A Membrane-proximal Tetracysteine Motif Contributes to Assembly of CD3δϵ and CD3γϵ Dimers with the T Cell Receptor

Assembly of the T cell receptor (TCR) with its dimeric signaling modules, CD3δϵ, CD3γϵ, and ζζ, is organized by transmembrane (TM) interactions. Each of the three assembly steps requires formation of a three-helix interface involving one particular basic TCR TM residue and two acidic TM residues of the respective signaling dimer. The extracellular domains of CD3δϵ and CD3γϵ contribute to assembly, but TCR interaction sites on CD3 dimers have not been defined. The structures of the extracellular domains of CD3δϵ and CD3γϵ demonstrated parallel β-strands ending at the first cysteine in the CXXCXEXXX motif present in the stalk segment of each CD3 chain. Mutation of the membrane-proximal cysteines impaired assembly of either CD3 dimer with TCR, and little complex was isolated when all four membrane-proximal cysteines were mutated to alanine. These mutations had, however, no discernable effect on CD3δϵ or CD3γϵ dimerization. CD3δϵ assembled with a TCRα mutant that lacked both immunoglobulin domains, but shortening of the TCRα connecting peptide reduced assembly, consistent with membrane-proximal TCRα-CD3δϵ interactions. Chelation of divalent cations did not affect assembly, indicating that coordination of a cation by the tetracysteine motif was not required. The membrane-proximal cysteines were within close proximity but only formed covalent CD3 dimers when one cysteine was mutated. The four cysteines may thus form two intrachain disulfide bonds integral to the secondary structure of CD3 stalk regions. The three-chain interaction theme first established for the TM domains thus extends into the membrane-proximal domains of TCRα-CD3δϵ and TCRβ-CD3γϵ. Assembly of the T cell receptor (TCR) with its dimeric signaling modules, CD3δϵ, CD3γϵ, and ζζ, is organized by transmembrane (TM) interactions. Each of the three assembly steps requires formation of a three-helix interface involving one particular basic TCR TM residue and two acidic TM residues of the respective signaling dimer. The extracellular domains of CD3δϵ and CD3γϵ contribute to assembly, but TCR interaction sites on CD3 dimers have not been defined. The structures of the extracellular domains of CD3δϵ and CD3γϵ demonstrated parallel β-strands ending at the first cysteine in the CXXCXEXXX motif present in the stalk segment of each CD3 chain. Mutation of the membrane-proximal cysteines impaired assembly of either CD3 dimer with TCR, and little complex was isolated when all four membrane-proximal cysteines were mutated to alanine. These mutations had, however, no discernable effect on CD3δϵ or CD3γϵ dimerization. CD3δϵ assembled with a TCRα mutant that lacked both immunoglobulin domains, but shortening of the TCRα connecting peptide reduced assembly, consistent with membrane-proximal TCRα-CD3δϵ interactions. Chelation of divalent cations did not affect assembly, indicating that coordination of a cation by the tetracysteine motif was not required. The membrane-proximal cysteines were within close proximity but only formed covalent CD3 dimers when one cysteine was mutated. The four cysteines may thus form two intrachain disulfide bonds integral to the secondary structure of CD3 stalk regions. The three-chain interaction theme first established for the TM domains thus extends into the membrane-proximal domains of TCRα-CD3δϵ and TCRβ-CD3γϵ. The T cell receptor (TCR) 3The abbreviations used are: TCR, T cell receptor; TM, transmembrane; SBP, streptavidin-binding peptide; PC, Protein C-derived peptide; HA, hemagglutinin peptide; IP, immunoprecipitation; snIP, sequential nondenaturing immunoprecipitation; TBS, Tris-buffered saline; ER, endoplasmic reticulum; WT, wild type; mAb, monoclonal antibody; BisTris, 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)propane-1,3-diol. controls T cell development and function and is assembled from six distinct component polypeptides into an eight-chain complex. The TCR heterodimer is responsible for ligand recognition but lacks cytoplasmic signaling domains. Instead, it associates through non-covalent interactions with three dimeric signaling modules, CD3δϵ, CD3γϵ, and ζζ (1Oettgen H.C. Terhorst C. Crit. Rev. Immunol. 1987; 7: 131-167PubMed Google Scholar, 11Weissman A.M. Frank S.J. Orloff D.G. Mercep M. Ashwell J.D. Klausner R.D. EMBO J. 1989; 8: 3651-3656Crossref PubMed Scopus (93) Google Scholar). The ζ chain only has a short nine-amino acid extracellular domain and forms covalent dimers through a cysteine at position 2 of the predicted transmembrane (TM) domain (3Samelson L.E. Harford J.B. Klausner R.D. Cell. 1985; 43: 223-231Abstract Full Text PDF PubMed Scopus (222) Google Scholar, 12Sussman J.J. Bonifacino J.S. Lippincott-Schwartz J. Weissman A.M. Saito T. Klausner R.D. Ashwell J.D. Cell. 1988; 52: 85-95Abstract Full Text PDF PubMed Scopus (270) Google Scholar, 13Weissman A.M. Baniyash M. Hou D. Samelson L.E. Burgess W.H. Klausner R.D. Science. 1988; 239: 1018-1021Crossref PubMed Scopus (202) Google Scholar). The CD3γ, -δ, and -ϵ chains have more substantial extracellular components, and their Ig domains form noncovalent heterodimers through parallel β-strands, which create a β-sheet along the dimer interface. In each CD3 chain, a nine-amino acid segment with a highly conserved CXXCXEXXX motif connects this β-strand to the TM domain (1Oettgen H.C. Terhorst C. Crit. Rev. Immunol. 1987; 7: 131-167PubMed Google Scholar, 14Krissansen G.W. Owen M.J. Fink P.J. Crumpton M.J. J. Immunol. 1987; 138: 3513-3518PubMed Google Scholar, 18Gold D.P. Clevers H. Alarcon B. Dunlap S. Novotny J. Williams A.F. Terhorst C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7649-7653Crossref PubMed Scopus (82) Google Scholar). Assembly of the TCR heterodimer with the three dimeric signaling modules requires proper placement of a total of nine ionizable TM residues, the three basic TM residues of the TCR heterodimer (7Blumberg R.S. Alarcon B. Sancho J. McDermott F.V. Lopez P. Breitmeyer J. Terhorst C. J. Biol. Chem. 1990; 265: 14036-14043Abstract Full Text PDF PubMed Google Scholar, 19Alcover A. Mariuzza R.A. Ermonval M. Acuto O. J. Biol. 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Wucherpfennig K.W. Cell. 2002; 111: 967-979Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). of the CD3γ extracellular domains to of assembly was shown by domain a CD3γ mutant with the TM domain of was into TCR-CD3 complexes, but a CD3γ mutant with the extracellular domain of to (4Call M.E. Pyrdol J. Wiedmann M. Wucherpfennig K.W. Cell. 2002; 111: 967-979Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 22Dietrich J. Neisig A. Hou X. Wegener A.M. Gajhede M. Geisler C. J. Cell Biol. 1996; 132: 299-310Crossref PubMed Scopus (66) Google Scholar, A.M. Hou X. J. Geisler C. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). The the physical nature of TCR-CD3 extracellular domain interactions have in a of Immunity. Full Text Full Text PDF PubMed Scopus Google Scholar). interactions between Ig domains of TCR and CD3 dimers. a more the that the CD3 may located close to the cell the TCR constant domains Proc. Natl. Acad. Sci. U. S. 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Cell. 2002; 111: 967-979Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). were by were as with a acid The streptavidin-binding peptide was Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), with and cysteine to that or cysteine not into the were as and peptide In was from a and and Assembly of of acid cysteine and of of of each of and of These were on from a as (4Call M.E. Pyrdol J. Wiedmann M. Wucherpfennig K.W. Cell. 2002; 111: 967-979Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). and assembly were at of was by a assembly for TCR-CD3 complexes, 2 for assembly the of to were as for with sequential nondenaturing and and assembly were by with of TBS, and were and were in of with when mAb was by and for at in a total of of were for with and were at were in of with for mAb of was by with of with for at and were with subsequent and Protein for 2 at and as with was as (4Call M.E. Pyrdol J. Wiedmann M. Wucherpfennig K.W. Cell. 2002; 111: 967-979Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). were for at with of on to and to was the in the Chelation of TCR-CD3 were for to of into was to the to all divalent cations is present in the of was to was to formation of disulfide a assembly were and in with TCR-CD3 were isolated by the and were as The of CD3γϵ and membrane-proximal of CD3γ, -δ, and -ϵ a highly conserved CXXCXEXXX and the two cysteines within this motif located at and to the predicted TM The and structures of CD3γϵ and CD3δϵ demonstrated that the of the two chains form a β-sheet at the dimer interface Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, T. J. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Reinherz Cell. Full Text Full Text PDF PubMed Scopus Google Scholar), as in for CD3γϵ and The cysteines were not in these or not in these but the that the four cysteines located within close proximity in the stalk of both CD3 dimers. located close to the of the β-strands, and the stalk with the first cysteine of this motif The four cysteines may form two intrachain disulfide bonds that the secondary structure of the CD3 stalk or may a divalent as The CD3 that were used to the of these cysteine residues to assembly of CD3 dimers with TCR in The for CD3δϵ or CD3γϵ the assembly of CD3 dimers and their interaction with TCR an in with isolated from a cell and and have shown that this interactions in the with in (4Call M.E. Pyrdol J. Wiedmann M. Wucherpfennig K.W. Cell. 2002; 111: 967-979Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, J.B. J. PubMed Scopus (82) Google Scholar, J. Immunol. Google Scholar). of this that a of mutations in parallel and that of highly M.E. Pyrdol J. Wucherpfennig K.W. EMBO J. PubMed Scopus Google Scholar). on that the membrane-proximal cysteines may contribute to formation of CD3 dimers Reinherz Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. Alarcon B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and the of CD3 for in which both membrane-proximal cysteines of CD3γ, -δ, -ϵ were by CD3 were isolated with the mAb that binds to an of mAb is for this because it CD3γϵ and CD3δϵ dimers and CD3 dimers into TCR-CD3 (4Call M.E. Pyrdol J. Wiedmann M. Wucherpfennig K.W. Cell. 2002; 111: 967-979Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. Immunol. Google Scholar, J. Terhorst C. J. Immunol. PubMed Scopus Google Scholar). substitution of the two membrane-proximal cysteines of CD3ϵ did not reduce of dimers with CD3γ or and in which the three chains were and to of with and demonstrated that dimer formation in and not mutations of both cysteines in either CD3γ or and respectively, and did not dimer formation with and substitution of all four membrane-proximal cysteines did not reduce either CD3γϵ or CD3δϵ and 3The membrane-proximal cysteines of CD3δϵ contribute to assembly with CD3ϵ and in which both membrane-proximal cysteines were by and were for their to into a three-chain TCRα-CD3δϵ complex. were isolated by the to TCRα and the The of TCRα was to the with of the two membrane-proximal cysteines of to reduced assembly with TCRα and substitution of all four membrane-proximal cysteines assembly to to and controls in which TCRα and CD3δϵ were in that were to of of of to demonstrated of of were for their to form CD3δϵ dimers and to into TCRα-CD3δϵ The two membrane-proximal cysteines were either both by 2 and or and or by and and CD3δϵ dimer formation was by the on CD3ϵ and the on whereas TCRα-CD3δϵ were isolated the to TCRα and the CD3ϵ specific mAb substitution of the two CD3δϵ cysteines by reduced assembly with TCRα but did not affect CD3δϵ Mutation of membrane-proximal cysteines in formation of covalent CD3δϵ dimers and indicating that the cysteines the tetracysteine motif within close of of to demonstrated of TCRα mutant both Ig domains with TCRα assembly with the CD3δϵ dimer was for a mutant the two TCRα Ig domains and a mutant in which the connecting peptide was to six residues these TCRα and CD3δϵ were isolated by the on TCRα and the The both TCRα Ig domains assembled with assembly at reduced with the The peptide was in the first and was present in the isolated but this short peptide was to at the isolated in this peptide of was of the no in the The in in which TCRα and CD3δϵ chains were and to demonstrated of cysteine with the TCRα as with assembly were with and TCR or and three-chain were isolated as in B. of one or both cysteine residues by and or reduced assembly of either TCR or with The of CD3ϵ in with these was with with of tetracysteine in formation of the assembly of the complex was in with and CD3γ, -δ, and TCR both CD3γϵ and CD3δϵ were isolated by the and to CD3γ and The and to of all by of the two membrane-proximal cysteines by in only CD3γ or CD3ϵ reduced assembly of the but of these mutations into both CD3γ and CD3ϵ chains and in a assembly the cysteine mutations not affect the assembly of the ζζ dimer. Assembly with all TCR-CD3 were with the ζ chain TCR-CD3 were isolated by the ζ chain and CD3ϵ whereas were isolated from the ζ chain by and CD3γ of divalent cations not with assembly of TCR-CD3 a that CD3 dimers not form the of the this of in and were by the of CD3 dimers were the mAb a assembly the of not assembly of TCRα with were isolated with the mAb when was the assembly an with an the of not with formation of TCR-CD3 of was demonstrated by the mAb and with an mAb Mutation of Assembly of CD3δϵ with first in the assembly of TCR-CD3 is the interaction of TCRα with the CD3δϵ dimer (4Call M.E. Pyrdol J. Wiedmann M. Wucherpfennig K.W. Cell. 2002; 111: 967-979Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 20Kearse K.P. Roberts J.L. Singer A. Immunity. 1995; 2: 391-399Abstract Full Text PDF PubMed Scopus (69) Google Scholar), and of the membrane-proximal cysteines of or -ϵ this assembly were isolated by the to TCRα in the first with CD3ϵ were isolated in the with the of the two cysteines at and of reduced assembly of CD3δϵ with TCRα to and substitution of all four membrane-proximal cysteines of the CD3δϵ dimer in an assembly to of the two cysteines in only CD3ϵ only a effect but these two cysteines to assembly, because of and CD3ϵ in a more assembly of to WT, controls and demonstrated of assembly, and parallel of of the of of on these in which the cysteines were by and and CD3δϵ as as assembly with TCRα mutations reduced assembly with TCRα to a as substitution of both cysteines to and of either one or both cysteine chains may formation of an intrachain disulfide and thus the of the stalk a mutant in which both cysteines were by this mutant the and of the chains at and but lacked the to form a disulfide at this mutant the assembly as the or of in which only a cysteine was demonstrated that the four cysteines in the stalk were within close of either cysteine in the formation of covalent dimers with CD3ϵ by the in and The used for this of CD3 because the streptavidin-binding peptide to CD3ϵ was used in the first with and the on was used in the TCRα the Ig with that interactions the membrane-proximal may in assembly of TCRα with the on the TCRα by a mutant that the connecting peptide but lacked both and constant Ig domains mutant assembled with CD3δϵ indicating that interaction TCR Ig domains and CD3δϵ was not for this of demonstrated that of TCRα and were present in these and mutant in which the TCRα connecting peptide was to six residues to CD3δϵ, but the of this three-chain complex was reduced to TCRα a with TCRα when the of in which the membrane-proximal cysteines were by or and the mutant a assembly with both and to indicating that of the TCRα Ig domains not in a of interaction with CD3δϵ for these Mutation of of CD3γϵ with TCR but of the ζζ The CD3γϵ dimer with only in the of the CD3δϵ dimer (4Call M.E. Pyrdol J. Wiedmann M. Wucherpfennig K.W. Cell. 2002; 111: 967-979Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar), indicating that of CD3γϵ formation of the assembly the effect of of the membrane-proximal cysteines in the CD3γϵ dimer in the of the assembly by the CD3γ and chains in a have shown that this of all three dimers M.E. Pyrdol J. Wucherpfennig K.W. EMBO J. PubMed Scopus Google Scholar). Mutation of the two cysteines in either CD3γ or CD3ϵ and reduced of the complex and but substitution of all four cysteines in a assembly to In this the CD3ϵ the interaction of both CD3δϵ and CD3γϵ with TCR, but the in demonstrated that the CD3ϵ only a effect in the of the CD3δϵ dimer as as was of to The in the of was thus to of the four cysteines in the CD3γϵ dimer. The for the CD3γϵ dimer were thus to CD3δϵ, because of all four cysteines a more substantial effect of cysteines in only one chain and Mutation of the membrane-proximal cysteines of CD3γ and -ϵ did not affect the assembly the of the ζζ dimer because the in of assembled the of complexes, indicating that was no effect on the assembly In the of with the CD3γ and -ϵ cysteine mutations was in the of ζζ of to CD3γ and in its of to that the ζζ dimer may that these mutated CD3 Chelation of the of TCR-CD3 of cysteine residues or cations T. J. Science. 1990; PubMed Scopus Google Scholar), and the for divalent cations assembly of TCR-CD3 as an to the formation of intrachain disulfide bonds a divalent cation is at a may and only of the complex. an in which divalent cations were by to of In the in and the is to of into to formation of disulfide dimers that into TCR-CD3 formation of disulfide and assembly thus not the of the that no CD3 dimers were formed the and and that the of little effect on the subsequent formation of CD3 dimers and a of of the and the of assembly reduce the of TCRα-CD3δϵ three-chain or TCR-CD3 The demonstrated that of divalent cations no substantial effect on assembly, the that the membrane-proximal tetracysteine a divalent cation for interaction of CD3 dimers with in of the assembled complex was to the that the of divalent cations disulfide These that the tetracysteine motif in the membrane-proximal stalk of CD3γϵ and CD3δϵ dimers is an for their assembly with Assembly was impaired or with when all four cysteines of a CD3 dimer were mutated to alanine. The interaction of CD3δϵ with TCRα was in and of the two membrane-proximal cysteines of to or was to reduce assembly Mutation of the two cysteines of CD3ϵ only interaction with but of and CD3ϵ in an assembly to more for the mutant of the two membrane-proximal cysteines of CD3γ reduced assembly with TCR but CD3γ and CD3ϵ mutations in a assembly In this the CD3ϵ mutant was into both CD3γϵ and CD3δϵ dimers, but it that the assembly was to the CD3γϵ because the CD3ϵ only the These the first mutations in the CD3 extracellular domains shown to assembly of CD3 dimers with that the contribute to CD3 dimer A. A. Alarcon B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) with CD3 in which the cysteines in the motif of CD3ϵ were mutated to alanine. CD3ϵ chains were with the mAb and CD3γ or were by A. A. Alarcon B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Reinherz Cell. Full Text Full Text PDF PubMed Scopus Google Scholar) but and with to and These mutated the cysteines of CD3ϵ to but did not a substantial in the of assembly with CD3γ of in to the by A. A. Alarcon B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) with a CD3ϵ of three mutations and located N-terminal to and of CD3ϵ impaired CD3γϵ dimer formation Reinherz Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). a in which the motif was mutated in all three CD3 chains and that of all four membrane-proximal cysteines did not reduce CD3γϵ or CD3δϵ dimer The cysteine residues thus not to for CD3 the dimer interface is by of is consistent with the that CD3γϵ and CD3δϵ from in that the T. J. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Reinherz Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). experiments that the cysteines form covalent CD3δϵ dimers when a cysteine in the motif is mutated to The cysteines of and CD3ϵ chains thus within proximity to form an disulfide and the of disulfide bonds in CD3γϵ and CD3δϵ dimers that the either to an ligand or that have formed intrachain disulfide bonds to CD3 dimerization. the that the tetracysteine motif may a cation as and cations in as the domain of the The receptor domain has two and each binds through four cysteine The first is formed by two and the the that binds in the of T. J. Science. 1990; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). assembly of the or TCR-CD3 was not reduced when cations were with The experiments an role of a cation in assembly of CD3 dimers with Mutation of the two cysteines in one chain of a CD3 dimer predicted to a coordination of the motif in CD3ϵ only reduced assembly of CD3δϵ with for the role of the is that the two membrane-proximal cysteines of a CD3 chain form an intrachain disulfide as by A. A. Alarcon B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Several lines of evidence this The close physical proximity of the two cysteines may formation of intrachain disulfide bonds of the chain into the of the ER, and formation of these disulfide bonds may more of the Ig domain and subsequent CD3 dimerization. formation of an intrachain disulfide may thus CD3γϵ or CD3δϵ dimers disulfide form the of and and the two cysteines form a disulfide at a particular in the indicating that cysteines in within proximity for disulfide The N-terminal cysteine in the motif of serves as a and forms a disulfide with the of this disulfide by the cysteine the reduced and in the formation of a disulfide between the two cysteines of the motif D. J. Rev. PubMed Scopus Google Scholar, Rev. 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Full Text Full Text PDF PubMed Scopus Google Scholar), but mutations that assembly not been that the stalk of the CD3 dimers for the interaction of CD3 dimers with is by the that a TCRα mutant the connecting peptide but the Ig domains assembled with CD3δϵ, whereas a mutant in which the connecting peptide was to six residues assembled at a reduced role of the CD3 stalk in assembly is consistent with the that mutations in the TCRα connecting peptide TCR signaling A. B. Immunity. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The membrane-proximal may contribute to the secondary structure of the CD3 stalk and may to position the CD3 TM domains for interaction with the TCR TM