Structure of a TCR with high affinity for self-antigen reveals basis for escape from negative selection
2011; Springer Nature; Volume: 30; Issue: 6 Linguagem: Inglês
10.1038/emboj.2011.21
ISSN1460-2075
AutoresYiyuan Yin, Yili Li, Melissa C. Kerzic, Roland Martinꝉ, Roy A. Mariuzza,
Tópico(s)Immunotherapy and Immune Responses
ResumoArticle4 February 2011free access Structure of a TCR with high affinity for self-antigen reveals basis for escape from negative selection Yiyuan Yin Yiyuan Yin Institute for Bioscience and Biotechnology Research, University of Maryland, WM Keck Laboratory for Structural Biology, Rockville, MD, USA Program in Molecular and Cell Biology, University of Maryland, College Park, MD, USA Search for more papers by this author Yili Li Yili Li Institute for Bioscience and Biotechnology Research, University of Maryland, WM Keck Laboratory for Structural Biology, Rockville, MD, USA Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, USA Search for more papers by this author Melissa C Kerzic Melissa C Kerzic Institute for Bioscience and Biotechnology Research, University of Maryland, WM Keck Laboratory for Structural Biology, Rockville, MD, USA Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, USA Search for more papers by this author Roland Martin Roland Martin Center for Molecular Neurobiology Hamburg, Institute for Neuroimmunology and Clinical Multiple Sclerosis Research, University Medical Center Eppendorf, Hamburg, Germany Search for more papers by this author Roy A Mariuzza Corresponding Author Roy A Mariuzza Institute for Bioscience and Biotechnology Research, University of Maryland, WM Keck Laboratory for Structural Biology, Rockville, MD, USA Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, USA Search for more papers by this author Yiyuan Yin Yiyuan Yin Institute for Bioscience and Biotechnology Research, University of Maryland, WM Keck Laboratory for Structural Biology, Rockville, MD, USA Program in Molecular and Cell Biology, University of Maryland, College Park, MD, USA Search for more papers by this author Yili Li Yili Li Institute for Bioscience and Biotechnology Research, University of Maryland, WM Keck Laboratory for Structural Biology, Rockville, MD, USA Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, USA Search for more papers by this author Melissa C Kerzic Melissa C Kerzic Institute for Bioscience and Biotechnology Research, University of Maryland, WM Keck Laboratory for Structural Biology, Rockville, MD, USA Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, USA Search for more papers by this author Roland Martin Roland Martin Center for Molecular Neurobiology Hamburg, Institute for Neuroimmunology and Clinical Multiple Sclerosis Research, University Medical Center Eppendorf, Hamburg, Germany Search for more papers by this author Roy A Mariuzza Corresponding Author Roy A Mariuzza Institute for Bioscience and Biotechnology Research, University of Maryland, WM Keck Laboratory for Structural Biology, Rockville, MD, USA Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, USA Search for more papers by this author Author Information Yiyuan Yin1,2, Yili Li1,3, Melissa C Kerzic1,3, Roland Martin4 and Roy A Mariuzza 1,3 1Institute for Bioscience and Biotechnology Research, University of Maryland, WM Keck Laboratory for Structural Biology, Rockville, MD, USA 2Program in Molecular and Cell Biology, University of Maryland, College Park, MD, USA 3Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, USA 4Center for Molecular Neurobiology Hamburg, Institute for Neuroimmunology and Clinical Multiple Sclerosis Research, University Medical Center Eppendorf, Hamburg, Germany *Corresponding author. Institute for Bioscience and Biotechnology Research, University of Maryland, WM Keck Laboratory for Structural Biology, 9600 Gudelsky Drive, Rockville, MD 20850, USA. Tel.: +1 240 314 6243; Fax: +1 240 314 6225; E-mail: [email protected] The EMBO Journal (2011)30:1137-1148https://doi.org/10.1038/emboj.2011.21 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The failure to eliminate self-reactive T cells during negative selection is a prerequisite for autoimmunity. To escape deletion, autoreactive T-cell receptors (TCRs) may form unstable complexes with self-peptide–MHC by adopting suboptimal binding topologies compared with anti-microbial TCRs. Alternatively, escape can occur by weak binding between self-peptides and MHC. We determined the structure of a human autoimmune TCR (MS2-3C8) bound to a self-peptide from myelin basic protein (MBP) and the multiple sclerosis-associated MHC molecule HLA-DR4. MBP is loosely accommodated in the HLA-DR4-binding groove, accounting for its low affinity. Conversely, MS2-3C8 binds MBP–DR4 as tightly as the most avid anti-microbial TCRs. MS2-3C8 engages self-antigen via a docking mode that resembles the optimal topology of anti-foreign TCRs, but is distinct from that of other autoreactive TCRs. Combined with a unique CDR3β conformation, this docking mode compensates for the weak binding of MBP to HLA-DR4 by maximizing interactions between MS2-3C8 and MBP. Thus, the MS2-3C8–MBP–DR4 complex reveals the basis for an alternative strategy whereby autoreactive T cells escape negative selection, yet retain the ability to initiate autoimmunity. Introduction The ability of the immune system to distinguish between non-self pathogens and self-antigens involves the elimination or inactivation of autoreactive T cells during T-cell ontogeny in the thymus, thereby avoiding immune responses to self. However, in autoimmune diseases such as multiple sclerosis (MS) and type 1 diabetes, the presence of autoreactive T cells in the periphery demonstrates that this filtering process is imperfect. Thymic selection is based on recognition of self-peptide–MHC complexes, whereby weak interactions with self-peptide–MHC promote T-cell survival (positive selection), whereas strong interactions induce apoptosis (negative selection) (Kappler et al, 1987; Goodnow et al, 2005; Daniels et al, 2006; Zehn and Bevan, 2006). Failure of negative selection may result from reduced T-cell receptor (TCR) affinity for self-peptide–MHC ligands, such that the complex with TCR is too short-lived to permit negative selection, yet sufficiently stable for activation in the periphery at high antigen concentrations (Ohashi, 2003; Goodnow et al, 2005) or activation by cross-reactive foreign peptide ligands (Hemmer et al, 1997). Alternatively, unusually weak binding of the self-peptide to MHC could effectively destabilize the complex with TCR (Anderton et al, 2001; Stadinski et al, 2010). Both these affinity-based mechanisms for escaping negative selection are supported by animal models of autoimmune disease (Ohashi, 2003; Goodnow et al, 2005), and by binding measurements which have demonstrated correlations between the longevity (half-life) of TCR–peptide–MHC complexes and selection outcome (Alam et al, 1996; Williams et al, 1999; Daniels et al, 2006). Structural studies of autoimmune TCRs bound to self-peptide–MHC ligands have begun to reveal how such TCRs escape negative selection in the thymus, but still retain the ability to productively engage self-antigens in the periphery (Deng and Mariuzza, 2007; Wucherpfennig et al, 2009). Five structures have been reported to date, involving two human TCRs from MS patients (Hahn et al, 2005; Li et al, 2005) and three mouse TCRs from the experimental autoimmune encephalomyelitis (EAE) model of MS (Maynard et al, 2005; Feng et al, 2007). The human TCRs are Ob.1A12, which recognizes myelin basic protein (MBP) peptide 85–99 in the context of HLA-DR2b (Hahn et al, 2005), and 3A6, which recognizes MBP 89–101 presented by HLA-DR2a (Li et al, 2005). Both Ob.1A12 and 3A6 engage their MBP–HLA-DR ligands with docking topologies that differ substantially from the way most TCRs specific for microbial or other foreign epitopes bind peptide–MHC. Whereas anti-microbial TCRs typically dock on peptide–MHC in a diagonal orientation over the centre of the antigenic peptide, Ob.1A12 and 3A6 are displaced towards the N-terminus of MBP (Wucherpfennig et al, 2009). This suboptimal binding mode results in fewer specific interactions with the self-peptide, which is manifested by the much lower affinities of Ob.1A12 and 3A6 compared with anti-microbial TCRs. Although anti-microbial TCRs usually adopt a central diagonal orientation, deviations from this topology have been described (Ely et al, 2008). For example, an MHC class I-restricted TCR (CF34) specific for an Epstein-Barr virus peptide presented by HLA-B8 was reported, in which the TCR is shifted towards the N-terminus of the bound peptide in a manner reminiscent of 3A6 (Gras et al, 2009). Moreover, TCR CF34 binds its ligand with reasonably high affinity. Nevertheless, an emerging pattern for autoimmune TCRs appears to be to form suboptimal interactions with self-ligands that ultimately permit escape from thymic deletion (Ely et al, 2008; Wucherpfennig et al, 2009). The three mouse TCRs (172.10, 1934.4 and cl19) recognize acetylated MBP 1–11 (MBP Ac1–11) presented by I-Au. The MBP–I-Au ligand is structurally defective in that the peptide only partially fills the MHC-binding groove (He et al, 2002). As a result, these mouse TCRs, like Ob.1A12 and 3A6, also exhibit suboptimal interactions with the MBP self-peptide, marked by a paucity of specific contacts (Maynard et al, 2005; Feng et al, 2007). Together, these studies suggest that structural alterations which destabilize the TCR–peptide–MHC recognition unit may enable certain self-reactive T cells to escape negative selection, without necessarily compromising their ability to cause autoimmune disease. Indeed, mice transgenic for TCR Ob.1A12 and HLA-DR2b developed central nervous system (CNS) inflammation (Madsen et al, 1999), and transfer into mice of T-cell clones recognizing MBP–I-Au induced EAE (Goverman, 2009). Despite these advances, the database of autoimmune TCR–peptide–MHC complexes is still too limited to describe the recognition properties of autoreactive TCRs at the repertoire level, or to fully assess the structural mechanisms underlying escape from negative selection (Wucherpfennig et al, 2009). Accordingly, we determined the structure of a human autoimmune TCR (MS2-3C8) in complex with a self-peptide from MBP (MBP 111–129) and the MS-associated MHC class II molecule HLA-DR4 (HLA-DRB1*0401). Notably, this peptide represents an immunodominant epitope of MBP in HLA-DR4-positive MS patients (Muraro et al, 1997; Sospedra and Martin, 2005). Unlike the MBP peptides recognized by TCRs Ob.1A12 and 3A6, which bind with high affinity to HLA-DR2 and elicit a diverse TCR repertoire, MBP 111–129 binds weakly to HLA-DR4 and is recognized by a restricted TCR repertoire. The MBP 111–129-specific T-cell clone MS2-3C8 was repeatedly isolated from the peripheral blood of a patient with relapsing-remitting MS over a 2-year period (Muraro et al, 1997). In addition, the clonal overrepresentation and persistent expansion of MS2-3C8 T cells in this patient suggested an involvement in the disease process. The pathogenic potential of TCR MS2-3C8 was demonstrated using transgenic mice expressing this autoimmune TCR and HLA-DR4 (Quandt et al, 2004). These humanized mice readily developed EAE in adoptive transfer experiments without antigen administration. Interestingly, the clinical phenotype of TCR MS2-3C8/HLA-DR4 transgenic mice with brain stem involvement reiterated clinical findings in the MS patient from whom the TCR was derived (Quandt et al, 2004). The structure of the MS2-3C8–MBP–DR4 complex revealed the basis for the weak binding of MBP to HLA-DR4, which likely enabled MS2-3C8 T cells to survive thymic deletion. Furthermore, the structure showed how, at the atomic level, an autoreactive TCR can compensate for an unstable self-antigen to achieve sufficiently high affinity for T-cell activation in the periphery and the induction of autoimmunity. Results Interaction of TCR MS2-3C8 with MBP–HLA-DR4 To characterize the interaction between MS2-3C8 (Vα4.1Jα32, Vβ2.1Jβ1.6) and MBP–DR4, we expressed recombinant TCR by in vitro folding from bacterial inclusion bodies. To produce soluble MBP–DR4, we first attempted folding the MHC class II α and β chains in vitro in the presence of MBP 111–129 or MBP 114–126, which is indistinguishable from MBP 111–129 in terms of MHC binding and T-cell stimulation (Muraro et al, 1997). However, yields were extremely low and the HLA-DR4 α and β chains dissociated during purification, presumably due to the weak interaction between peptide and MHC. Indeed, the low affinity of MBP 111–126 for HLA-DR4 is well documented in the literature. In one study, MBP 111–126 was found to bind HLA-DR4 ∼75-fold less tightly than influenza virus hemagglutinin peptide 307–319 (HA), a good binder (Muraro et al, 1997). In another study, the affinity of this MBP peptide for HLA-DR4 was estimated at 2000 nM, which is in the low range for peptide–MHC interactions (Valli et al, 1993). To permit in vitro folding, we covalently attached MBP 114–126 (FSWGAEGQRPGFG) to the N-terminus of the HLA-DR4 β chain via a 16-mer peptide linker (Fremont et al, 1996). We used surface plasmon resonance (SPR) to measure the affinity and kinetics of TCR MS2-3C8 binding to MBP–DR4. To permit directional coupling to a streptavidin-coated biosensor surface, a biotinylation sequence was added to the C-terminus of the DR4 α chain. Kinetic parameters (on- and off-rates) for the binding of soluble TCR MS2-3C8 to immobilized HLA-DR4 were kon=1.9 × 103 M−1 s−1 and koff=0.011 s−1, corresponding to a dissociation constant (KD) of 5.5 μM (Figure 1A). Under equilibrium binding conditions, a KD of 5.0 μM was obtained (Figure 1B), in close agreement with the KD from kinetic analysis. The affinity of MS2-3C8 for its self-antigen is therefore at the high end of the range for TCR interactions with peptide–MHC class I or peptide–MHC class II (1 to >100 μM) (van der Merwe and Davis, 2003; Cole et al, 2007). Indeed, MS2-3C8 binds MBP–DR4 about as tightly as the most avid anti-microbial class I-restricted TCRs bind their respective ligands, and considerably more tightly than do anti-microbial class II-restricted TCRs, such as HA1.7, which recognizes HA–DR4 with KD=40 μM (Cole et al, 2007). It also binds far more tightly than do human autoimmune TCRs 3A6 and Ob.1A12 (KD>100 μM) (Appel et al, 2000; Li et al, 2005), but similarly to mouse autoimmune TCR 172.10 (6 μM) (Garcia et al, 2001). Moreover, the off-rate of the interaction (0.011 s−1, corresponding to a half-life of 69 s) is exceptionally slow, compared with the off-rates of all other TCR–peptide–MHC interactions characterized to date (0.02 to >1 s−1) (Cole et al, 2007). For TCR 172.10, the off-rate of its interaction with MBP–I-Au is ∼20-fold faster (0.22 s−1) (Garcia et al, 2001). The slow off-rate of MS2-3C8 is counterbalanced by a very slow on-rate (1.9 × 103 M−1 s−1) relative to the on-rates of other TCRs (2 × 103 to >1 × 106 M−1 s−1), including 172.10 (3.7 × 104 M−1 s−1), which may imply conformational changes in MS2-3C8 and/or MBP–DR4 during complex formation. Figure 1.SPR analysis of the binding of TCR MS2-3C8 to MBP–DR4. (A) For kinetic measurements, TCR MS2-3C8 at concentrations of 2.9, 5.8, 11.5, 23 and 46 μM was injected over immobilized MBP–DR4 (320 RU). Sensograms were fitted to a 1:1 binding model to obtain on- and off-rates: kon=1.9 × 103±0.01 M−1 s−1 and koff=0.011±0.0003 s−1. KD was calculated as koff/kon. (B) For equilibrium measurements, TCR MS2-3C8 at concentrations of 1.2, 2.4, 4.7, 9.4 and 18.8 μM was injected over immobilized MBP–DR4 (400 RU). Inset shows the fitting curve for equilibrium binding that resulted in a KD of 5.0±0.1 μM. Equilibrium measurements for TCR MS2-3C8 mutants are also shown: (C) CDR1α T29A, (D) CDR3α K96A, (E) CDR2β E50A, (F) CDR2β T55A, (G) CDR3β S98A and (H) CDR3β N100A. For weakly binding mutants CDR3β S98A and CDR3β N100A, KDs were estimated at >300 and >100 μM, respectively. Download figure Download PowerPoint Although these results indicate that the intrinsic affinity of MS2-3C8 for MBP–DR4 is surprisingly high, it is important to note that SPR measurements were conducted using an engineered version of the ligand designed to overcome the low affinity of MBP for HLA-DR4. However, the instability of the natural MBP–DR4 ligand would effectively decrease the half-life of the overall complex with TCR, which most likely allowed MS2-3C8 T cells to survive thymic deletion. At the same time, the tight binding of MS2-3C8 to MBP–DR4 at least partially compensates for the weak binding of MBP to HLA-DR4, thereby explaining the ability of this autoreactive TCR to induce CNS inflammation in MS2-3C8–DR4 transgenic mice (Quandt et al, 2004). Overview of the MS2-3C8–MBP–DR4 complex To understand why the MBP self-peptide binds weakly to HLA-DR4, and to test the hypothesis that the canonical docking mode of anti-foreign TCRs has evolved to provide optimal binding to peptide–MHC (Rudolph et al, 2006; Marrack et al, 2008), we determined the structure of MS2-3C8 in complex with MBP–DR4 to 2.8 Å resolution (Table I; Figure 2A). The interface between TCR and peptide–MHC was in unambiguous electron density for each of the two molecules in the asymmetric unit of the crystal (Figure 2B). Several loops in the MHC α2β2 and TCR CαCβ modules lacked electron density; however, these disordered regions are distant from the interface. The 16-mer peptide linking MBP to the N-terminus of the HLA-DR4 β chain exhibited clear electron density in one of the two complexes in the asymmetric unit, probably due to crystal contacts with the Cβ domain of a neighbouring complex molecule; no density could be detected for the linker peptide in the other complex, indicating flexibility. The r.m.s. deviation in α-carbon positions for the TCR VαVβ and MHC α1β1 modules, including the MBP peptide, is 0.22 Å for the two complexes in the asymmetric unit. Based on this close similarity, the following description of TCR–peptide–MHC interactions applies to both complex molecules. Figure 2.Structure of the MS2-3C8–MBP–DR4 complex. (A) Side view of the MS2-3C8–MBP–DR4 complex (ribbon diagram), including the 16-mer peptide linking MBP to the HLA-DR4 β chain. TCR α chain, blue; TCR β chain, green; MHC α chain, grey; MHC β chain, yellow; MBP peptide (stick representation), magenta; linker peptide, orange. (B) Electron density in the interface of the MS2-3C8–MBP–DR4 complex. Density from the final 2Fo−Fc map at 2.8 Å resolution is contoured at 1 σ. Download figure Download PowerPoint Table 1. Data collection and refinement statistics Data collection Space group P21212 Resolution (Å) 49.2–2.80 Unit cell (Å) a=102.5, b=218.4, c=98.4 Unique reflections 55 156 Redundancya 14.2 (5.0) Completeness (%)a 100 (100) Mean I/σ (I)a 29.7 (5.2) Rmerge (%)a,b 8.8 (35.5) Refinement Resolution range (Å) 49.2–2.80 Rwork (%)c 23.9 Rfree (%)c 27.9 Protein atoms 12 876 Water molecules 27 R.m.s.d. from ideality Bond lengths (Å) 0.010 Bond angles (deg) 1.339 Ramachandran plot statistics Favoured (%) 92.0 Allowed (%) 7.8 Outliers (%) 0.2 a Values in parentheses correspond to the highest resolution shell (2.80–2.90). b Rmerge(I)=(Σ∣I(i)− ∣/ΣI(i)), where I(i) is the ith observation of the intensity of the hkl reflection and is the mean intensity from multiple measurements of the hkl reflection. c Rwork (Rfree)=Σ∣∣Fo∣−∣Fc∣∣/Σ∣Fo∣; 5% of data were used for Rfree. Compared with anti-microbial TCRs such as HA-specific TCR HA1.7 (Hennecke and Wiley, 2002), which typically dock over the central portion of the foreign peptide and interact symmetrically with the MHC α1 and β1 α helices (Figure 3A), the autoimmune TCRs Ob.1A12 and 3A6 (Hahn et al, 2005; Li et al, 2005) display substantially altered binding geometries, in which the TCR is shifted towards the N-terminus of the self-peptide and towards the MHC β1 α helix (Figure 3B and C). In sharp contrast to Ob.1A12 and 3A6, MS2-3C8 engages its self-ligand via a binding mode which closely resembles that of anti-foreign TCRs (Figure 3D), as exemplified by the HA1.7–HA–DR4 complex (Figure 3A). Thus, MS2-3C8 docks symmetrically over MBP–DR4 in a canonical diagonal orientation, with a crossing angle of TCR to peptide–MHC (Reinherz et al, 1999) of 65°, compared with 70° for the HA1.7–HA–DR4 complex. Importantly, mouse autoimmune TCR 172.10 also recognizes its self-ligand in a canonical manner (Maynard et al, 2005), implying that MS2-3C8 and 172.10 use similar mechanisms to escape negative selection (see Discussion). In addition, MS2-3C8 is positioned directly over the central P5 residue of MBP, rather than being displaced towards the N-terminus of the self-peptide, as in the Ob.1A12–MBP–DR2b and 3A6–MBP–DR2a complexes. As discussed later, this docking mode, along with a unique CDR3β conformation, optimizes interactions between MS2-3C8 and the self-peptide. Figure 3.Comparison of human anti-microbial and autoimmune TCR–peptide–MHC class II complexes. (A) Upper panel: top view of the anti-microbial HA1.7–HA–DR4 complex (PDB accession code 1J8H) (Hennecke and Wiley, 2002). Colours of TCR, MHC and peptide are the same as in Figure 2. The central P5 residue of the peptide is shown as a red sphere. Bottom panel: position of the TCR CDR3 loops over the HA peptide. The peptide is drawn in ball-and-stick representation with carbon atoms in pink, oxygen atoms in red and nitrogen atoms in blue. The CDR loops are positioned above the central P5 residue of the peptide. (B) The autoimmune Ob.1A12–MBP–DR2b complex (1YMM) (Hahn et al, 2005). (C) The autoimmune 3A6–MBP–DR2a complex (1ZGL) (Li et al, 2005). (D) The autoimmune MS2-3C8–MBP–DR4 complex. The comparison shows that autoimmune TCR MS2-3C8, like anti-microbial TCR HA1.7, docks symmetrically over peptide–MHC in a central diagonal orientation, whereas autoimmune TCRs Ob.1A12 and 3A6 are displaced towards the peptide N-terminus and the HLA-DR β1 helix. The CDR3 loops of HA1.7 and MS2-3C8 are centred over peptide residue P5, those of Ob.1A12 and 3A6 are positioned over P2. Download figure Download PowerPoint Interaction of MBP with HLA-DR4 Based on the structure, the primary anchor residues for MBP 114–126 bound to HLA-DR4 are Trp116 (P1) and Gln121 (P6); the secondary anchor residues are Glu119 (P4), Arg122 (P7) and Gly124 (P9). Although Trp116 fulfills the need for an aromatic residue at P1 for efficient binding to HLA-DR4 and Glu119 at P4 is a suitable anchor as well, Gln121 does not meet the requirement for a small residue at P6 (Hammer et al, 1993, 1995; Southwood et al, 1998). Secondary anchor residue P7 Arg122 also does not conform to the optimal binding motif for HLA-DR4, which calls for an aliphatic residue at this position. To understand the basis for the weak binding of MBP to HLA-DR4, the conformation of the MBP peptide was directly compared with those of two peptides that bind HLA-DR4 with high affinity: HA and collagen II peptide 1168–1180 (CII) (Hammer et al, 1993, 1995). HA and CII display very similar main-chain conformations in their corresponding complexes with HLA-DR4, from residues P1 to P9 (Dessen et al, 1997; Hennecke and Wiley, 2002) (Figure 4A). However, MBP diverges from HA and CII at residues P6 and P7, with the result that MBP sits less deeply in the peptide-binding groove than HA or CII. Figure 4.Basis for low-affinity binding of MBP to HLA-DR4. (A) Conformation of high- and low-affinity peptides bound to HLA-DR4. The α-carbon backbone of MBP (magenta) was compared with the α-carbon backbones of hemagglutinin peptide (HA) (green) and collagen II peptide (CII) (yellow) by superposing the α1β1 domains of HLA-DR4 in the MBP–DR4, HA–DR4 (1J8H) (Hennecke and Wiley, 2002) and CII–DR4 (2SEB) (Dessen et al, 1997) complexes. The peptides are viewed from the side of the β1 helix. HA and CII bind HLA-DR4 with much higher affinity than MBP. MBP diverges from HA and CII at residues P6 and P7. (B, C) Interactions between MBP (B) or HA (C) and the β-sheet floor of the HLA-DR4-binding groove (grey). The peptides are oriented the same as in (A). Hydrogen bonds are shown as dotted red lines. The arrow in (C) indicates the hydrogen bond between P7 and Tyr30β in the HA–DR4 complex that is absent in the MBP–DR4 complex, where the corresponding atoms are too distant (4.9 Å) for hydrogen bond formation (B). (D, E) Interactions between MBP (D) or HA (E) and the α1 and β1 helices of HLA-DR4. The arrows in (E) indicate three additional hydrogen bonds in the HA–DR4 complex not present in the MBP–DR4 complex (D). Download figure Download PowerPoint In HA and CII, anchor residues P6 and P7 have short side chains (HA: Thr-Leu; CII: Ala-Ala), whereas in MBP these residues have long side chains (Gln-Arg). The shallower binding of MBP is mainly attributable to these longer side chains, but for different reasons. Thus, the side chain of P6 Gln pushes up the MBP main chain by pointing directly into the P6 pocket of HLA-DR4 (Figure 4B), in the same direction as the shorter side chain of HA P6 Thr (Figure 4C). By contrast, the long side chain of P7 Arg projects out towards the top of the DR4 β1 helix, rather than into the P7 pocket, in order to avoid colliding with the α1β1 platform if it pointed in the same direction as the side chain of HA P7 Leu (Figure 4B and D). Therefore, P7 Arg is not positioned as a typical anchor in MBP–DR4. Instead, this residue interacts extensively with TCR (see below). In the HA–HLA-DR4 structure, the HA peptide forms four hydrogen bonds with β-sheet residues on the floor of the peptide-binding groove (Figure 4C). However, the elevation of MBP results in loss of the hydrogen bond between the main-chain nitrogen of P7 and the Oη atom of Tyr30β (Figure 4B). In addition, a twist in MBP eliminates a hydrogen bond between P7 and DR4 Asn69α found in the HA–DR4 complex (Figure 4D and E). The MBP–DR4 complex also lacks two side-chain–side-chain hydrogen bonds to Glu55α and Asn62α, due to the absence of suitable hydrogen bond donors at P-1 and P3. This net loss of four hydrogen bonds, along with 35 fewer van der Waals between peptide and MHC, likely explains the 75-fold lower affinity of MBP than HA for HLA-DR4 (Muraro et al, 1997). Interaction of TCR MS2-3C8 with HLA-DR4 The MS2-3C8–MBP–DR4 complex buries a total solvent-accessible surface of 2010 Å2, comparable to that in other MHC class II complexes (Rudolph et al, 2006). The buried surface area on Vβ (670 Å2, 66%) is nearly twice that on Vα (350 Å2, 34%) (Figure 5A). Such dominance by Vβ is unusual among TCR–peptide–MHC complexes, in which Vα and Vβ typically contribute roughly equal buried surfaces, as in the HA1.7–HA–DR4 complex (Vα: 47%; Vβ: 53%) (Figure 5B), or in which Vα dominates. Indeed, only three other complexes displaying a similar degree of Vβ dominance as MS2-3C8–MBP–DR4 have been reported, involving the HLA-A2-restricted TCR JM22 (67%) (Stewart-Jones et al, 2003), the H-2Kb-restricted TCR BM3.3 (63%) (Reiser et al, 2000) and the MBP-specific TCR 3A6 (61%) (Li et al, 2005). Overall, Vβ makes 57 van der Waals contacts with HLA-DR4, compared with only 19 by Vα. These contacts are mediated by 11 Vβ and 6 Vα residues and involve 15 MHC residues (Table II), of which 12 are contacted by HA1.7 and 10 by 3A6 (Supplementary Table S1). Figure 5.Comparison of TCR footprints and interactions by CDR1 and CDR2 loops of MS2-3C8 and HA1.7. (A) Footprint of autoimmune TCR MS2-3C8 on MBP–DR4. The top of the MHC molecule is represented as a grey surface; the bound peptide (pink) is shown in stick format under the transparent surface. The areas contacted by individual CDR loops are colour-coded: CDR1α, violet; CDR2α, purple; CDR3α, blue; CDR1β, yellow; CDR2β, orange; CDR3β, green. The contact areas of the V domains are contoured: Vα, yellow; Vβ, red. (B) Footprint of anti-microbial TCR HA1.7 on HA–DR4. (C, D) Conserved interactions between the DR4 β1 helix and the CDR1α/2α loops of MS2-3C8 (C) or HA1.7 (D). CDR1α, violet; CDR2β, purple; DR4 β chain, yellow. (E, F) Conserved interactions between the DR4 α1 helix and the CDR1β/2β loops of MS2-3C8 (E) or HA1.7 (F). CDR1β, yellow; CDR2β, orange; DR4 α chain, grey. Van der Waals contacts are shown as dotted black lines, hydrogen bonds as dotted red lines and salt bridges as solid red lines. Download figure Download PowerPoint Table 2. Interactions between TCR MS2-3C8 and MBP–HLA-DR4 TCR Hydrogen bonds Number of van der Waals contacts TCR-DR4 contacts CDR1α Ser 27α Oγ His 81β Nε2 1 Gly 28α His 81β 3 Thr 29α O Thr 77β Oγ 3 His 81β 6 CDR2α Leu 50α Thr 77β 1 CDR3α Asn 95α Gly 58α 1 Ala 61α 1 Lys 96α Nη Gln 57α Oε 3 CDR1β Thr 29β Ala 61α 1 Ala 64α 3 Val 65α 6 Thr 30β Ala 61α 1 CDR2β Asn 49β Leu 60α 2 Ala 61α 6 Ala 64α 1 Glu 50β Oε Lys 67α Nη 1 Thr 55β Oγ Lys 39α Nη Gln 57α 1 CDR3β Arg 95β Ala 61α 1 Gly 97β Val 65α 2 Ser 98β Gly 58α 1 Ala 61α 5 Oγ Asn 62α Oδ 4 Val 65α 1 Tyr 99β Phe 54α 2 Gly 58α 9 Ala 59α 1 Asn 62α 2 Asn 100β O Gln 70β Nε 6 Pro 102β Asp 66β 1 TCR-MBP contacts CDR1α Ser 27α Ser P-1 3 CDR3α Gly 92α Trp P1 1 Gly P2 3
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