998). Two-

site interaction of nuclear factor of activated T cells with activated

calcineurin. J. Biol. Chem. 273, 23877–23883.

Goddard, T.D., and Kneller, D.G. (2006). SPARKY 3—NMR

Assignment and Integration Software (San Francisco: University of

California).

Goto, N.K., Gardner, K.H., Mueller, G.A., Willis, R.C., and Kay, L.E.

(1999). A robust and cost-effective method for the production of Val,

Leu, Ile (delta 1) methyl-protonated 15N-, 13C-, 2H-labeled proteins.

J. Biomol. NMR 13, 369–374.

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). X-ray structure of calcineurin inhibited by the immunophilin-

immunosuppressant FKBP12-FK506 complex. Cell 82, 507–522.

Gross, J.D., Gelev, V.M., and Wagner, G. (2003). A sensitive and robust

method for obtaining intermolecular NOEs between side chains in

large protein complexes. J. Biomol. NMR 25, 235–242.

Hemenway, C.S., and Heitman, J. (1999). Calcineurin. Structure, func-

tion, and inhibition. Cell Biochem. Biophys. 30, 115–151.

Hogan, P.G., and Li, H. (2005). Calcineurin. Curr. Biol. 15, R442–R443.

Huai, Q., Kim, H.Y., Liu, Y., Zhao, Y., Mondragon, A., Liu, J.O., and Ke,

H. (2002). Crystal structure of calcineurin-cyclophilin-cyclosporin

shows common but distinct recognition of immunophilin-drug com-

plexes. Proc. Natl. Acad. Sci. USA 99, 12037–12042.

Jain, J., McCaffrey, P.G., Miner, Z., Kerppola, T.K., Lambert, J.N.,

Verdine, G.L., Curran, T., and Rao, A. (1993). The T-cell transcription

factor NFATp is a substrate for calcineurin and interacts with Fos

and Jun. Nature 365, 352–355.

Jin, L., and Harrison, S.C. (2002). Crystal structure of human calci-

neurin complexed with cyclosporin A and human cyclophilin. Proc.

Natl. Acad. Sci. USA 99, 13522–13526.

Kiani, A., Rao, A., and Aramburu, J. (2000). Manipulating immune re-

sponses with immunosuppressive agents that target NFAT. Immunity

12, 359–372.

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., et al. (1995). Crystal structures of human calcineurin and the

human FKBP12-FK506-calcineurin complex. Nature 378, 641–644.

Klee, C.B., Crouch, T.H., and Krinks, M.H. (1979). Calcineurin: a cal-

cium- and calmodulin-binding protein of the nervous system. Proc.

Natl. Acad. Sci. USA 76, 6270–6273.

Koradi, R., Billeter, M., and Wuthrich, K. (1996). MOLMOL: a program

for display and analysis of macromolecular structures. J. Mol. Graph.

14, 51–55, 29–32.

Laskowski, R.A., Rullmannn, J.A., MacArthur, M.W., Kaptein, R., and

Thornton, J.M. (1996). AQUA and PROCHECK-NMR: programs for

ts reserved

http://www.structure.org/cgi/content/full/15/5/587/DC1/

http://www.structure.org/cgi/content/full/15/5/587/DC1/

Structure


checking the quality of protein structures solved by NMR. J. Biomol.

NMR 8, 477–486.

Li, H., Rao, A., and Hogan, P.G. (2004). Structural delineation of the

calcineurin-NFAT interaction and its parallels to PP1 targeting interac-

tions. J. Mol. Biol. 342, 1659–1674.

Lohr, F., Katsemi, V., Hartleib, J., Gunther, U., and Ruterjans, H. (2003).

A strategy to obtain backbone resonance assignments of deuterated

proteins in the presence of incomplete amide 2H/1H back-exchange.

J. Biomol. NMR 25, 291–311.

Macian, F. (2005). NFAT proteins: key regulators of T-cell development

and function. Nat. Rev. Immunol. 5, 472–484.

Michael, H.A.R. (2004). Structural and Chemical Genetic Insights into

Protein-Protein Interaction of Phosphate-Dependent Cell Signaling:

The Cases of Calcineurin and Protein Kinase C Iota (Boston: Harvard

University).

Mondragon, A., Griffith, E.C., Sun, L., Xiong, F., Armstrong, C., and Liu,

J.O. (1997). Overexpression and purification of human calcineurin

alpha from Escherichia coli and assessment of catalytic functions of

residues surrounding the binuclear metal center. Biochemistry 36,

4934–4942.

Rao, A., Luo, C., and Hogan, P.G. (1997). Transcription factors of the

NFAT family: regulation and function. Annu. Rev. Immunol. 15, 707–

747.

Reibarkh, M., Malia, T.J., Hopkins, B.T., and Wagner, G. (2006). Iden-

tification of individual protein-ligand NOEs in the limit of intermediate

exchange. J. Biomol. NMR 36, 1–11.

Rodriguez, A., Martinez-Martinez, S., Lopez-Maderuelo, M.D., Ortega-

Perez, I., and Redondo, J.M. (2005). The linker region joining the cata-

lytic and the regulatory domains of CnA is essential for binding to

NFAT. J. Biol. Chem. 280, 9980–9984.

Roehrl, M.H., Kang, S., Aramburu, J., Wagner, G., Rao, A., and Hogan,

P.G. (2004a). Selective inhibition of calcineurin-NFAT signaling by

blocking protein-protein interaction with small organic molecules.

Proc. Natl. Acad. Sci. USA 101, 7554–7559.

Roehrl, M.H.A., Wang, J.Y., and Wagner, G. (2004b). Discovery of

small-molecule inhibitors of the NFAT-calcineurin interaction by

Structure 15, 5

competitive high-throughput fluorescence polarization screening.

Biochemistry 43, 16067–16075.

Rusnak, F., and Mertz, P. (2000). Calcineurin: form and function. Phys-

iol. Rev. 80, 1483–1521.

Stewart, A.A., Ingebritsen, T.S., Manalan, A., Klee, C.B., and Cohen, P.

(1982). Discovery of a Ca2+- and calmodulin-dependent protein phos-

phatase: probable identity with calcineurin (CaM-BP80). FEBS Lett.

137, 80–84.

Takahashi, H., Miyazawa, M., Ina, Y., Fukunishi, Y., Mizukoshi, Y.,

Nakamura, H., and Shimada, I. (2006). Utilization of methyl proton

resonances in cross-saturation measurement for determining the

interfaces of large protein-protein complexes. J. Biomol. NMR 34,

167–177.

Takahashi, H., Nakanishi, T., Kami, K., Arata, Y., and Shimada, I.

(2000). A novel NMR method for determining the interfaces of large

protein-protein complexes. Nat. Struct. Biol. 7, 220–223.

Takeuchi, K., Ng, E., Malia, T.J., and Wagner, G. (2007). 1-(13)C amino

acid selective labeling in a (2)H(15)N background for NMR studies of

large proteins. J. Biolmol. NMR, in press. Published online March 28,

2007. 10.1007/s10858-007-9152-z.

Wolfe, S.A., Zhou, P., Dotsch, V., Chen, L., You, A., Ho, S.N., Crabtree,

G.R., Wagner, G., and Verdine, G.L. (1997). Unusual Rel-like architec-

ture in the DNA-binding domain of the transcription factor NFATc.

Nature 385, 172–176.

Wouters, M.A., and Curmi, P.M.G. (1995). An analysis of side chain

interactions and pair correlations within antiparallel beta-sheets: the

differences between backbone hydrogen-bonded and non-hydrogen-

bonded residue pairs. Proteins Struct. Funct. Genet. 22, 119–131.

Zhou, P., Sun, L.J., Dotsch, V., Wagner, G., and Verdine, G.L. (1998).

Solution structure of the core NFATC1/DNA complex. Cell 92, 687–

696.

Accession Numbers

A set of structures of the Cn-VIVIT complex has been entered into the

Protein Data Bank under the accession number 2JOG.



structure of the calcineurin-nfat complex: defining a t cell activation switch using solution nmr...

Documents