Structural Biology of the T-cell Receptor: Insights into ...cshperspectives.cshlp.org/content/2/4/a005140.full.pdfInsights into Receptor Assembly, Ligand Recognition, and Initiation

Structural Biology of the T-cell Receptor:Insights into Receptor Assembly, LigandRecognition, and Initiation of Signaling

Kai W. Wucherpfennig1,2,3, Etienne Gagnon1, Melissa J. Call1, Eric S. Huseby4, andMatthew E. Call5

1Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts 021152Department of Neurology, Harvard Medical School, Boston, Massachusetts 021153Program in Immunology, Harvard Medical School, Boston, Massachusetts 021154Department of Pathology, University of Massachusetts Medical School, Worchester, Massachusetts 016555Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston,Massachusetts 02115

Correspondence: [email protected]

The T-cell receptor (TCR)-CD3 complex serves as a central paradigm for general principlesof receptor assembly, ligand recognition, and signaling in the immune system. There is noother receptor system that matches the diversity of both receptor and ligand components.The recent expansion of the immunological structural database is beginning to identifykey principles of MHC and peptide recognition. The multicomponent assembly of theTCR complex illustrates general principles used by many receptors in the immune system,which rely on basic and acidic transmembrane residues to guide assembly. The intrinsicbinding of the cytoplasmic domains of the CD31 and z chains to the inner leaflet of theplasma membrane represents a novel mechanism for control of receptor activation:Insertion of critical CD31 tyrosines into the hydrophobic membrane core prevents theirphosphorylation before receptor engagement.

LIGAND BINDING BY THEEXTRACELLULAR DOMAINS OF T-CELLRECEPTORS: STRUCTURAL BASIS OF ab

AND gd TCR BINDING TO A DIVERSEGROUP OF LIGANDS

ThestructuresofmanyT-cellreceptors(TCRs)in their ligand-bound state have now been

determined, allowing some of the general

rules of antigen recognition to be distilled.Though the best studied interaction is the bind-ing of abTCR with peptide-MHC complexes,it is highly instructive to compare the proto-typical binding to peptide-MHC with therecognition of lipid antigen-CD1 complexesand engagement of nonclassical MHC class Imolecules by gdTCRs. These comparisonsshow that the recognition strategies for these

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three classes of TCR ligands are strikingly differ-ent (Fig. 1).

Many structures of human and mouse com-plexes have shown a typical binding mode,which maximizes abTCR interaction with theMHC-bound peptide (Garboczi et al. 1996; Gar-cia et al. 1996; Rudolph et al. 2006). This bindingmode places the hypervariable loops of the TCR,the CDR3a and CDR3b loops, over the center ofthe bound peptide (Fig. 1A,D). Two other TCRloops, CDR1a and CDR1b, frequently also con-tribute to peptide recognition by binding overamino-terminal and carboxy-terminal peptide

segments, respectively. Most contacts to theMHC molecule are typically made by thegermline-encoded CDR1 and CDR2 loops ofboth TCRa and b, but the CDR3 loops canalso contribute to MHC binding. Thus,six TCR loops can be involved in peptide andMHC recognition and usually provide substan-tial specificity for both components (Fig. 1A,D).

abTCRs not only recognize MHC-boundpeptides, but also CD1-bound lipid antigens.CD1 molecules have a similar overall fold asMHC class I molecules, but their groove issubstantially wider and more hydrophobic to

A B C

D Eβ2

α1α3

β2 α3

β3

α2

α1

β1

β3

α2

β3 β1

α3

α1

α2

HLA-A2, Tax P6A, A6 CD1d, α-gal-ser, NKT15 T22, γδ TCR G8

F

Figure 1. TCR structures with different classes of ligands show distinct binding solutions. (A–C). The dockingtopologies are compared for the abTCR A6 bound to a peptide-MHC class I complex (HLA-A2 with Tax P6Apeptide; PDB entry 1QRN) (A), the abTCR NKT15 bound to the complex of CD1d and the glycolipida-galactosylceramide (PDB entry 2PO6) (B), and the gdTCR G8 bound to the nonclassical MHC class Ibmolecule T22 (PDB entry 1YPZ) (C). (D–F) The placement of the TCR loops is compared for the samecomplexes. (D) The A6 TCR uses all six loops to contact the MHC molecule and the bound peptide. Thefour germline-encoded loops (a1, a2, b1, and b2) contribute to MHC binding, while the two CDR3 loopsare positioned over the peptide (a3 and b3). (E) The NKT15 TCR contacts the CD1d-bound glycolipid onlythrough germline elements encoded in the invariant TCRa chain (a1 and a3 loops). (F) The gd TCR G8inserts the CDR3d chain loop into the hydrophobic groove of T22; other TCR loops do not appear to beessential for T22 binding.

K.W. Wucherpfennig et al.

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accommodate a diverse group of lipids, glyco-lipids, and lipopeptides (Barral and Brenner2007). NK T cells recognize lipid antigens boundto CD1d, with a-galactosylceramide beingthe best characterized lipid antigen (Bendelacet al. 2007). In the structure of a human NKTCR, the TCR binds over the extreme end ofthe CD1d binding cleft (Borg et al. 2007). Fur-thermore, the TCR is positioned approximatelyparallel to the long axis of the CD1d bindinggroove, in contrast to the typical diagonal foot-print for most abTCRs on peptide-MHC(Fig. 1 B,E). NK T cells use a limited TCR reper-toire for CD1d-lipid binding, in particular aninvariant TCRa chain and a specific Ja segment(Va24-Ja18) (Bendelac et al. 2007). In the struc-ture only the glycosyl head group of the lipidantigen is exposed and it is contacted solelyby germline-encoded loops, CDR1a (encodedby Va24) and CDR3a (encoded by Ja18)(Fig. 1E). Only germline-encoded segmentsthus confer specificity for the glycolipid antigenby this NK TCR (Borg et al. 2007). This is instriking contrast to the central role of the hyper-variable CDR3a and b loops in discriminat-ing between MHC-bound peptides.

The ligands for most gd T cells remain un-known, but in mice the closely related nonclas-sical MHC class Ib molecules T10 and T22 serveas ligands for a sizable population of gd T cellsin nonimmunized mice (Shin et al. 2005). Thestructure of the G8 gdTCR bound to T22 showsa binding mode that is entirely different fromabTCR engagement of peptide-MHC (Adamset al. 2005). The G8 TCR binds T22 sideways at ahighly tilted angle (Fig. 1C), contrasting withthe essentially parallel alignment of the longaxes of the abTCR and peptide-MHC. T22does not have a bound peptide and the segmentthat corresponds to the amino-terminal part ofthe a2 helix of classical MHC class I moleculesis actually unwound, exposing the hydrophobicgroove. The CDR3 loop of TCRd inserts intothis hydrophobic groove and contributes a sub-stantial fraction of the buried surface (Fig. 1F).Interestingly, transfer of this CDR3d loop intoan ab TCR resulted in T22 binding (Adamset al. 2008). Thus, a single loop of this gdTCRappears to be sufficient for T22 binding.

The overall binding mode thus differs greatlyfor all three classes of ligands: abTCRs typicallybind in a diagonal orientation over most of theMHC-bound peptide, whereas the NK TCR sitsover only part of the CD1d binding groove par-allel to the long axis of the groove. The mostextreme case is the gdTCR that binds sidewaysto T22 and inserts one loop into the exposedT22 groove.

Structural Insights Into the Mechanism ofMHC Restriction

There has been much debate on the fascinatingproblem of why abTCRs are “MHC restricted,”and the structural database is now large enoughto extract key principles. The overall bindingmode of most crystallized abTCRs with pep-tide-MHC is similar, even though there is sub-stantial variation in the binding angle relativeto the long axis of the MHC molecule (Rudolphet al. 2006; Garcia et al. 2009). Importantly,the general location of the TCR chains on thepeptide-MHC ligand is similar in all studiedstructures: The TCRb chain is positioned overthe a1 helix of the MHC molecule and theTCRa chain over the other MHC helix (a2 inMHC class I, which corresponds to b1 in MHCclass II). TCRs have a similar overall fold to anti-body Fab segments, but if there were no inherentrules to TCR binding to peptide-MHC, theopposite placement (TCRa rather than TCRbover the MHC a1 helix) should have been seenby now.

Genes of the MHC are extremely polymor-phic. In man, there are .800 MHC class I allelesand .600 alleles of MHC class II (Robinsonet al. 2009). If TCRs are prebiased to interactwith MHC proteins, essential genetic elementshave to be shared between all MHC proteins.It has been appreciated for some time that theoverall structure of MHC proteins and theirbound peptide is quite similar (Stern and Wiley1994). Likely because of selective pressure bypathogens, the vast majority of allelic variationbetween MHC proteins is located within thepeptide binding groove (Bjorkman et al. 1987).Conventional alignment of the a1 domain ofMHC class I and II molecules failed to show

Structural Biology of the T-cell Receptor

Cite this article as Cold Spring Harb Perspect Biol 2010;2:a005140 3



substantial homology in solvent-exposed resi-dues that could be contacted by the TCR.However, if the alignment is shifted by a singleturn of an a-helix substantial homology is evi-dent among solvent exposed residues availablefor interaction with TCRs (Huseby et al. 2005;Huseby et al. 2008). For example, when thesurface exposed side chains of H2-Kb and I-Ab

are compared, there is substantial conservationbetween these MHC class I and class II proteins(Table 1). Interestingly, these conserved aminoacids are located within TCR binding footprints(Fig. 2). Thus, conserved/chemically similaramino acids on the helices of all MHC proteinsmay be the elements that the TCR V-domainsare genetically encoded to bind.

The diagonal binding orientation on thepeptide-MHC surface is similar among mostcrystallized ab TCRs, but because of variationin this binding angle, it has not been possibleto identify MHC residues contacted by allTCRs (Baker et al. 2001; Rudolph et al. 2006).Rather, the growing structural database suggests

an alternative hypothesis: Individual V-genes orgroups of related V-genes have coevolved withMHC, which has resulted in preferred contactsby these V-domains with MHC (Garcia et al.2009). This hypothesis is supported by a sub-stantialnumberofcrystalstructures involvingsixdifferent Vb8.2 TCRs and one Vb8.1 TCR incomplex with mouse I-A molecules. These struc-tures show close convergence of the CDR1b andCDR2b contacts with the I-A a1 chain helix(Reinherz et al. 1999; Feng et al. 2007; Dai et al.2008; Garcia et al. 2009). This Vb8 interactionmotif has been seen in structures with differentI-A alleles, Va segments, and peptides, and mut-ation of these TCRb residues reduced or abol-ished activation by a panel of T cells. However,at least some V-gene segments can use morethan one set of interactions to bind a givenMHC molecule. Forexample, two Vb2-contain-ing TCRs with different peptide specificitiesshow distinct docking footprints on the H-2Kb

a1 helix (Reiser et al. 2000; Reiser et al. 2003).The hypervariable CDR3 loops of the TCR and

Table 1. Alignment of solvent accessible murine MHC class I and class II residues

Alpha 1 domain Beta 1 domain

Alpha 2 domainAlpha 1 domain

55 57 61 64 68 72 75 81 77 73 70 69 66 64IA b, u IA b, dIA d, g7, k IA g7, f, k, q Y Q

IE b, k, s, uIE d, k IE d

H T A R E E QH T A R E 67-

H T A Q E E QH T A D E E Q

IE u

D Q Q A H V KE Q Q A H I K

IA s D Q Q A Y I K IA u Y T A R E 67-Y QIA f D Q Q A H I K IA s H T A Q E 67-Y NIA q D Q Q A H G K

E Q A A A V EE Q A A A V K

MHC class I

MHC class II

58 61 62 65 69 72 76 79 166 163 158 155 154 152 150 149Kb Kb E T A R E E A QKd Kd E E A Y E D A QKk Kk E T A R E D A QDb Db E E A H E A S QDk Dk E T A R E A A QLd, q

E E R Q G Q V RE E E Q S Q V RE E R Q G Q V RE E R Q G Q V RE E R Q G Q V RE E R Q G Q V R Ld, q E E A Y E A A Q

Helical residues of murine MHC class I and class II proteins were aligned based on a previously reported approach that takes

a shift in the MHC class I and class II a1 helices into account (Huseby et al. 2005). Identical residues are highlighted in yellow,

structurally similar residues in orange. In some MHC class II molecules, there is a deletion corresponding to one amino acid

and residues 66/67 located in the same position of the b1 domain are indicated.





the MHC-bound peptide are probably involvedin determining which binding interactions withthe MHC helices are possible and energeticallymost favorable.

The identification of the conserved Vb8interaction motif with I-A proteins providedan opportunity to examine if these germline-encoded TCR amino acids are required forT-cell development. Particularly prominent inthis interaction motif are the contributions ofthe CDR2b loop residues Y46, Y48, and E54.In mice expressing single, rearranged TCRbchains, individual mutation of these CDR2bresidues to alanine substantially reduced devel-opment of the entire TCR repertoire (Scott-Browne et al. 2009). The phenotype was par-ticularly strong for mutation of the two tyro-sines (Y46 and Y48) in the CDR2b loop. Oneof these tyrosines (Y46) is conserved in anothermouse Vb segment (Vb6) and thymic CD4T cells were again significantly reduced whenY46 was mutated to alanine. Interestingly, thelowered Vb affinity appeared to be compen-sated by selection of TCRa chains with higheraffinity for MHC. These results show that

thymic selection is controlled by germline-encoded MHC contact points.

This conclusion is supported by the find-ing that a substantial fraction of T cells frommice with limited negative selection are highlyMHC and peptide cross-reactive (Huseby et al.2005). Interestingly, some of these TCRs are pos-itively selected on both MHC class I and class II,which shows that some T cells can recognizeconserved features of the MHC class I and IIhelices. Structural comparison of highly specificand highly cross-reactive TCRs from these miceshowed that the highly cross-reactive TCR had amuch smaller concentrated interaction surfacethat was more hydrophobic (Dai et al. 2008).The interaction with the MHC became highlyfocused on a limited number of TCR aminoacids, which included the CDR2b residuesY46, Y48, and E54 discussed above. The germ-line-coded TCR contacts with MHC can thusyield T cells with a high degree of MHC and pep-tide cross-reactivity, which are normally elimi-nated by negative selection (Huseby et al. 2008).

An alternative hypothesis to explain thediagonal binding mode of the TCR with

BBM3.3 binding H2-KbA B3K506 binding IAb

.

Figure 2. Conserved helical residues of MHC class I and class II proteins are often contacted by CDR1 and CDR2residues of TCRs. Conserved MHC helical residues are highlighted in green for structures of H-2Kb (A) and I-Ab

(B). The BM3.3 TCR (H-2Kb; PDB entry 1NAM) and the B3K506 TCR (I-Ab; PDB entry 3C5Z) contact theseconserved helical MHC residues. TCR CDR1b and CDR2b are colored red, CDR3a and CDR3b orange, andCDR1a and CDR2a blue. Peptide residues are colored yellow. Colored cyan are the H2-Kb a1 and IAb a1domains, and magenta H2-Kb a2 and IAb b1 domains.





peptide-MHC proposes that this geometry isdriven by the requirement to accommodate thecoreceptors CD4/CD8 or by a requirement forformation of TCR dimers/oligomers before ini-tiation of signaling (Ding et al. 1998). No struc-tural data are yet available to test the validity ofthese ideas. It is important to keep in mind thatthese hypotheses are not mutually exclusive. Forexample, the requirement for formation of ahigher-order structure could have defined thestructural boundaries for coevolution of TCRwith MHC.

A common theme emerging from all of thesestructural studies is that particular germlineelements are used in different ways for ab orgdTCR recognition of peptide-MHC, lipid-CD1 complexes, or nonclassical MHC class Imolecules. In the case of the G8 gdTCR, a singleTCR loop appears to be sufficient for binding tothe nonclassical MHC class Ib molecule T22(Adams et al. 2005). In contrast, abTCRs typi-cally use four germline-encoded loops for bind-ing to the MHC helices, which enables thehypervariable CDR3 loops to probe the con-tents of the peptide binding groove (Rudolphet al. 2006). The recognition mode is yet againdifferent for ab TCRs binding to CD1d-lipidcomplexes (Borg et al. 2007). In this specializedcase, the germline encoded Va and Ja segmentsof the invariant TCRa chain are used to bind toa CD1 embedded glycolipid antigen.

ASSEMBLY OF T-CELL RECEPTOR-CD3COMPLEXES

How is an encounter with appropriate peptide-MHC or lipid-CD1 complexes actually “read”into a T cell? The mature TCR proteins carryno signaling motifs, but rather transmit signalsvia tyrosine-based ITAM motifs in the cytoplas-mic portions of the TCR-associated CD3g1,CD3d1, and zz modules (Germain and Stefa-nova 1999; Samelson 2002). The precise mecha-nisms linking MHC binding outside the cell toearly biochemical events inside the cell remainan area of vigorous investigation and signifi-cant controversy. Proposed triggering modelsrange from receptor clustering and corecep-tor recruitment to an array of conformational

change models in which TCR proteins are pro-posed to physically impinge on the CD3 mod-ules to transmit signals (Kuhns et al. 2006;Choudhuri and van der Merwe 2007). Our abil-ity to distinguish among these different modelsdepends critically on a solid understanding ofthe spatial organization of the complete TCR-CD3 complex. However, no high-resolutionstructure of an intact complex is yet available.As such, we must rely on three types of avail-able experimental data to inform our workingmodels: (1) measurements of the stoichiomet-ric relationships among subunits, (2) availableatomic-resolution structures of folded do-mains, and (3) biochemical data identifyingthe molecular surfaces involved in intersubunitcontacts.

TCR-CD3 Stoichiometry

TheabTCRisnoncovalentlyassociatedwiththreedimeric signaling modules: CD3d1, CD3g1, andzz (Call and Wucherpfennig 2005). On ligandbinding by the TCR, cytoplasmic ITAM motifsin the CD3 and z chains become phosphorylatedby the Src-family kinase Lck (Weiss and Littman1994), constituting the earliest detectable bio-chemical consequence of TCR ligation. Earlygenetic, biochemical, and immunofluorescence-based experiments established that the intactTCR-CD3 complex contains two copies ofCD31 (Blumberg et al. 1990b; de la Hera et al.1991), consistent with a model in which bothCD3d1 and CD3g1 are incorporated into eachabTCR complex. Similar approaches have failedto detect more than one TCRaor TCRb chain inintact TCR-CD3 complexes (Punt et al. 1994;Call et al. 2002), indicating that the receptor ismonovalent with respect to the ligand-bindingmodule. This model is supported by direct bio-chemical measurements of the TCR-CD3 stoich-iometry inER-assembledcomplexes, inwhichthecomposition was found to be TCRab:CD3d1:CD3g1:zz (Call et al. 2004).

The composition of thegdTCR andpre-TCRcomplexes differ from abTCR. Mice deficientin CD3g, CD31, or z subunits show a near-complete arrest of thymocyte development atthe CD42CD82 (DN) stage, the point at which





pre-TCR signaling is required for further matu-ration (reviewed in Dave 2009). However, inCD3d-deficient mice, thymocyte developmentproceeded to the CD4þCD8þ (DP) stage, sug-gesting that pre-TCR function was intact, andthe gd T-cell compartment was unperturbed(Dave et al. 1997). Thus, whereas CD3g, CD31,and z are required for all developmental stagesand T-cell lineages, CD3d may be dispensablefor gd and pre-TCR function in mice. A recentreport has indeed shown that surface-expressedTCR-CD3 complexes on CD3d-sufficient mu-rine gd T cells do not contain CD3d1 modules,and quantitative flow cytometry measurementsyielded a stoichiometry of TCRgd:CD3g1(2):zz(Hayes and Love 2006). We note that this appearsto be a difference between mouse and humangdTCR, which does incorporate CD3d1 (Siegerset al. 2007). Both ab and gdTCR thus requiretwo CD3 modules, and based on our currentunderstanding of TCR-CD3 assembly mecha-nisms (see the following discussion), the sameis likely to be true for pre-TCR.

Mechanisms Directing TCR-CD3 ComplexAssembly

Like the TCR, CD3d1 and CD3g1 heterodimersare formed through interactions between theirextracellular Ig domains. The subunit interfaceis formed primarily through an extended con-tact along two b strands that terminate in thestalk regions connecting the Ig domains to theTM domains (Sun et al. 2001; Arnett et al.2004; Kjer-Nielsen et al. 2004; Sun et al. 2004).In contrast, the disulfide-linked zz homodimer,having only a very small (nine amino acid) ecto-domain, forms through contacts predomi-nantly within the TM domain (Rutledge et al.1992; Call et al. 2002; Call et al. 2006). A majorremaining challenge has been to identify themolecular surfaces responsible for stabilizingcontacts among these four dimeric modules.

Soluble ectodomain fragments of TCR andCD3 dimers show no measurable affinity forone another, and we now understand that thedeterminants for assembly are contained pri-marily within the TM and juxtamembraneregions. The TCR proteins have three basic

amino acids within the TM domains: Two inTCRa and one in TCRb. Similarly, CD3 and z

proteins each contain a single aspartic or gluta-mic acid in their TM domains, creating a pairof acidic residues in each dimeric module. Cellu-lar transfection studies established that at leastsome of these ionizable residues participatein assembly (Alcover et al. 1990; Blumberget al. 1990a; Cosson et al. 1991; Manolios et al.1991). A more comprehensive mutagenesisanalysis using in vitro-assembled complexesrevealed that each of the basic TM residues inthe TCR specifically recruits one of the threesignaling modules in contacts requiring bothacidic TM residues (Call et al. 2002; Call et al.2004). The picture that emerges from thesestudies is that of an ordered assembly processorganized around three trimeric intramembraneinteractions: CD3d1 associates with TCRa, andCD3g1 with TCRb through the centrally placedlysine residues, and then the zz module associ-ates with TCRa through an arginine residuein the upper third of the TM domain (Fig. 3A).Alanine substitution at any one of these ninepositions prevents formation of a completeTCR-CD3 complex.

The identity and placement of the basic TMresidues are conserved among abTCR, gdTCR,and pre-TCR sequences, implying that theassembly mechanism described for abTCRapplies to all three complexes. This wouldaccount for the presence of two CD3 modulesin gdTCR (and possibly pre-TCR) even whenCD3d is not available. Selectivity for specificTCR chains in the assembly process is likelydetermined by sequences in the extracellularportions of the CD3 proteins, because achimericCD3g bearing the CD3d TM domain can beincorporated into complete abTCR-CD3 com-plexes and rescues surface TCR expression inCD3g-deficient T cells (Wegener et al. 1995;Call et al. 2002). Other lines of evidence alsopoint to important interactions among ectodo-mains. Mutations in the TCRa connecting pep-tide have adverse effects on assembly andsignaling (Werlen et al. 2000), and a specific“CxxC” motif in the stalk regions of the CD3 pro-teins is required for association with TCR (Xuet al. 2006). The nine amino acid extracellular





CD3δε

D6

L9

Y12

T17

TCRαβ

CD3γε

ξξ

A

B

Figure 3. Assembly of the TCR-CD3 complex. (A) The TCR-CD3 complex is composed of four dimeric modules:TCRab (or gd), CD3d1, CD3g1, and zz, which associate through intramembrane contacts to form the intactcomplex. Each dimeric signaling module associates with the TCR through a pair of acidic TM residues that binda specific basic TM residue in the TCR. The result is an octameric complex with the stoichiometry as shown.Ribbon structures were generated from PDB entries 1MI5 (LC13 abTCR), 1XMW (CD3d1), and 1SY6(CD3g1). (B) NMR structure of the zz TM homodimer in detergent micelles (PBD entry 2HAC). Theaspartic acid pair (D6-D6) that mediates assembly with TCR is located at the helix dimer interface andstabilized by interhelical H-bonds between side-chain oxygens and backbone amide protons (indicated bydotted lines). Further interface contacts include methyl and aromatic packing (such as L9-L9) and twotyrosine-threonine H-bonds (between Y12 and T17) that are critical for dimer formation.





stalks of the zz dimer also appear to play a role inassociation with TCR, because mutations inthis region adversely affect assembly (Xu andWucherpfennig, unpubl.). Likewise, mutationsin loop regions in both TCRa and TCRb con-stant domains have been implicated in receptorcomplex stability as well as signaling functions(Kuhns and Davis 2007; Beddoe et al. 2009).

Conservation of Membrane-based ReceptorComplex Assembly

The zz dimer is one of four homodimeric mod-ules known to provide the signaling capacityto many activating receptors in the immunesystem (Call and Wucherpfennig 2007). TheFc receptor g (Fcg) subunit associates with asubset of Fc receptors, natural killer (NK) cellreceptors, and activating receptors expressedon osteoclasts and platelets. DAP10 and DAP12associate with NK cell receptors (Lanier 2009).These four signaling proteins share three com-mon features: They are all disulfide-linkedhomodimers, all bear an aspartic acid pair inthe dimeric TM domains, and all carry tyrosine-based cytoplasmic motifs that are phosphory-lated on receptor triggering.

With very few exceptions, the receptors thatassociate with these signaling modules have abasic residue in the TM domain that is requiredfor assembly. In a series of biochemical studiesthat included a broad representation of recep-tor complexes, a set of common features wasidentified (Feng et al. 2005; Garrity et al. 2005;Feng et al. 2006; Feng et al. 2007). First, in everycase investigated, the association required bothaspartic acid residues; alanine substitutionof only one in the pair invariably resulted indramatic assembly defects. Second, the deter-minants for intramembrane assembly are con-tained almost entirely within this focusedcontact site, because the TM domains of recep-tors could be substituted with poly-leucineor poly-valine sequences without disruptingassembly, as long as the basic residue remainedin its native position (Feng et al. 2006). Finally,extracellular and intracellular domains werenot required for the intramembrane assembly,because short peptides encompassing TM and

juxtamembrane sequences were sufficient to buildreceptor complexes.

Structural Basis for IntramembraneAssembly

Precise structural information is required tounderstand why this particular intramembranearrangement is desirable. The only atomic-reso-lution structure to emerge so far is the solutionNMR structure of the zz TM dimer (Call et al.2006). Consistent with the requirement for apair of acidic TM residues, the sidechains ofthe two aspartic acids are colocalized at the helixdimer interface (Fig. 3B). The close appositionof two acidic groups is stabilized by a hydrogen-bonding network that includes both intra-and interhelical hydrogen bonds. The specificgeometry of the di-aspartate site seems to becritical for binding to the receptor, becauseeven the chemically conservative substitutionof glutamic acid can result in major assemblydefects (Call et al. 2002; Call et al. 2006).

Functional Ramifications of UnderstandingReceptor Assembly

At the level of TCR-CD3 complex formationwithin the ER, the intramembrane assemblymechanism provides an important quality con-trol checkpoint. The basic and acidic residuesthat organize and stabilize the complex alsoact as signals for degradation of subunits thatremain unassembled (Bonifacino et al. 1990;Call and Wucherpfennig 2005; Call et al.2006). Assembly and destruction are thereforedirectly competing processes that functiontogether to ensure that only intact, functionalreceptor complexes reach the T-cell surface.

The sequestration of the most critical stabi-lizing contacts within the membrane may pro-vide a requisite degree of conformationalfreedom allowing rearrangements among extra-cellular and intracellular domains to transmitsignals across the plasma membrane. The highlyfocused and polar nature of these contacts(especially in the hydrophobic bilayer interiorwhere competing ions are lacking) may evenallow for reorientations of TM helices around





fixed points to mechanically communicate con-formational changes to the intracellular signal-ing domains (Engelman 2003). These aredifficult hypotheses to test, and will requiresophisticated new experimental approaches formonitoring molecular motions in living cellsreceiving activating signals.

LIPID BINDING BY THE CD31 AND z

CYTOPLASMIC DOMAINS

Each ITAM has two tyrosines and two aliphaticresidues (YxxL/Ix6-12YxxL/I) and phosphory-lation of both tyrosines by Lck or Fyn is requiredfor binding of the tandem SH2 domains ofZAP-70 (Reth 1989; Weiss and Littman 1994;Hatada et al. 1995). Most textbooks show thecytoplasmic domains of the CD3 subunits asflexible chains in the cytosol, a rendering thatsuggests that they would be continuously acces-sible to tyrosine kinases. Is this model correct?

Lipid Binding of ITAMS

The inner leaflet of the plasma membrane hasa negative charge because phosphatidylserine(PS) is almost exclusively localized to the innerleaflet in live cells. Phosphatidylserine is themost abundant anionic lipid in cellular mem-brane (representing �20% of inner leafletlipids), and its asymmetric distribution is main-tained by an ATP-dependent lipid flippase re-ferred to as aminophospholipid translocase(Devaux 1991; Fridriksson et al. 1999; Devauxet al. 2008). This lipid asymmetry is lost whencells become apoptotic, enabling detection ofsuch cells by labeling with the PS binding pro-tein annexin V. Other anionic lipids presentat significantly lower densities contribute tothe negative charge, including phosphatidyl-glycerol, phosphatidic acid, and a variety ofphosphoinositides in different phosphorylationstates (Fridriksson et al. 1999).

The cytoplasmic domains of both CD31and z chains have a net positive charge, whichraised the question whether they could bind tothe inner leaflet of the plasma membranethrough electrostatic interactions. Using in vitroassays with synthetic lipid vesicles, the z chain

cytoplasmic domain was shown to bind tovesicles with a net negative charge, but not tovesicles lacking such a charge (Aivazian andStern 2000). Binding resulted in a substantialincrease in a-helical content detected by circu-lar dichroism measurements, and these authorsproposed that the three ITAMs fold into aa-helical structure upon lipid binding. Bindingto synthetic lipid vesicles was later also shownfor the CD31 cytoplasmic domain as well asthe cytoplasmic domain of Fcg, which servesas a signaling module for activating Fc receptors(Sigalov et al. 2006; Xu et al. 2008; Deford-Wattset al. 2009). CD31 has a higher net positivecharge than z and bound acidic lipid vesicleswith higher affinity. Mutation of two clustersof basic residues in the amino-terminal part ofthe CD31 cytoplasmic domain abrogated lipidbinding, confirming the importance of electro-static interactions (Xu et al. 2008). Little or nobinding was detected for the cytoplasmicdomains of the CD3g and CD3d chains inthese in vitro assays, which lack a net positivecharge, although it remains possible that theybind cooperatively with z and CD31 to cellularmembranes.

It was critical to establish that such lipidbinding actually occurs in cells because all bind-ing studies had been performed in vitro withsynthetic lipid vesicle preparations. A cellularfluorescence resonance energy transfer (FRET)assay was developed to directly address thisquestion (Xu et al. 2008). In a fully extendedconformation, the 57 amino acid cytoplasmicdomain of CD31 is at a substantial distancefrom the plasma membrane (�200 A). This dis-tance is outside of the range for FRET becauseenergy transfer from donor to acceptor fluoro-phore is highly distance dependent, with theupper limit being �100 A (Kenworthy 2001).Efficient FRET between a fluorescent proteinattached to the carboxy-terminus of CD31and a fluorescent dye in the plasma membraneis therefore only expected in the lipid-boundstate of CD31, but not when the cytoplasmicdomain has dissociated from the membrane.The validity of this FRET-based approach wastested with constructs using flexible linkers ofincreasing length (3, 25, and 50 amino acids).





A high FRET signal was observed with the wild-type CD31 cytoplasmic domain, but not with amutant that failed to show binding in the invitro assay described above (Sigalov et al. 2006;Xu et al. 2008).

Structure of the Membrane-bound Itam

Determination of the structure of the CD31cytoplasmic domain required a lipid bilayersurface sufficiently large in size to enable properbinding of the cytoplasmic domain. Detergentmicelles with the appropriate lipid headgroupswould not be suitable because of their highcurvature. The solution to this problem was touse bicelles, which represent flat discs formedusing a mixture of short- and long-chainlipids. The long-chain lipids form the bilayercore of such bicelles, whereas the short chainlipids associate at the rim. Bicelles have a sizesimilar to small proteins and thus tumblequickly enough for solution NMR studies(Prosser et al. 2006). This approach enableddetermination of the structure of the CD31cytoplasmic domain in a lipid-bound state(Xu et al. 2008). A surprising finding was thatthe cytoplasmic domain was actually inserted

into the bilayer, with the peptide backbonebeing located at the interface between thehydrophilic headgroup region and the hydro-phobic acyl chain layer. This overall positionof the backbone enabled insertion of all hydro-phobic side chains into the hydrophobic core ofthe bilayer. The structure of the ITAM itselfshows insertion of all four key residues, thetwo tyrosines, and the two aliphatic residues,into the acyl chain region of the bilayer(Fig. 4). Helical structure was confined to shortsegments centered on the tyrosines, and circulardichroism experiments confirmed the presenceof a small amount of a-helical structure thatwas lost when the tyrosines and aliphaticresidues of the ITAM were mutated. Interest-ingly, the amino-terminal part of the CD31cytoplasmic domain is rather flexible, indicatingthat the ITAM is connected to the TM domainthrough a membrane-bound flexible linker(Xu et al. 2008). Functional studies showedthat lipid binding by CD31 or z preventedphosphorylation by Lck (Aivazian and Stern2000; Xu et al. 2008). This means thatdissociation of the ITAMs from the membraneis required as one of the initial events in TCRactivation.

1stGlycerol

2ndGlycerol

Acylchain

Y38 Y49

L52

Defined planes

OO

HP

OH

O

OO

OO

OO

HI41

Hyd

rop

hili

cH

ydro

ph

ob

ic

Figure 4. Structure of the CD31 ITAM in the lipid-bound state. The two tyrosines (Y38 and Y49) and the twoaliphatic residues (I41 and L52) of the ITAM protrude into the hydrophobic acyl chain region of the lipid bilayer.The peptide backbone of the ITAM resides primarily at the interface between the hydrated lipid headgroupregion and the hydrophobic bilayer interior. The hydrophobic layer of the bilayer is shaded light blue and thehydrated lipid headgroup region in dark blue. The POPG structure graphic to the right shows the location ofthe hydrophilic headgroup and the hydrophobic acyl chains of the lipid (PDB entry 2K4F).





Possible Mechanisms for ITAM Release fromthe Membrane

The mechanisms resulting in dissociation of theCD31 and z cytoplasmic domains from themembrane on TCR triggering remain unknown.It is unlikely that subtle conformational changein the extracellular domains of the TCR-CD3complex during peptide-MHC binding aresufficient due to the flexible nature of theN-terminal part of the cytoplasmic domain(Aivazian and Stern 2000; Xu et al. 2008). Othermechanisms must therefore account for thechange in tyrosine accessibility during TCRtriggering.

It has been proposed that the continuedmovement of T cells across antigen presentingcells results in a mechanical force on the TCR-peptide-MHC recognition unit (Ma et al. 2008;Kim et al. 2009). Such a mechanical force couldpossibly result in dissociation of the ITAM fromthe membrane but would have to be strongor sustained enough to act through the flexi-ble amino-terminal part of the cytoplasmicdomain on the ITAM. A second possibility isthat clustering of TCR-CD3 complexes in earlymicroclusters at the immunological synapseresults in competition among the cytoplasmicdomains for membrane surface (Aivazian andStern 2000). TCR microclusters can form beforeinitiation of signaling because they are observedeven in the presence of a Src kinase inhibitorthat blocks Lck activity (Campi et al. 2005). Athird hypothesis is that TCR clustering changesthe lipid environment in the vicinity of theclustered TCRs, reducing the affinity of thecytoplasmic domains for the membrane. Lipidbinding is highly sensitive to the density of neg-atively charged phospholipids due to the essen-tial role of basic residues in the cytoplasmicdomains for lipid binding.

CONCLUDING REMARKS

Structural data are now available not only forthe extracellular domains, but also some of thetransmembrane and cytoplasmic domains ofthe TCR-CD3 complex. These structural studiescreate a solid framework for understanding

key functional aspects of the TCR-CD3 com-plex and many other activating receptors inthe immune system.

ACKNOWLEDGMENTS

This work was supported by grants to K.W.W.to the National Institutes of Health (RO1AI054520 and AI064177). E.S.H. was supportedin part by Beckman Young Investigator andSearle Scholar awards.

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