doi:10.1016/j.jmb.2007.04.032 J. Mol. Biol. (2007) 369, 1296–1306

Structure of Calcineurin in Complex with PVIVITPeptide: Portrait of a Low-affinity Signalling Interaction

Huiming Li1,2, Lan Zhang3, Anjana Rao1,2, Stephen C. Harrison3,4

and Patrick G. Hogan1⁎

1The CBR Institute,for Biomedical Research,200 Longwood Avenue,Boston, MA 02115, USA2Department of Pathology,Harvard Medical School,200 Longwood Avenue,Boston, MA 02115, USA3Laboratory of MolecularMedicine, Children's Hospital,320 Longwood Avenue,Boston, MA 02115, USA4Howard Hughes MedicalInstitute, Harvard MedicalSchool, 250 Longwood Avenue,Boston, MA 02115, USA

Abbreviations used: CNA, calcinecalcineurin B.E-mail address of the correspondi

hogan@cbr.med.harvard.edu

0022-2836/$ - see front matter © 2007 E

The protein phosphatase calcineurin recognizes a wide assortment of sub-strates and controls diverse developmental and physiological pathways ineukaryotic cells. Dephosphorylation of the transcription factor NFAT andcertain other calcineurin substrates depends on docking of calcineurin at aPxIxITconsensus site.We describe here the structural basis for recognition ofthe PxIxIT sequence by calcineurin. We demonstrate that the high-affinitypeptide ligand PVIVIT adds as a β-strand to the edge of a β-sheet ofcalcineurin; that short peptide segments containing the PxIxIT consensussequence suffice for calcineurin-substrate docking; and that sequence varia-tions within the PxIxIT core modulate the Kd of the interaction within thephysiological range 1 μMto 1mM.Calcineurin can adapt to awide variety ofsubstrates, because recognition requires only a PxIxIT sequence and becausevariation within the core PxIxIT sequence can fine-tune the affinity to matchthe physiological signalling requirements of individual substrates.

© 2007 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: calcineurin; NFAT; VIVIT peptide; crystal structure; docking site

Introduction

Calcineurin is a protein phosphatase of similarsequence and biochemical features in fungi, multi-cellular invertebrates, and vertebrates.1,2 It has a cat-alytic subunit, calcineurinA (CNA), and a regulatorysubunit, the EF-hand protein calcineurin B (CNB).Active calcineurin also includes tightly boundcalmodulin, which displaces the CNA autoinhibi-tory segment from the catalytic site. Thus calcineurintranslates an elevation in intracellular Ca2+ concen-tration into dephosphorylation of specific substrates.Calcineurin is the target of the clinically important

immunosuppressants CsA and FK506.3–5 Depho-sphorylation of the transcription factors NFAT bycalcineurin initiates their nuclear import and DNAbinding and has a major role in T cell activation.6

Efficient dephosphorylation of NFAT in cellsdepends on a docking sequence, PRIEIT in NFAT1

urin A; CNB,

ng author:

lsevier Ltd. All rights reserve

and NFAT2 and the closely related PSIRIT andPSIQIT in NFAT3 and NFAT4.7–10 Blocking thisprotein−protein interaction prevents activation ofNFAT in Tcells.7,11 This finding has been extended tocalcineurin-NFAT signalling in other cell types12,13

and to the K+ channel TRESK, a calcineurin substratethat uses the related docking site PQIIIS.14,15

The PxIxIT motifs of individual NFAT proteinshave not evolved for tight binding. The Kd forcalcineurin binding to the docking peptide of NFAT1is ∼25 μM. Replacing the native docking site with aconsensus peptide having Kd 0.5 μM results inconstitutive activation of NFAT in cells.11 Conver-sely, a TNA substitution in the PRIEIT sequence ofNFAT1, which produces a tenfold change in Kd for amodel peptide,16 prevents activation.7 These resultssuggest that the strength of calcineurin-NFATbinding, or a related variable, such as the dissocia-tion rate of the complex, is tuned in a narrowwindow. Several yeast calcineurin substrates (Crz1,Slm1, Slm2, and Hph1) utilize the PxIxIT dockingsite,17–19 and normal signalling in the calcineurin-Crz1 pathway depends on the precise affinity of theinteraction.20 The lessons emerging from observa-tions on mammalian and yeast substrates are that

d.

Figure 1. PxIxIT recognition sequences. The corerecognition peptides occurring in human NFAT1, humanand mouse TRESK, and the yeast calcineurin substratesCrz1, Slm1, Slm2, and Hph1 are shown with consensusresidues highlighted. The Kd for binding of each peptide tocalcineurin, estimated from its ability to compete withfluorescent PVIVIT peptide (Li et al.,16 Roy et al.,20 andunpublished results), is listed at right. Kds for the yeastpeptides from Crz1, Slm1, Slm2, and Hph1 weremeasured with yeast calcineurin. Other established PxIxITdocking sequences are in human NFAT2 (PRIEIT), NFAT3(PSIRIT), and NFAT4 (PSIQIT). The sequence of the 14merPVIVIT consensus peptide used for crystallization isshown with its residue numbering at the bottom of theFigure.

1297Calcineurin-PVIVIT Complex

there is considerable diversity in recognition motifs;that rather different motifs can sometimes confersimilar affinity, as illustrated by NFAT1 (PRIEIT)and Slm2 (PEFYIE); and that biologically relevantaffinities cover a broad range and extend to ratherweak interactions (Figure 1).We have previously derived an approximate

picture of 6mer PVIVIT docking onto the surfaceof calcineurin A based on calcineurin-peptide cross-linking, binding studies with mutated calcineurinsand substituted peptides, and in silico dockingsimulations.16 We report here the X-ray crystal-lographic structure of human calcineurin in complexwith a 14mer PVIVIT peptide, together with anexperimental analysis of factors that determine theaffinity of the transient, reversible interactionsbetween calcineurin and its substrates.

Results

Structure overview

A complex of CNA-CNB with PVIVIT peptidecrystallized in the P212121 space group with unit celldimensions a=86.10 Å, b=89.16 Å, and c=157.69 Å.The asymmetric unit contains two copies of theCNA-CNB heterodimer, with the second heterodi-mer (molecules C and D) related to the first(molecules A and B) by a non-crystallographic2-fold rotation plus a one-residue translation,about an axis roughly coincident with the PVIVITpeptide (Figure 2(a)). Although we used purifiedCNA-CNB-PVIVIT complex as starting material,

there is only one copy of the 14-mer PVIVIT peptidein the asymmetric unit. The peptide is sandwichedbetween two CNA molecules, in an extendedconformation parallel to β-strand 14 of bothmolecules (Figure 2(a) and (b)). The sheets com-posed of β-strands 4, 3, 2, 13, and 14 of the two CNAmolecules are integrated into one continuous sheetby the intercalated PVIVIT peptide. The density forthe core residues of PVIVIT peptide, Val7 to Thr11, isextremely well defined with an average B factor of33 Å2 (Figure 3(a)). (The single-letter code is used forCNA, and the triple-letter code is used for thepeptide. The peptide residues are numbered 3−16for consistency with previous publications11,16)Outside its core region, the peptide follows thesurface of CNA molecule A closely, and evidencediscussed below indicates that the interactionbetween PVIVIT and CNA molecule A is the onerelevant to calcineurin−NFAT docking. Because ofthe extended conformation of the peptide and therotation-translation operation that relates the twocalcineurins, the IVIT sequence docks againstmolecule C in essentially the same way as the VIVIsequence docks against molecule A (Figure 2(c)).Moreover, the succession of five β-branched resi-dues (VIVIT) causes even the details of van derWaals contacts in these two interactions to be verysimilar. Thus, the interaction of the peptide withmolecule C informs us about the basis for the effectsof (hypothetical) I–NV and V–NI mutations on theprincipal docking mode with molecule A.

Contacts between PVIVIT peptide andcalcineurin A

The peptide augments the 4-3-2-13-14 β-sheet ofCNA by backbone hydrogen bonds with Val7, Val9,and Thr11. This central interaction is supplementedby van der Waals contacts and hydrogen bondsbetween the core PVIVIT segment and CNA, alongthe edge of the protein that contains strands β14 andβ11, and by contacts extending into the regionbounded by the β13-β14 and β11-β12 loops. Theside-chains of Pro6, Ile8 and Ile10 each fit into welldefined pockets on CNA (Figure 4(a)−(c)). The N330side-chain amide nitrogen donates hydrogen bondsboth to the carbonyl of Val9 and to Oγ of Thr11,nucleating a hydrogen-bond network that includesHis14 and R332 (Figure 4(d)). Van der Waalscontacts between Cγ of Thr11 and Cγ1 of Val9 andbetween Cγ2 of Val9 and Cγ1 of Val328 create a chainof non-polar interactions that may help account forthe enhanced binding of peptides with a β-branchedside-chain at position 9.Contacts outside the core PVIVIT docking se-

quence include, on the N-terminal side, a hydrogenbond to the backbone carbonyl of Pro4 from the side-chain of N327 and, on the C-terminal side, a set ofnon-polar interactionswith Pro13. There is an abruptbackbone turn in the peptide, facilitated by the ϕ−ψtorsion angles available to Gly12 and the cisconfiguration of Pro13, which positions the His14backbone amide to hydrogen bond with the Q333

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

1298 Calcineurin-PVIVIT Complex

Figure 3. (a) Electron density of the core PVIVIT docking sequence and adjacent residues of CNA molecules C (atleft) and A (at right). (b) Electron density of the central region of PVIVIT peptide, with the slab taken in a differentorientation, showing the proximity of the Ile8 and Ile10 side-chains to M290 of CNA. Synthetic PVIVIT peptide withp-benzoylphenylalanine inserted either at position 8 or at position 10 can be photo-crosslinked to M290 of CNA.16

1299Calcineurin-PVIVIT Complex

backbone carbonyl and enables the histidine side-chain to pack against the guanidinium group of R332and to hydrogen bond with Thr11 (Figure 4(d)).The side-chains directed into the β11-β14 groove

of molecule C are those that point away from theβ11-β14 groove of molecule A. Thus, Val7 projectsshallowly into the “Pro6” pocket of molecule C,Val9, into the “Ile8” pocket, and Thr11, into the“Ile10” pocket. The N and C-terminal segments ofthe peptide bend away from molecule C, but Ile8and Ile10 augment the 4-3-2-13-14 β-sheet with fourbackbone hydrogen bonds, essentially equivalent tothose made by Val7 and Val9 with strand β14 ofmolecule A, and the backbone carbonyl and amideof Gly12 are linked through ordered water mole-cules to the I331 and Q333 carbonyl oxygen atoms,respectively. An Ile10 carbonyl-N330 side-chainhydrogen bond with molecule C mimics the Val9backbone-N330 side-chain contact with molecule A.

The two modes of interaction

Various structural features support the conclusionthat the PVIVIT:CNA-A contact is the correctrepresentation of NFAT docking. First, this modeof contact can accommodate the polar Arg7 andGlu9 side-chains of the native PRIEIT motif inNFAT1, while the PVIVIT:CNA-C contact wouldhave these polar side-chains projecting into non-polar recesses on the CNA surface. Second, thePVIVIT:CNA-A contact has the experimentally

demonstrated Thr11−N330 interaction16 (Figure4(d)), whereas Thr11 is on the opposite side of theβ sheet from the N330 side-chain of molecule C.Third, the proximity of the Ile8 and Ile10 side-chainsto M290 of molecule A (Figure 3(b)) is consistentwith the experimental finding that PVIVIT peptidesubstituted with p-benzoylphenylalanine at either ofthese two positions can crosslink efficiently tocalcineurin A.16 Finally, the PVIVIT:CNA-A contactis likely to be the stronger interaction, with tenH-bonds between the peptide and molecule A and1486 Å2 of buried surface, compared with fiveH-bonds with molecule C and 902 Å2 of buriedsurface (Figure 5).The PVIVIT:CNA-C contact represents an alter-

native mode of docking that might be utilized bysome substrates. Binding of PVIVIT in this confi-guration is weak, since PVIVIT peptides withp-benzoylphenylalanine replacing Val9 or Thr11do not crosslink to the reactive M290 site ofcalcineurin, even when calcineurin and peptide arepresent at concentrations exceeding the calcineurinand substrate concentrations in cells (Li et al.16 andunpublished results). Correspondingly, bound PVI-VIT does not induce significant dimerization ofcalcineurin in solution, as indicated by the findingsthat the calcineurin−peptide binding reaction is first-order in calcineurin concentration16,21 and thatcochromatography with PVIVIT does not alter themigration of calcineurin on a size-exclusion column(see Materials and Methods).

Figure 4. Details of the contacts between PVIVIT and calcineurin. (a) The pocket occupied by Pro6. The peptide is instick representation, and calcineurin A in surface representation with the contacting surface residues shaded orange and,in some cases, labelled. The N-terminal residues of the peptide, Pro4 and His5, have been omitted for clarity. (b) Thehydrophobic surface occupied by Ile8 and Ile10. The proline pocket is at the bottom of the panel, with the Pro6 side-chainlargely obscured by the His5 carbonyl oxygen. (c) Packing interaction between the Ile10 side-chain of the peptide andY288, M290, and I331 of calcineurin A. Ile8 is in the foreground, but a part of the calcineurin surface that contacts Ile8 hasbeen cut away. (d) A part of the hydrogen-bonding network connecting CNA and the C-terminal region of the peptide.Backbone hydrogen bonds of Val7, Val9, and Thr11 are not depicted.

1300 Calcineurin-PVIVIT Complex

Effect on affinity of variations in the core motif

The calcineurin-PVIVIT structure shows contactswith both core and flanking regions of the peptide.We have examined binding of the consensus PVIVITpeptide and of the native docking peptide from theyeast calcineurin substrate Hph1, two examples thatspan the full range of affinities observed in a bindingassay with yeast calcineurin.20 The peptides differ inthree residues of the core sequence and havedissimilar flanking regions (Figure 6(a)). Replace-ment of only the three core residues within the14mer PVIVIT context reduces its affinity to that ofthe native Hph1 peptide (Figure 6(b)). In fact, thePVIAVN core with PVIVIT flanking regions hasslightly lower affinity than with its native flankingsequences. Thus, variations in the core sequencealone can make a decisive contribution to affinityvariation in the physiological range.We examined the effect of substituting the single

residues that differentiate the tight-binding PVIVITfrom the less preferred PVIAVN. The flankingregions were in all cases those of the consensusPVIVIT peptide. Replacement of threonine byasparagine at position 11 of the PVIVIT peptideresulted in an approximately fivefold shift of Kd to

higher concentration, and the symmetrical replace-ment of asparagine by threonine in PVIAVNpeptide resulted in an approximately fivefoldshift of Kd to lower concentration (Figure 6(c)). Interms of free energy, binding of the peptides withThr11 is more favorable by ∼1 kcal/mol thanbinding of the corresponding peptides with Asn11.In contrast, replacement of isoleucine by valine, orvaline by isoleucine, at position 10 caused at most asmall change in either case (Figure 6(d)). Compar-ison of the contacts between Ile10 and CNA-A andthose between Thr11 and CNA-C indeed suggeststhat side-chain rotamer adjustments within theIle10 pocket can minimize the effect of removingthe γ-methyl group.The reproducible competition by the standards

PVIVIT and PVIAVN in each experiment allowed usto compare the competition curves of the pairsPVIVVT/PVIAVT and PVIAVN/PVIVVN obtainedin separate experiments. Replacement of valineby alanine at position 9 caused an approximately15-fold shift in Kd, or ∼1.6 kcal/mol reduction in thefree energy of binding, and replacement of alanineby valine caused a comparable shift in the oppositedirection (Figure 6(c)−(e)). The γ-methyl groups ofVal9 mediate a chain of van der Waals contacts,

Figure 5. The interactions ofPVIVIT peptide with CNA mole-cule A and with CNA molecule C.The peptide is in stick representa-tion, and calcineurin is in surfacerepresentation with the contactingsurface residues shaded orange.The placement of consensus PxIxITresidues is indicated in the paneldepicting CNA molecule A. Thealternative residues that occupythe same non-polar pockets onthe surface of calcineurin are indi-cated for CNA molecule C. Thecentral β-strand contact is similar inthe two cases. Outside its centralregion, the peptide follows the CNAmolecule A surface more closely,and the surface engaged (shadedorange) is distinctly larger for mole-cule A than for molecule C.

1301Calcineurin-PVIVIT Complex

described above; these interactions may account forthe loss of affinity on substitution with alanine. Insummary, the residues at positions 9 and 11 togethercan contribute most of the 100-fold difference inaffinity between PVIVIT and PVIAVN, and bothconsensus and non-consensus residues in the coreare relevant.The signature proline residue, which is common to

both peptides, contributes, as expected, to peptiderecognition. In an experimental comparison of the12mer peptides HPVIVITGPHEE and HAVIVITG-PHEE, where the first proline (Pro4) was omitted toavoid any ambiguity in the register of binding tocalcineurin, replacingPro6with alanine caused a shiftof ∼100-fold in Kd (Figure 6(f)). There was none-theless detectable competition by the Ala6 peptide.

Discussion

Our crystallographic results establish the struc-tural basis for recognition of PxIxIT dockingsequences by calcineurin. The calcineurin−peptidebinding studies help to define how this structuretranslates into the weak, reversible interactions thattypify calcineurin−substrate recognition, and showhow the strength of the specific binding interactioncan be adjusted in the physiological range.

The calcineurin-PVIVIT structure

The calcineurin-PVIVIT 14mer structure, showingthe core region of the peptide aligned along β strand14 and specific contacts of consensus PxIxIT residues

with the surface of CNA, agrees well with theintermediate-resolution picture from computationaldocking of 6mer PVIVIT.16 The orientation andprotein−peptide contacts within the core PVIVITsegment are identical, but the crystal structureshows specific side-chain contacts of the corepeptide and defines interactions in regions of thepeptide flanking the core PxIxIT sequence, whichwere not modelled in the earlier work.There are two changes in calcineurin relative to

the published calcineurin structure 1TCO, the modelused for the docking simulations. First, the R332side-chain is displaced by His14 of the peptideligand, which was not represented in the shorterPVIVIT(6-11) ligand of the docking simulations, andthe side-chain of N330 is slightly re-oriented so thatthe two side-chains are integrated into a moreextended hydrogen-bond network connecting calci-neurin and peptide. Second, the backbone config-uration of the β13-β14 loop of calcineurin is alteredwithout any substantive change in the position ofthe residues forming the proline pocket, other thanN327. Comparison of molecule Awith molecule C orwith the published calcineurin structures 1AUI and1TCO indicates that the β13-β14 loop is flexible, but,because crystal packing contacts in molecule A andin the 1AUI and 1TCO apoenzyme structures mayaffect the position of the loop, we cannot relate thechange in configuration solely to binding of PVIVITpeptide.The structural role of β-sheet augmentation in the

interaction of PDZ and PTB domains with theirpeptide targets and in the assembly of other protein-protein complexes has been recognized.22,23 For

Figure 6. Competitive binding experiments to assess the effect of specific substitutions within the PxIxIT coremotif on calcineurin−peptide binding. (a) Sequences of 14mer PVIVIT peptide, 14mer PVIAVN peptide, and Hph1peptide. (b) Increasing concentrations of the unlabelled peptides PVIVIT, Hph1, and PVIAVN displace fluorescentPVIVIT from calcineurin. Hph1 peptide shows relatively weak binding to human calcineurin, and a peptide with thePVIAVN core shows similarly weak binding even when it is flanked by the consensus N-terminal and C-terminalregions. (c) Competition by pairs of peptides differing at position 11. (d) Competition by pairs of peptides differingat position 10. (e) Diagram summarizing the effect of the substitutions at positions 9−11 of the peptide. The arrow atleft indicates ranking from low to high affinity. (f) Truncation to HPVIVITGPHEE, that is, to PVIVIT(5-16), has onlya small effect on the ability of the unlabelled peptide to compete with fluorescent PVIVIT. PNA replacement atposition 6 of PVIVIT(5-16) shifts Kd approximately 100-fold.

1302 Calcineurin-PVIVIT Complex

PDZ domains, the question has been which proteinsare recognized and incorporated into complexes,with the strongest interactions considered mostrelevant to signalling. Here we explore β-sheetaugmentation as the common structural under-pinning of calcineurin−substrate interactions, forwhich the strength of the interaction must vary fromsubstrate to substrate.

Basis for a weak, reversible interaction

Calcineurin, like some other signalling enzymes,does not typically form stable complexes with itssubstrates. For cellular calcineurin concentrationsfrom 0.1 to 10 μM, Kds for individual substratesranging from 1 μM to 1 mM could give effectiverecognition with only a small fraction of each

1303Calcineurin-PVIVIT Complex

substrate in complexwith calcineurin at a given time.The structural foundation for this low-affinity bind-ing in the calcineurin-PxIxIT complex is a limitedregion of contact and the inherently low stability of aparallel β-strand interaction.The requirement for weak, transient interactions

in intracellular signalling sets up a tension betweenlow affinity and specificity. For example, in SH3domain−peptide interactions, which involve recog-nition of proline-rich segments, two key features area propensity for the target peptide to form apolyproline-II helix and a contact pocket for prolineCδ.24,25 Proline is also a key conserved residue in thecalcineurin−substrate contact examined here. Theenzyme discriminates between proline and alanine,but not because of polyproline-II helix formation,and the geometry of recognition differs from proline−SH3 contacts in more complete burial of the prolineside-chain. The detectable binding of HAVIVITG-PHEE indicates that non-proline residues can sub-stitute in an otherwise favorable sequence context.We noted previously the possibility that AKAP79/150 (PIAIIIT), a calcineurin targeting and regulatoryprotein,26–28 docks on calcineurin with its firstisoleucine occupying the proline pocket.16

Basis for variations in calcineurin−substratebinding affinity

The findings that calcineurin-NFAT1 and calci-neurin-Crz1 signalling are disrupted by either anincrease or a decrease in the affinity of the dockinginteraction7,11,20 imply that evolution has selectednot just for specific recognition of these substrates,but also for an appropriate affinity. We have shownhere that side-chain contributions from both con-sensus and non-consensus positions of the xIxITsegment contribute to modulating affinity over awide range. For consensus positions, the notablepoint is the extent of sequence diversity that has beenexploited at position 11, where six different hydro-philic residues occur in the small sample of sub-strates so far examined carefully (Figure 1). It will benecessary to examine a larger sample of recognitionsequences to ascertain what variations can beaccepted at consensus positions 8 and 10. Abundantvariability was already evident at the non-consensuspositions, just from the sequences in Figure 1 and thesequences of NFAT1-4, but an important new findingis that residue replacement at a non-consensusposition can make a substantial contribution toaffinity variation.There is a close parallel to our findings in a

quantitative study of peptide binding to the AF6,ERBIN, and SNA1 PDZ domains.29 For these threePDZ domains, the four C-terminal residues ofpeptide make nearly independent contributions tothe free energy of binding; the spread in Kds that canbe produced by substitutions at individual positionsis fivefold tomore than 20-fold; and themaximal shiftin Kd due to substitutions at non-consensus positionscan be as large as that caused by replacements atconsensus positions. These data complement our

observations on calcineurin, and support the conclu-sion that coarse and fine gradations in binding can beachieved within the context of β-sheet augmentationby replacing single core motif residues.Both the calcineurin-PVIVIT structural model

presented here and peptide selection experiments11

are consistent with a further contribution from non-conserved residues immediately flanking the PxIxITsegment. The effect of altering flanking residues isexpected to be less than that of changing residues inthe core motif, based on the lower preference scoresin the peptide selection experiments11 and theinsensitivity of PVIAVN binding to interchangingthe Hph1 and consensus PVIVIT flanking regions.The absence of a consensus sequence in the flankingsegments indicates that the detailed contacts varywith the individual substrate. Indeed, in somesubstrates one or both of the flanking regions mayhave no direct role in contacting calcineurin.

Connecting specific substrate recognition todephosphorylation

The minimal requirement for efficient dephosphor-ylation of many calcineurin substrates is recognitionof the PxIxIT peptide segment.7,11,15,17,18,19 Preciseplacement of the PxIxIT segment relative to the site ofdephosphorylation may not be necessary as long asthere is sufficient flexibility to position the targetedphosphorylated residues in the catalytic site ofcalcineurin. The role of a flexible spacer has beenexamined in the case of the phospho-CDK2/cyclin Ainteraction with its substrates.30–32 There, binding ofsubstrate at a cyclin A recruitment site distant fromthe phospho-CDK2 catalytic site reduces the Km forphosphorylation, and correspondingly the structureof phospho-CDK2-cyclin Awith a modified substratefragment reveals interactions at the recruitment siteand the catalytic site, with the intervening spacerpeptide unstructured and invisible in the model.32

Binding through a PxIxIT recognition sequencecould be combined with additional contacts toprovide higher affinity or to restrict the orientationof the partner protein relative to calcineurin.Simultaneous use of PxIxIT and another contacthas not been documented for calcineurin substrates,but there are such additional contacts in calcineurinregulatory proteins.33,34 The strategy is more fullyexploited by the related protein phosphatase PP1,which is primarily delivered to substrates by stablybound targeting subunits. PP1 recognizes an RVxFmotif in its targeting subunits through binding at asite cognate to the PxIxIT-binding site of calci-neurin.35–38 To effect the more stable binding ofPP1 targeting subunits, the RVxF contact is supple-mented by additional contacts, as illustrated by thestructure of the PP1-MYPT1 complex.36

Conclusion

We describe the structural basis for calcineurin−substrate recognition for those substrates that utilize

1304 Calcineurin-PVIVIT Complex

the PxIxIT-binding site. The simplicity of theinteraction and the ease with which its affinity canbe adjusted in the range from 1 μM to 1 mM providea partial explanation for the success of calcineurin inadapting to new substrates in the course of evolu-tion. The PxIxIT segment is probably unstructuredor part of a flexible loop before binding calcineurin,placing minimal constraints on the ability of an in-frame insertion in DNA to add a PxIxIT recognitionsequence to the encoded protein, or on the ability ofa point mutation to vary its affinity. Further study ofcalcineurin−substrate recognition in this light mayafford insight into the evolution of intracellularsignalling pathways.

Materials and Methods

Protein expression and purification

Because our previous biochemical and in silico dockingstudies mapped the PVIVIT docking site to a region ofCNA surface occupied, in published structures of calci-neurin, by the N-terminal portion of CNB from aneighboring asymmetric unit, we removed residues 2−15of CNB. The CNA subunit was truncated at residue 380,C-terminal to the CNB-binding helix, to improve proteinstability. The crystallized protein was therefore a hetero-dimer of CNA(1-380), with a residual tripeptide linkerpreceding residue 1, and CNB(16-170). Hexahistidine-tagged CNA was expressed together with CNB inEscherichia coli strain BL21(DE3) from a tandem CNA-CNB expression construct in pET15b based on theconstruct previously used for the full-length proteins.39

Bacteria were grown at 37 °C to an A600nm of 0.5, inducedwith 0.5 mM IPTG after the temperature had been reducedto 18 °C, and harvested 48 h later. CNA-CNB proteincomplex was initially purified over a Ni-NTA column(Qiagen). After the hexahistidine tag was cleaved byovernight digestion with thrombin at 4 °C, the proteinsolution was again passed through a Ni-NTA column toremove the cleaved peptide tag, then further purified on aphenyl Sepharose column (Phenyl Sepharose High Perfor-mance, GE Healthcare) and an S200 size-exclusion column(Superdex 200, Prep Grade, GE Healthcare). The proteinpeak eluting at a position corresponding to a CNA-CNB

Table 1. Data collection and refinement statistics

Data collection

Space group P212121Cell dimensions

a, b, c (Å) 86.104, 89.155, 157.685α, β, γ (°) 90.00, 90.00, 90.00

Wavelength (Å) 0.9789Resolution (Å) 2.3Rsym

a 0.104 (0.518)b

I/σ 15.1(1.9)Completeness (%) 96.2(90.7)Redundancy 4.7(2.6)

a Rsym=∑hkl∑i|Ii(hkl) − I(hkl)|/ ∑hkl∑iIi(hkl) where I(hkl) is the averb Numbers in the parentheses correspond to the highest resolutionc Rwork=(∑hkl||Fobs|−κ|Fcalc||)/ ∑hkl|Fobs|, where Fobs and F

respectively, and κ is a scaling factor.d Rfree is the same as Rwork but over randomly selected reflections (

heterodimer was subjected to a final purification step on aQ column (Q Sepharose, High Performance, GE Health-care). PVIVIT 14-mer peptide (GPHPVIVITGPHEE-amide,in which the peptide residues are numbered 3−16 forconsistency with previous publications11,16) was synthe-sized and HPLC-purified at Tufts University Core Facility,Boston, MA. Peptide was mixed at a 1.2:1 ratio withpurified CNA-CNB complex and subjected to size-exclu-sion chromatography. The peptide copurified with theprotein complex as confirmed by matrix-assisted laserdesorption ionization-time of flight (MALDI-TOF) massspectrometry. The position where the CNA-CNB-PVIVITcomplex elutedwas essentially the same as for the complexof CNA and CNB alone.

Crystallization and structure determination

The calcineurin-PVIVIT complex was crystallized by thesitting-drop method at 20 °C. Purified CNA-CNB-PVIVITcomplex (7 mg/ml) in 20 mM Tes (pH 7.0), 50 mM NaCl,0.1 mM EGTA, was mixed at a 1:1 ratio with reservoirbuffer containing 100mMTes (pH 8.0), 100mMCaCl2, 12%(w/v) PEG 4000, 1 mM DTT. Small plate-shaped crystalsappeared overnight and continued to grow to about0.4 mm×0.15 mm×0.03 mm in a week. Crystals wereflash-frozen in cryo-loops using reservoir buffer plus 30%MPD in liquid nitrogen. X-ray diffraction data werecollected at the Advanced Photon Source ID-19 beamlineand processed with HKL-2000 software (HKL Research,Charlottesville, VA).40 The crystals belong to the P212121space group, with unit cell dimensions a=86.10 Å,b=89.16 Å, and c=157.69 Å. The structure was solved bymolecular replacement using the CNA-CNB complexstructure (PDB entry 1AUI, omitting the autoinhibitorypeptide) as a search model in Phaser,41 and was refined to2.3 Å resolution using CNS.42 Data collection and refine-ment statistics are listed in Table 1. TheR factor andRfree forthe finalmodel are 19.2%and 25.4%, respectively. There aretwo copies of the CNA-CNB heterodimer in the asym-metric unit, giving a solvent content of 50%. The finalmodel contains CNA (residues 14 to 370 for both copies,molecules A and C), CNB (residues 15 to 160 for moleculeB, and residues 15 to 167 for molecule D) and PVIVITpeptide (residues 4−16, with only backbone and Cβ atomsmodelled for Glu16) CNB residues in the model arenumbered as in the structures 1AUI and 1TCO, disregard-ing the initial methionine that is removed by post-translational processing in mammalian cells. Hydrogen

Refinement

Resolution (Å) 2.3No. reflections 51,123Rwork

c/Rfreed 19.2(30.7)/25.4(32.6)

No. atomsProtein 8308Water 565Other 22

Averaged B-factors(Å2) 39.8R.m.s deviationsBond lengths (Å) 0.005Bond angles (°) 1.2

age intensity.shell.calc are the observed and calculated structure factor amplitudes,

5%) excluded from the refinement.

1305Calcineurin-PVIVIT Complex

bonds between the peptide ligand and calcineurin wereidentified visually and verified using the programHBPLUS.43 Buried solvent-accessible surfaces were calcu-lated using the programAREAIMOL in CCP4.44 The valuespecified in each case is the sum of buried protein surfaceand buried peptide surface. As reported by other authors,some regions of CNB, including both the N and C termini,the loop connecting helices 2 and 3, and the loopconnecting helices 4 and 5, exhibit rather high B-factor,probably due to chain flexibility. Figures were made usingthe programs MolScript,45 O,46 and Chimera.47

Calcineurin-peptide binding assays

Additional peptides with C-terminal amide were syn-thesized and HPLC-purified at Tufts University CoreFacility. Peptide concentrations were determined initiallyby BCA assay (Pierce, Rockford, IL) and confirmed byamino acid analysis at Yale University Core Facility. Theability of peptides to compete with fluorescent PVIVITpeptide for binding to calcineurin was measured asdescribed.16

Protein Data Bank accession code

The structure factors and final refined atomic coordi-nates of the calcineurin-PVIVIT complex have beendeposited in the RCSB Protein Data Bank and are availableunder accession code 2P6B.

Acknowledgements

The authors thank L. Pelletier for sharing herexperience in crystallizing calcineurin, X. Lu and J.Zimmer for assistance with X-ray data collection,and W. Li for helpful suggestions on structuredetermination. We acknowledge the AdvancedPhoton Source at the Argonne National Laboratory.This work was supported by NIH grant AI40127 (toA.R). L.Z. acknowledges a fellowship from theHelenHay Whitney Foundation; S.C.H. is an investigatorof the Howard Hughes Medical Institute.

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Edited by M. Guss

(Received 10 February 2007; received in revised form 4 April 2007; accepted 9 April 2007)Available online 19 April 2007