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DOI: 10.1126/science.1256679, 1620 (2014);345Science

et al.Thomas Spatzalreactivated nitrogenaseLigand binding to the FeMo-cofactor: Structures of CO-bound and

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ACKNOWLEDGMENTS

We thank K. Kaibuchi, M. Mishina, T. Tanabe, K. Doya, J. Wickens,

T. Toyoizumi, and K. Kasai for helpful discussion; K. Deisseroth

for the channelrhodopsin-2 plasmids; M. Z. Lin for the Camui-CR

and AKAR2-CR plasmids; K. Iwamoto and S. Jinde for confocal

microscopy; M. Ikeda for technical assistance; and M. Ogasawara

and M. Tamura for laboratory facility. This work was supported

by the Strategic Research Program for Brain Sciences

(Bioinformatics for Brain Sciences to H.K. and S.I.) and

Grants-in-Aid (2000009 and 26221011 to H.K.) from the Ministry of

Education, Culture, Sports, Science, and Technology and by the

National Institutes of Health GM53395, DA035612, and NS069720

(G.C.R.E.-D.). This work was also supported by the Human

Frontier Science Program (H.K.). G.C.R.E.-D. has a patent in the

United States for the synthesis of dinitroindolinyl-caged

neurotransmitters.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/345/6204/1616/suppl/DC1

Materials and Methods

Figs. S1 to S11

References (3139)

1 May 2014; accepted 29 August 2014

10.1126/science.1255514

NITROGEN FIXATION

Ligand binding to the FeMo-cofactor:Structures of CO-bound andreactivated nitrogenaseThomas Spatzal,1*Kathryn A. Perez,1 Oliver Einsle,2,3 James B. Howard,1,4 Douglas C. Rees1*

The mechanismof nitrogenaseremains enigmatic, witha major unresolved issue concerning

how inhibitors and substrates bind to the active site. We report a crystal structure of

carbon monoxide (CO)inhibited nitrogenase molybdenum-iron (MoFe)protein at

1.50 angstrom resolution, which reveals a CO molecule bridging Fe2 and Fe6 of theFeMo-cofactor. The m2binding geometry is achieved by replacing a belt-sulfur atom (S2B)

and highlights the generation of a reactive iron species uncovered by the displacement

of sulfur.The CO inhibition is fully reversible as established by regain of enzyme activity

and reappearance of S2B in the 1.43 angstrom resolution structure of the reactivated

enzyme. The substantial and reversible reorganization of the FeMo-cofactor accompanying

CO binding was unanticipated and provides insights into a catalytically competent

state of nitrogenase.

B iological nitrogen fixation is natures path-wayto convert atmospheric dinitrogen (N2)into a bioavailable form, ammonia (NH3).Nitrogenase, the only known enzyme ca-pable of performing this multielectron

reduction, consists of two component metallo-

proteins, the iron (Fe)

protein (Av2) and themolybdenum-iron (MoFe)protein (Av1) (13). TheFe-protein, containing a [4Fe:4S]-cluster, medi-atesthe adenosine triphosphate (ATP)dependentelectron transfer to the MoFe-protein to supportdinitrogen reduction (4). The MoFe-protein is ana2b2heterotetramer with one catalytic unit perabheterodimer (5). To achieve the elaborate re-dox properties required for reducing the NNtriple bond, two metal centers are present inthe MoFe-protein: the P-cluster and the FeMo-cofactor. The P-cluster, an [8Fe:7S] entity, is theinitial acceptor for electrons, donated from theFe-protein during complex formation betweenthe two proteins (68). Electrons are subsequently

transferred to theFeMo-cofactor,a [7Fe:9S:C:Mo]-R-homocitrate cluster that constitutes the activesite for substrate reduction and is the most com-

plex metal center known in biological systems(5, 912).

Substrates and inhibitors bind only to formsofthe MoFe-protein reduced by two to four elec-trons relative to the resting, as-isolated state,

which can only be generated in the presence

of reduced Fe-protein and ATP (1). Mechanisticstudies must take into account the dynamic na-

ture of the nitrogenase system, which requiresmultiple association and dissociation events be-tween the twocomponent proteins,as well as theubiquitous presence of protons that are reducedto dihydrogen even in competition with othersubstrates (1,1315). The resulting distributionof intermediates under turnover conditions com-plicates the structural and spectroscopic investi-gation of substrate interactions. Hence, even thefundamental question of whether molybdenumor iron represents the site for substrate bindingat the FeMo-cofactor is still under debate, and asa consequence, a variety ofmechanistic pathways

have been proposed based on either molybdenumor iron as the catalytic center, mainly followingChatt-type chemistry (16).

Inhibitors are potentially powerful tools forthe preparation of stably trapped transient statesthat could provide insight into the multielectronreduction mechanism. In this regard, carbonmonoxide (CO), a noncompetitive inhibitor forall substrates except protons (17,18), has a num-

ber of attractive properties; CO is isoelectronicto the physiological substrate, is a reversibleinhibitor, and only binds to partially reducedMoFe-protein generated under turnover condi-

tions. Although noncompetitive inhibitors atraditionally considered to bind at distinct sifrom the substrate, for complex enzymessuas nitrogenasewith multiple oxidation staand potential substrate-binding modes, this dtinction is not required(19). More recently, it halso been shown that CO is a substrate, albei

very poor one, whose reduction includes cocomitant CC bond formation to generate and longer-chain hydrocarbons, in a reaction reiniscent of the Fischer-Tropsch synthesis (20, 2Therefore, CO binding as inhibitor or substrmust involve important active-site propertcommon to the reduction of the natural sustrate dinitrogen. For this reason, CO bindihas been investigated by a variety of spectrscopic methods, most notably electron paramnetic resonance and infrared spectroscopy, adepending on the partial pressure, multiple C

bound species have been observed; yet, a structally explicit description of any CO-binding shas been elusive (18, 2227).

Building on these observations, we have dtermined a high-resolution crystal structurea CO-bound state of the MoFe-protein froAzotobacter vinelandii. This necessitated ovcoming several obstacles. First, the experimensetup for all protein-handling steps, includicrystallization, was deemed to require the cotinuous presence of CO. Second, because inhi

tion requires enzyme turnover, a prerequisite wthe ability to obtain crystals of the MoFe-protefrom activity assay mixtures, rather than froisolated protein (see supplementary material assay details), conditions that are typically cotradictory to crystallization requirements. Finly, rapid MoFe-protein crystallization (5 hou

was crucial and was achieved on the basispreviously developed protocols in combinati

with seeding strategies and in the presenceCO (10).

Crystals of the inhibited MoFe-protein (Av1-Cyielded structural data at 1.50 resolutiowhich allowed clear identification of a CO land (Fig. 1, A and C, and fig. S1, A to D). T

Av1-CO structure directly demonstrates tbinding of one molecule of CO per active siteam2-bridging mode between Fe2 and Fe6 thforms one edge of the trigonal six-iron pri(Fe2-3-4-5-6-7) of the FeMo-cofactor (Fig. 1, A,and D). CO binding is accompanied by a dplacement of one of the belt sulfur atoms (S2although it retains the essentially tetrahedcoordination spheres for Fe2 and Fe6. As a resuthe two Fe arecoordinated by two sulfur andtwcarbon atomsa geometry, to our knowledge, npreviously observed in metalloclusters [althouhighercoordination number geometries ha

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1Howard Hughes Medical Institute and Division of Chemistryand Chemical Engineering, MailCode 114-96, CaliforniaInstitute of Technology, Pasadena, CA 91125, USA. 2 Institutfr Biochemie, Albert-Ludwigs-Universitt Freiburg, 79104Freiburg, Germany. 3BIOSS Centre for Biological SignallingStudies, Albert-Ludwigs-Universitt Freiburg, 79104 Freiburg,Germany. 4Department of Biochemistry, Molecular Biologyand Biophysics, University of Minnesota, Minneapolis, MN55455, USA.*Corresponding author. E-mail: [email protected] (T.S.);

[email protected] (D.C.R.)

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been observed in FeFe-hydrogenases (28)]. Con-firmation of the S2B displacement was provided

by anomalous difference Fourier maps calculatedwith diffraction data measured at 7100 eV (Fig.

1B); this energy is just below the Fe K-edge sothat the anomalous scattering from S is subs-tantially enhanced relative to Fe. The carbonatom of CO is located at a distance of 1.86

from each of the irons (Fe2 and Fe6), comparwith a previous distance of 2.2 for S2B (Fig. 1The altered ligand environment results in a smadjustment of the FeMo-cofactor geometry, wthe Fe2-Fe6 distance (2.5 ) slightly shortenrelative to the unchanged Fe4-Fe5 and Fe3-Fdistances (2.6 ) (Fig. 1C).

Given the complete displacement of S2B, wassessed whether the CO-inhibited protein cou

be reactivated or if it was irreversibly modifiCrystals of CO-inhibited MoFe-protein were tive when dissolved in an assay mixture in tabsence of CO. Furthermore, when we newassayed CO-inhibited MoFe-protein from toriginal inhibition preparations after remoof CO (see supplementary material for assdetails), a quantitative recovery (94 T 4%)the initial activity was obtained (Table 1). Treactivated MoFe-protein was subsequently isolated from activity assay mixtures and crtallized, which yielded a structure at 1.43resolution. The structural data of the prote(Av1 reactivated) revealed that S2B is regain

by replacing the previously bound CO liganwhich results in the recovery of the resting stFeMo-cofactor. The full occupancy of the sulfat the S2B site in the reactivated enzyme is edent by inspecting the 2Fobs Fcalcelectron desity map, as well as the anomalous electrdensity map verifying the anomalous scattericontribution expected for S2B (Fig. 2, A and B

The finding that S2B can be reversibly placed by CO raises the question of where thatom is located in the CO-inhibited state. If tS2B binding site is ordered, candidate locatioshould be evidentby an inspection of the 7100anomalous difference Fourier map. In this maner, one sitepercatalytic unitwas identified wanomalous density compatible with sulfur. Th

potential sulfur binding site (SBS) is position~22 away from the S2B position in the FeMcofactor and consists of a small protein pocketthe interface of the a andb subunits, formedthe side chains of residues a-Arg93, a-Thr10a-Thr111, a-Met112,b-Asn65, b-Trp428, b-Phe4andb-Arg453 (Fig. 3, A and B). The positive suface charge of the cavity is suited to accommdatean anionic species suchas HS or S2 (Fig. 3In previous structures of the resting state ezyme [Protein Data Bank (PDB) IDs: 1M1N a3U7Q], this site has been assigned as water; nothat the density at this site is also decreasedthe structure of reactivated Av1 (fig. S2). Tpotential SBS is connected to the FeMo-cofacto

binding pocket by a noncontinuous water chanel, and conformational rearrangements woube needed to accommodate the reversible migtion of S2B from and to the active site. Althou

we have observed a correlation between densat the potential SBS and the CO-inhibited stof the MoFe-protein, the identity of this sitethe displaced S2B cannot be achieved sole

based on crystallographic data. In assessing trelevance of this site, it should be noted ththe residues forming the pocket are poorly coserved, the site seems rather remote from tFeMo-cofactor, and because sulfur and chlori

SCIENCE sciencemag.org 26 SEPTEMBER 2014 VOL 345 ISSUE 6204 16

Fe2

Fe6

6

2.5

2.6

-Cys275

-His442

-Val70

-His195

Fe2/6

S3A

S5A

S4AS4B

S2A

S3B

R-homocitrate

S1A

S1B Fe4/5

Fe3/7

CO Fe2/6

S3A

S5A

S4AS4B

S2A

S3B

R-homocitrate

S1A

S1B Fe4/5

Fe3/7

CO

R-homocitrate

Fig. 1. CO-inhibited MoFe-protein (Av1-CO). Refined structure of the CO-bound FeMo-cofactor at aresolution of 1.50 . (A) View along the Fe1-C-Mo direction. The electron density (2Fobs Fcalc) map iscontoured at 4.0 s and represented as blue mesh. The density at the former S2B site is substantiallydecreased and in excellent agreement with bound CO [see also (C)]. ( B) Same orientation as (A)superimposed with the anomalous density map calculated at 7100 eV (green) at a resolution of 2.1 contoured at 4.0s, showing the absence of anomalous electron density at the CO site. (C) Side view ofFeMo-cofactor highlighting them2binding geometry of CO.The electron density (2Fobs Fcalc) map (bluemesh) surrounding CO-Fe2-Fe6-C is contoured at 1.5 s. (D) Same orientation as (C) highlighting theligand environment of the metal center. The catalytically important side-chain residues a-Val70 anda-His195 are in close proximity to the CO-binding site. Iron atoms are shown in orange, sulfur in yellow,molybdenum in turquoise, carbon in gray, nitrogen in blue, and oxygen in red.

Table 1. Nitrogenase activity. The acetylene reduction activity of as-isolatedAv1, Av1-CO, and Av1reactivated was measured by the quantification of ethylene production. Nitrogenase activity is quan-titatively recovered upon reactivation. Errors represent standard deviations of three measurements.

SampleSpecific reduction activity

Amount [nmol(acetylene) min1 mg(Av1)1] Percent (%)

Av1 as isolated 1930 T90 100T5Av1-CO < 2 T2 < 0.1 T0.1Av1 reactivated 1820 T80 94 T4

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have similar anomalous scattering propertiesat 7100 eV, it is possible that this is a generalanion-binding site in Av1.

The crystallographic characterization of CO-bound FeMo-cofactor of the MoFe-protein hasimportant implications for the mechanism ofsubstrate reduction by nitrogenase: The CO-

binding site is close to the side chains of resi-dues a-His195 (2.8 , NE2OC distance) and

a-Val70 (3.4 , closest methyl

OC distance).Modifications to both side chains were reportedto substantially alter the catalytic propertiesof the enzyme. Ana-His195 toa-Gln195 muta-tion resulted in the loss of N2 reduction activity,

while ana-Val70 toa-Ala/Gly70 alteration wasreported to enable the reduction of longer carbon-chain substrates and highlighted the poten-tial involvement of Fe6 in substrate reduction(2933). In the structure presented here, a-His195is in hydrogen bonding distance to the oxygenof CO, and a-Val70 directly flanks the bindingsite (Fig. 1D).

The displacement of S2B could be facilitatedby a proton donation from a-His195 to yieldeither HS or H2S, which would generate a betterleaving group than S2. Although the dissocia-tion of a sulfur may seem surprising, it opensup the ligand binding site, because the largeradius of S2 effectively shields the cofactor Featoms in the resting state from substrate and/orinhibitor attack (2). The more general implication

that binding of exogenous ligands can be accom-panied by the reversible dissociation of at leastone belt sulfur from the metal sites of the FeMo-cofactor changes the present view of the struc-tural inertness of the [7Fe:9S:C:Mo]-R-homocitratecluster toward ligand exchange. The relative lackof reactivity of the resting state is a strikingproperty of the FeMo-cofactor, and the require-ment for more highly reduced forms to bindsubstrate and inhibitors may reflect the needto dissociate sulfur ligands from Fe sites.

The displacement of the belt sulfur S2B bycarbon monoxide causes the FeMo-cofactor scaf-

fold to lose its intrinsic three-fold symmetAdditionally, Fe1, the interstitial carbon, amolybdenum are no longer aligned, creatian asymmetry in the resulting [7Fe:8S:C:Mcluster (Fig. 1C). The modest adjustments of tremaining scaffold upon CO binding are sugestive of an important role for the interstitcarbon in stabilizing the cofactor during arrangements and substitutions to the coornation environment of the irons (34, 35).

The experimental manipulations used generate the CO-inhibited structure are distinfrom those reported in previous spectroscopstudies; hence, it is not possible to unambuously assign the structure to one of the maannotated spectroscopic states. Note that maof the previously identified states undergo dnamic interchanges, including photoinductransitions between states (25). Like the struture presented here, the spectroscopically idetified lo-COstate has been proposed to invo

one molecule of CO bound to the active sitea bridging mode (22, 26). A state with two Cbound to Fe2 and Fe6 could correspond the high-COform (22,23) and might represean intermediate relevant to the CC couplireaction.

The generation and successful stabilizatiof CO-bound MoFe protein under turnover coditions has culminated in a crystal structure thprovides a detailed view of a ligand bound to tnitrogenase active site. The observations that Cis isoelectric to N2, is a potent yet reversible hibitor of substrate reduction without impedproton reduction to dihydrogen, and is boundclose proximity to previously determined ca

lytically important residues emphasize trelevance of the CO-bound structure toward uderstanding dinitrogen binding and reductioThis sheds light on N2activation based on a iron edge of theFeMo-cofactor and in thisrespresembles the Haber-Bosch catalyst that aluses an iron surface to break the NN tripleboThe demonstrated structural accessibility of C

bound MoFe-protein opens the door for coparable studies on a variety of inhibitors asubstrates, with the goal of understanding tdetailed molecular mechanism of dinitrogreduction by nitrogenase.

1622 26 SEPTEMBER 2014 VOL 345 ISSUE 6204 sciencemag.org SCIEN

Fig. 2. Reactivated MoFe-protein (Av1 reacti-

vated).Refined structure of the FeMo-cofactor ata resolution of 1.43 . (A) View along the Fe1-C-Modirection.The electron density (2Fobs Fcalc) mapis contoured at 4.0 sand represented as blue mesh.Electron density at the S2B site is in excellent agree-ment with a regained sulfur. (B) Same orientationas (A) superimposed with the anomalous densitymap (green) at a resolution of 2.15 contouredat 4.0s, showing the presence of anomalous den-sity at the S2B site. Color scheme is accordingto Fig. 1.

Fe2/6

S3A

S5A

SS4B

S2A

S3B

R-homocitrate

S1A

S1B Fe4/5

Fe3/7

S2BFe2/6

S3A

S5A

S4AS4B

S2A

S3B

R-homocitrate

S1A

S1B Fe4/5

Fe3/7

S2B

A B

-subunit

-subunit

FeMo-co

P-cluster

PotentialSBS

-Met112

-Thr104

-Arg93

-Thr111

-Trp428

-Asn65

-Phe450

-Arg453

PotentialSBS

Fig. 3. Overview of the potential sulfur binding site (SBS) in the CO-inhibited MoFe-protein

(Av1-CO).(A) Location of the potentially bound sulfur in a protein cavity on the interface between the aandb subunits of thea2b2 MoFe-protein.Thepotential SBS is located 22 away from itsformer positioninthe FeMo-cofactor (S2B-site). (B) Close-up view of the binding cavity. Positive surface charge is rep-

resented in blue, negative surface charge in red. The anomalous density map at a resolution of 2.1 isrepresented as green mesh and contoured at 4.0 s, showing the presence of anomalous density at thepotential SBS.The side-chain sulfur of a-Met112 provides an internal standard for full occupancy. Alternateconformations forb-Arg453 are indicated.The color scheme is according to Fig. 1.

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REFERENCES AND NOTES

1. B. K. Burgess, D. J. Lowe, Chem. Rev. 96, 29833012 (1996).

2. J. B. Howard, D. C. Rees,Proc. Natl. Acad. Sci. U.S.A. 103,

1708817093 (2006).

3. L. C. Seefeldt, B. M. Hoffman, D. R. Dean,Annu. Rev. Biochem.

78, 701722 (2009).

4. M. M. Georgiadiset al., Science 257, 16531659 (1992).

5. J. Kim, D. C. Rees,Nature360, 553560 (1992).

6. H. Schindelin, C. Kisker, J. L. Schlessman, J. B. Howard,

D. C. Rees, Nature387, 370376 (1997).

7. J. W. Peters, K. Fisher, W. E. Newton, D. R. Dean,J. Biol. Chem.

270, 2700727013 (1995).

8. D. J. Lowe, K. Fisher, R. N. F. Thorneley,Biochem. J. 292,

9398 (1993).

9. O. Einsleet al., Science 297, 16961700 (2002).

10. T. Spatzal et al., Science 334, 940 (2011).

11. K. M. Lancasteret al., Science334, 974977 (2011).

12. Y. Hu, M. W. Ribbe, Coord. Chem. Rev. 255, 12181224 (2011).

13. D. C. Rees et al., Philos. Trans. A Math. Phys. Eng. Sci. 363,

971984 (2005).

14. D. J. Lowe, R. N. F. Thorneley,Biochem. J. 224, 895901 (1984).

15. R. N. F. Thorneley, D. J. Lowe,Biochem. J. 215, 393403 (1983).

16. J. Chatt, J. R. Dilworth, R. L. Richards,Chem. Rev.78, 589625

(1978).

17. J. C. Hwang, C. H. Chen, R. H. Burris,Biochim. Biophys. Acta

292, 256270 (1973).

18. L. M. Cameron, B. J. Hales,Biochemistry37, 94499456 (1998).

19. L. C. Davis, M. T. Henzl, R. H. Burris, W. H. Orme-Johnson,

Biochemistry18 , 48604869 (1979).

20. C. C. Lee, Y. Hu, M. W. Ribbe, Science329, 642642 (2010).

21. Y. Hu, C. C. Lee, M. W. Ribbe,Science333, 753

755 (2011).22. H.-I. Lee, L. M. Cameron, B. J. Hales, B. J. Hoffman, J. Am.

Chem. Soc. 119, 1012110126 (1997).

23. L. Yan et al., Eur. J. Inorg. Chem.2011, 20642074 (2011).24. S. J. George, G. A. Ashby, C. W. Wharton, R. N. F. Thorneley,

J. Am. Chem. Soc.119, 64506451 (1997).25. Z. Maskos, B. J. Hales, J. Inorg. Biochem. 93, 1117 (2003).26. R. C. Pollock et al., J. Am. Chem. Soc. 117, 86868687 (1995).27. L. C. Davis, M. T. Henzl, R. H. Burris, W. H. Orme-Johnson,

Biochemistry18 , 48604869 (1979).28. D. W. Mulder et al., Structure19, 10381052 (2011).29. P. M. C. Benton et al., Biochemistry42 , 91029109 (2003).30. C. H. Kim, W. E. Newton, D. R. Dean, Biochemistry34,

27982808 (1995).31. P. C. Dos Santos, S. M. Mayer, B. M. Barney, L. C. Seefeldt,

D. R. Dean, J. Inorg. Biochem. 101 , 16421648 (2007).32. D. J. Scott, H. D. May, W. E. Newton, K. E. Brigle, D. R. Dean,

Nature343, 188190 (1990).33. J. Christiansen, V. L. Cash, L. C. Seefeldt, D. R. Dean, J. Biol.

Chem. 275 , 1145911464 (2000).34. J. Rittle, J. C. Peters, Proc. Natl. Acad. Sci. U.S.A. 110,

1589815903 (2013).35. I. Dance, Chem. Asian J. 2, 936946 (2007).

ACKNOWLEDGMENTS

We thank J. Peters, L. Zhang, J. Rittle, C. Morrison, B. Wenke, and

K. Drner for informative discussions. This work was supported by

NIH grant GM45162 (D.C.R.), Deutsche Forschungsgemeinschaft

grants EI-520/7 and RTG 1976, and the European Research Council

N-ABLE project (O.E.). We gratefully acknowledge the Gordon

and Betty Moore Foundation, the Beckman Institute, and the

Sanofi-Aventis Bioengineering Research Program at Caltech for

their generous support of the Molecular Observatory at Caltech

and the staff at Beamline 122, Stanford Synchrotron Radiation

Lightsource (SSRL), for their assistance with data collection.

SSRL is operated for the U.S. Department of Energy and supported

by its Office of Biological and Environmental Research and by

the NIH: National Institute of General Medical Sciences (P41GM103393)

and the National Center for Research Resources (P41RR001209).We thank the Center for Environmental Microbial Interactions for its

support of microbiology research at Caltech. Coordinates and

structure factors have been deposited in the Protein Data Bank of

the Research Collaboratory for Structural Bioinformatics, with IDs

4TKV (Av1-CO) and 4TKU (Av1 reactivated).

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/345/6204/1620/suppl/DC1

Materials and Methods

Figs. S1 and S2

Tables S1 and S2

References (3643)

29 May 2014; accepted 31 July 2014

10.1126/science.1256679

IMMUNODEFICIENCY

Immune dysregulation in humansubjects with heterozygous germlinemutations inCTLA4Hye Sun Kuehn,1* Weiming Ouyang,2* Bernice Lo,3,4* Elissa K. Deenick,5,6 Julie E. NiemelDanielle T. Avery,5 Jean-Nicolas Schickel,7Dat Q. Tran,8 Jennifer Stoddard,1 Yu Zhang,4,9

David M. Frucht,2 Bogdan Dumitriu,10 Phillip Scheinberg,10 Les R. Folio,11 Cathleen A. Frein

Susan Price,3,4 Christopher Koh,13 Theo Heller,13 Christine M. Seroogy,14Anna Huttenlocher,14,

V. Koneti Rao,3,4Helen C. Su,4,9 David Kleiner,16 Luigi D. Notarangelo,17Yajesh Rampertaap

Kenneth N. Olivier,18 Joshua McElwee,19 Jason Hughes,19 Stefania Pittaluga,16 Joao B. Oliveira

Eric Meffre,7 Thomas A. Fleisher,1Steven M. Holland,4,18 Michael J. Lenardo,3,4

Stuart G. Tangye,5,6 Gulbu Uzel18

Cytotoxic T lymphocyte antigen4 (CTLA-4) is an inhibitory receptor found on immune

cells. The consequences of mutations in CTLA4 in humans are unknown. We identified

germline heterozygous mutations in CTLA4 in subjects with severe immune

dysregulation from four unrelated families. Whereas Ctla4 heterozygous mice have

no obvious phenotype, human CTLA4 haploinsufficiency caused dysregulation of FoxP3

regulatory T (Treg) cells, hyperactivation of effector T cells, and lymphocytic infiltrationof target organs. Patients also exhibited progressive loss of circulating B cells, associat

with an increase of predominantly autoreactive CD21lo B cells and accumulation of B ce

in nonlymphoid organs. Inherited human CTLA4haploinsufficiency demonstrates a critic

quantitative role for CTLA-4 in governing T and B lymphocyte homeostasis.

Immune tolerance is controlled by multiplemechanisms (1,2), including regulatoryT (Treg)cells (35) and inhibitory receptors (6, 7). Tregcells constitutively express the inhibitory re-ceptor CTLA-4, which confers suppressive

functions (8, 9). CTLA-4, also known as CD152,is also expressed by activated T cells and, uponligation, inhibits their proliferation (10). Homo-

zygous deficiency of Ctla4 in mice causes fatalmultiorgan lymphocytic infiltration and destruc-tion (1113); hence, CTLA-4 functions at a keycheckpoint in immune tolerance. CTLA-4immunoglobulin(Ig)fusionproteinandneutralizingCTLA-4 antibody are used to modulate immunityinautoimmune and cancer patients (14, 15), respec-tively. Studies have given conflicting results regard-ing the association of CTLA4 single-nucleotide

variants (SNVs) with organ-specific autoimmunity(16). The consequences of genetic CTLA-4 defi-ciency in humans are unknown.

Ourindexpatienta 22-year-old female (A.II.1)developed brain, gastrointestinal (GI), and lunglymphocytic infiltrates, autoimmune thrombo-

cytopenia, and hypogammaglobulinemia in earlychildhood (Fig. 1A and table S1). Her 43-year-oldfather (A.I.1) manifested lung and GI infiltrates,hypogammaglobulinemia, and clonally expandedgd-CD8+ T cells infiltrating and suppressing the

bone marrow (fig. S1A). Four additional casesfrom three unrelated families (families B, C,and D) (fig. S1 and table S1) were identifiedamong a cohort of 23 patients with autoimmunecytopenias, hypogammaglobulinemia, CD4 T celllymphopenia, and lymphocytic infiltration of non-lymphoid organs. Patient B.I.1, previously diag-nosed with common variable immunodeficiency

SCIENCE sciencemag.org 26 SEPTEMBER 2014 VOL 345 ISSUE 6204 16

1Department of Laboratory Medicine, Clinical Center, NatioInstitutes of Health, Bethesda, MD 20892, USA. 2Laboratoof Cell Biology, Division of Monoclonal Antibodies, Office oBiotechnology Products, Center for Drug Evaluation andResearch, U.S. Food and Drug Administration, Bethesda,MD 20892, USA. 3Molecular Development of the ImmuneSystem Section, Laboratory of Immunology, NationalInstitute of Allergy and Infectious Diseases, Bethesda, MD20892, USA. 4NIAID Clinical Genomics Program, NationalInstitute of Allergy and Infectious Diseases, Bethesda, MD20892, USA. 5 Immunology and Immunodeficiency Group,Immunology Division, Garvan Institute of Medical ResearchSydney, NSW 2010, Australia. 6St. Vincents Clinical SchooFaculty of Medicine, University of New South Wales, SydneNSW 2010, Australia. 7Department of Immunobiology, YaleUniversity School of Medicine, New Haven, CT 06511, USA8Department of Pediatrics, University of Texas MedicalSchool, Houston, TX 77030, USA. 9Immunological DiseaseUnit, Laboratory of Host Defenses, National Institute ofAllergy and Infectious Diseases, Bethesda, MD 20892, USA10Hematology Branch, National Heart, Lung and BloodInstitute, Bethesda, MD 20892, USA. 11Radiology andImaging and Sciences, Clinical Center, National Institutes Health, Bethesda, MD 20892, USA. 12Clinical ResearchDirectorate, Clinical Monitoring Research Program, LeidosBiomedical Research Inc., Frederick National Laboratoryfor Cancer Research, Frederick, MD 21702, USA. 13LiverDiseases Branch, National Institute of Diabetes and

Digestive and Kidney Diseases, Bethesda, MD 20892, USA14Department of Pediatrics, University of Wisconsin, MadisWI 53706, USA. 15Department of Medical Microbiology andImmunology, University of Wisconsin, Madison, WI 53706,USA. 16Laboratory of Pathology, National Cancer Institute,Bethesda, MD 20892, USA. 17Division of Immunology andManton Center for Orphan Disease Research, ChildrensHospital, Harvard Medical School, Boston, MA 10217, USA18Laboratory of Clinical Infectious Diseases, National Institof Allergy and Infectious Diseases, Bethesda, MD 20892,USA. 19Merck Research Laboratories, Merck & Co., BostonMA 02130, USA. 20Instituto de Medicina Integral Prof.Fernando FigueiraIMIP, 50070 Recife-PE, Brazil.*These authors contributed equally to this work.

Corresponding author. E-mail: [email protected] (T.A.F.);

[email protected] (M.J.L.); [email protected] (G.U.)

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