Membrane Binding by the tyrosine-based signaling motif of the T cell receptor CD3 epsilon chain

NMR structure of the lipid bound state of the CD3e cytoplasmic domain.  All four hydrophobic residues of the ITAM (Y38, I41, Y49 and L52) are inserted into the core of the membrane, rendering the tyrosines inaccessible to kinases prior to receptor triggering.

Many immune system receptors signal through cytoplasmic tyrosine-based motifs (ITAMs), but how receptor ligation results in ITAM phosphorylation remained unknown.  Using a novel live cell imaging approach, we showed for the first time that the CD3e cytoplasmic domain of the T cell receptor (TCR) is bound to the inner leaflet of the plasma membrane in live T cells.  Membrane binding was visualized through fluorescence resonance energy transfer (FRET) between a C-terminal fluorescent protein and a membrane fluorophore.  Electrostatic interactions between basic CD3e residues and acidic phospholipids enriched in the inner leaflet of the plasma membrane were required for binding.  In collaboration with Dr. James Chou, we determined the nuclear magnetic resonance (NMR) structure of the lipid-bound state of the CD3e cytoplasmic domain.  The structure showed deep insertion of the two key tyrosines into the hydrophobic core of the lipid bilayer. Receptor ligation thus needs to result in unbinding of the CD3e ITAM from the membrane to render these tyrosines accessible to Src kinases. Sequestration of key tyrosines into the lipid bilayer represents a novel mechanism for control of receptor activation.  We are now examining how receptor triggering results in release of the ITAM from the membrane.

Unconventional topology of self-peptide/MHC binding by a human autoimmune T cell receptor

We determined the first crystal structure of a T cell receptor from a human autoimmune disease bound to its self-peptide/MHC target (Hahn et al., Nat. Immunol. 2005).  This TCR (termed Ob.1A12) originated from a patient with relapsing-remitting multiple sclerosis (MS), and transgenic mice that express this TCR and the human MHC class II molecule (HLA-DR2) develop spontaneous CNS autoimmunity (Madsen et al., Nat. Genetics 1999).  The structure showed a highly unusual topology of peptide/MHC binding that had not been observed for human TCRs directed against infectious agents.  All other human TCRs that had been crystallized with their peptide-MHC ligand had shown a very similar topology in which the TCR is positioned in a diagonal orientation over the center of the peptide/MHC surface.  In contrast, the Ob.1A12 TCR contacts only the N-terminal segment of the human myelin basic protein peptide and makes highly asymmetrical interactions with the MHC helices. 

Crystal structure of the Ob.1A12 TCR (left) isolated from a patient with relapsing-remitting MS, compared to the HA1.7 TCR (right) specific for influenza HA peptide.  The MS-patient derived TCR binds with a highly unusual topology to the self-peptide/MHC complex. 

 

This highly unusual binding mode is dominated by the hypervariable CDR3 loops of the TCR which make the majority of contacts to the MHC helices as well as the peptide. The figure shows the footprint of the Ob.1A12 TCR on the peptide/MHC surface. The hypervariable loops of TCR alpha and TCR beta are colored in yellow and red, respectively. The major MHC (DR beta 81 His) and peptide (P2 His) residues contacted by the hypervariable TCR loops are also indicated. In other TCR structures, the interaction with the MHC helices is dominated by the germline-encoded CDR1 and CDR2 loops, which had led to the hypothesis that all TCRs would have a similar diagonal topology. Unconventional topologies are therefore possible because of the sequence diversity of the hypervariable TCR loops created during rearrangement. 

The unconventional topology for this autoimmune TCR is probably the result of distinct selection pressures exerted on autoimmune T cells.  During repertoire development in the thymus, T cells with an optimal fit for a self-peptide/MHC complex are the most likely to be deleted.  In contrast, T cells whose TCR has an optimal fit for a microbial peptide bound to MHC are not systematically deleted in the thymus and have a competitive advantage over other T cells in an anti-microbial immune response.  Our structure thus provides an explanation for the finding that autoreactive T cells are present in every subject, despite elaborate mechanisms for the elimination of such cells.

Hahn M, Nicholson MJ, Pyrdol J, Wucherpfennig KW
Unconventional topology of self-peptide/MHC binding by a human autoimmune T-cell receptor
Nat Immunol. 2005 May;6(5):490-6. PubMed

 

Molecular mechanism for the assembly of the T cell receptor and other activating immune receptors

Organization of TCR-CD3 Assembly, above

The T cell receptor (TCR) is critical for T cell development in the thymus and the induction of T cell effector functions in an immune response. It is one of the most complex membrane receptors, being composed of six different polypeptides. The T cell receptor heterodimer is responsible for ligand recognition and assembles with three signaling dimers, CD3 delta/epsilon, CD3 gamma/espilon and zeta/zeta. We found that assembly of TCR with these three signaling dimers is organized by the same mechanism in the membrane environment. In each of these major assembly steps, a basic transmembrane residue of TCR interacts with a pair of acidic transmembrane residues of the relevant signaling dimer. This unusual three-helix motif thus guides the higher-order assembly steps of this important receptor. Mutation of any one of these nine basic/acidic transmembrane residues interferes with assembly, demonstrating the importance of these interactions in the formation of the correct receptor structure. Future efforts will examine the mechanism of TCR triggering based on the insights we have gained on the interactions among the subunits.

Call ME, Wucherpfennig KW
The T cell receptor: Critical role of the membrane environment in receptor assembly and function
©Annual Review of Immunol. 2005;23:101-25.(PDF)

Call ME, Wucherpfennig KW
Stoichiometry of the T cell receptor-CD3 complex and key intermediates assembled in the ER
EMBO J. 2004;12: 2348-57(PDF)

Call ME, Pyrdol J, Wiedmann M, Wucherpfennig KW
The organizing principle in the formation of the T cell receptor-CD3 complex
Cell, 2002; 111: 967-79, (PubMed)

 

Structural features of the zeta chain transmembrane dimer important for assembly with the T cell receptor

The biochemical studies on TCR assembly raised an important question: what is the structural arrangement of the two acidic transmembrane residues that interact with a basic TCR side chain? We expressed the transmembrane segment of the zeta/zeta dimer as a representative example and determined the NMR structure, in collaboration with the Chou lab at Harvard Medical School. The structure showed several polar features important for dimerization of the zeta chain and its interaction with the TCR. The two aspartic acids (D6) are located at the dimer interface and form a single structural unit. This arrangement is stabilized by hydrogen bonds between the D6 carboxyl group of one strand and the D6 backbone amide of the opposite strand. Interestingly, there is direct experimental evidence for water molecule(s) in the vicinity of D6, suggesting that structural water molecule(s) may stabilize this unusual arrangement. Two lateral hydrogen bonds formed between threonine 12 and tyrosine 17 (T12-Y17) represent a second polar feature that is important for zeta formation. The structure thus establishes a general framework for understanding the assembly of the signaling modules with the TCR.

Call ME, Schnell JR, Xu Chenqi, Lutz RA, Chou JJ, Chou JJ, Wucherpfennig KW.
The structure of the zeta zeta transmembrane dimer reveals features essential for its assembly with the T cell receptor.
Cell 2006; 127, 355-68. Pubmed

 

The membrane-based assembly mechanism is relevant for diverse immune receptors

We have recently found that the same assembly mechanism is also relevant for the formation of a large number of other activating immune receptors.  Each cell type of hematopoetic origin expresses at least one receptor that triggers a cell-type specific activation program leading to calcium flux, cytokine secretion, cytotoxicity and/or proliferation.  These activating receptors have short cytoplasmic domains and instead transmit activation signals through associated signaling modules, the DAP12, DAP10 or Fcg signaling dimers which carrying tyrosine-based phosphorylation motifs in their cytoplasmic domains.  The TM domains of all of these signaling modules have a pair of acidic residues (D-D), just like the signaling components of the TCR-CD3 complex.  We showed that these signaling dimers have a high degree of specificity for a particular basic side chain, DAP12 for a lysine (K) in the center and Fcg for an arginine (R) in the N-terminal third of the TM domain of the interacting receptor.  These results explain how these signaling modules can specifically assemble with a diverse group of receptors.

Feng J, Call ME, Wucherpfennig KW
The assembly of diverse immune receptors is focused on a polar membrane-embedded interaction site.
PLoS Biol. 2006 May;4(5):e142 (PubMed)

Garrity D, Call ME, Feng J, Wucherpfennig KW
The activating NKG2D receptor assembles in the membrane with two signaling dimers into a hexameric structure.
Proc Natl Acad Sci USA. 2005 May 24;102(21):7641-6 (PDF)

Feng J, Garrity D, Call ME, Moffett H, Wucherpfennig KW
Convergence on a distinctive assembly mechanism by unrelated families of activating immune receptors.
Immunity. 2005 Apr;22(4):427-38 (PubMed)

 

A novel approach for the identification of autoantibodies in human diseases

Autoantibodies are thought to contribute to the pathogenesis of MS and other demyelinating CNS diseases, but it has been difficult to study such antibodies in humans. We developed a novel, sensitive technique that permits conformation-sensitive autoantibodies to be detected. Pathogenic autoantibodies directed against cell surface antigens can have a low binding affinity and we therefore generated a tetrameric form of a self-antigen that has been implicated in the pathogenesis of MS. Myelin oligodendrocyte glycoprotein (MOG) is located on the surface of the myelin and is thus accessible to autoantibodies. Injection of such antibodies in mouse models can induce myelin damage (demyelination), but their role in human diseases is controversial. We fused the extracellular domain of MOG to the streptavidin sequence and observed spontaneous formation of tetramers. These tetramers permitted sensitive detection of autoantibodies, and we were able to show that autoantibodies to MOG are present in a subset of patients with acute demyelinating encephalomyelitis (ADEM). We are now using this approach to identify of novel autoantibody targets.

O'Connor KC, McLaughlin KA, De Jager PL, Chitnis T, Bettelli E, Xu C, Robinson WH, Cherry SV, Bar-Or A, Banwell B, Fukaura H, Fukazawa T, Tenembaum S, Wong SJ, Tavakoli NP, Idrissova Z, Viglietta V, Rostasy K, Pohl D, Dale RC, Freedman M, Steinman L, Buckle GJ, Kuchroo VK, Hafler DA, Wucherpfennig KW.
Self-antigen tetramers discriminate between myelin autoantibodies to native or denatured protein.
Nature Medicine. 2007 Feb;13(2):211-7. Epub 2007 Jan 12. 2007 (PubMed)

 

Structural basis for the presentation of self-peptides in autoimmune diseases to T cells

Structure of HLA-DR2 with the human myelin basic protein peptide, above

Autoimmune diseases are caused by self-reactive T cells that escaped negative selection during development in the thymus. Susceptibility to many human autoimmune diseases is linked to the MHC locus, indicating that antigen presentation to T cells is critical. In many of these diseases, the MHC is by far the most important susceptibility locus and the disease-associated MHC alleles differ only at a limited number of positions in the peptide binding site from non-associated alleles. We have determined the crystal structure of the multiple sclerosis (MS) associated HLA-DR2 molecule (DRA, DRB1*1501) with a bound peptide from human myelin basic protein that is immunodominant for human T cells. The structure demonstrated that HLA-DR2 differs from HLA-DR molecules associated with other autoimmune diseases in a centrally located pocket of the binding site (P4 pocket). In HLA-DR2, this pocket is large and hydrophobic, permitting binding of an aromatic residue from the MBP peptide. In the rheumatoid arthritis associated HLA-DR4 molecule, this pocket instead has a positive charge and thus a preference for small and acidic side chains.

Structure of HLA-DQ8 with the Insulin B (9-23) Peptide, above

Susceptibility to type 1 diabetes is associated with HLA-DQ8 and HLA-DQ2. The crystal structure of HLA-DQ8 with a peptide from human insulin demonstrated how the disease-associated beta 57 polymorphism, described by Hugh McDevitt’s laboratory, affects the peptide binding specificity. The absence of a negative charge at this polymorphic position in HLA-DQ8 profoundly changes the peptide repertoire that can be presented to T cells compared to HLA-DQ molecules with a negative charge at this position. We are now examining how these disease-associated MHC polymorphisms affect the T cell repertoire in animal models. Work in the NOD mouse model of type 1 diabetes has demonstrated a surprising degree of expansion of potentially pathogenic T cells in the thymus. Based on these data, we pursue the hypothesis that MHC genes that confer susceptibility to autoimmune profoundly shape the T cell repertoire such that potentially pathogenic T cells are already enriched in the naïve repertoire that leaves the thymus.

Lee KH, Wucherpfennig KW, Wiley DC
Structure of a human insulin peptide/HLA-DQ8 complex and susceptibility to type 1 diabetes
Nature Immunology 2001, 2: 501-7. (PDF)

Smith KJ, Pyrdol J, Gauthier L, Wiley DC, Wucherpfennig KW
Crystal structure of HLA-DR2 (DRA*0101, DRB1*1501) complexed with a peptide from human myelin basic protein
J. Exp. Med. 1998; 188: 1511-20 © Rockefeller University Press.(PDF)

Development of a novel approach for the generation of MHC class II tetramers: a tool for ex vivo analysis of the T cell repertoire

Analysis of the naïve T cell repertoire in the thymus requires an approach with which T cells directed against a particular peptide can be isolated. Since conventional assays of T cell function are not suitable for analysis of immature T cells, we have developed a novel approach for the generation of MHC class II tetramers with which a single protein preparation can be used for the creation of a series of tetramers. This approach is based on the natural peptide loading mechanism in which the MHC class II associated invariant chain is proteolytically cleaved so that only the CLIP peptide remains in the peptide binding site. Exchange of CLIP with other peptides is then catalyzed by HLA-DM, a MHC-like protein that promotes dissociation of MHC class II bound peptides. We expressed MHC class II molecules with a covalently linked CLIP peptide which can be exchanged with any peptide following cleavage of the linker with thrombin. With these tetramers, we have been able to isolate T cells of defined specificity in human diseases and in animal models. In ongoing studies, we are using this approach to characterize the T cell repertoire both in the thymus and the target organ in animal models of autoimmunity.

Jang M-H, Seth N, Wucherpfennig KW
Ex vivo analysis of thymic CD4 T cells in NOD mice with tetramers generated from I-Ag7/class II-associated invariant chain peptide precursors
J. Immunol., 2003; 171: 4175-86.(PubMed)

Day CL, Seth N, Lucas M, Appel H, Gauthier L, Lauer GM, Robbins GK, Szczepiorkowski ZM, Casson DR, Chung RT, Bell S, Harcourt G, Walker BD, Klenerman P, Wucherpfennig KW
Ex vivo analysis of human memory CD4 T cells specific for hepatitis C virus using MHC class II tetramers
J. Clin. Invest., 2003; 112: 831-42.(PubMed)

Mechanism for the activation of self-reactive T cells in
autoimmune diseases

A large body of literature indicates an important link between infection and the development of autoimmune diseases. We have shown that T cell receptors can recognize not only a single peptide, but rather a number of distinct peptides that have only limited sequence similarity. These findings have provided experimental support to the idea that autoimmune diseases can be caused by “molecular mimicry” – sequence similarity between pathogen-derived proteins and self-antigens.

Such a degree of crossreactivity is a general property of TCR recognition, and is relevant in several other contexts. During T cell development in the thymus, T cells are “positively selected” by recognition of crossreactive self-peptide/MHC complexes and recognition of such crossreactive complexes also supports the survival of naïve T cells in the periphery. Crossreactivity also decreases the likelihood that mutations within a particular viral epitope will result in escape from immune recognition, and increases the likelihood that other viral strains are recognized by previously expanded T cells. We are now examining the structural mechanisms by which T cells can recognize peptides with limited sequence similarity.

 

Wucherpfennig KW
Structural basis of molecular mimicry
J. Autoimm. 2001; 16: 293-302.(PubMed)

Hausmann S, Martin M, Gauthier L, Wucherpfennig KW
Structural features of autoreactive T-cell receptors that determine the degree of degeneracy in peptide recognition
J. Immunol. 1999; 162: 338-44.(PubMed)

Wucherpfennig KW, Strominger JL
Molecular mimicry in T cell mediated autoimmunity: Viral peptides activate human T cell clones specific for myelin basic protein
Cell 1995; 80: 695-705.(PubMed)