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• W2162622550 abstract "Molecular mimicry is discussed as a possible mechanism that may contribute to the development of autoimmune diseases. It could also be involved in the differential association of the human major histocompatibility subtypes HLA-B*2705 and HLA-B*2709 with ankylosing spondylitis. These two subtypes differ only in residue 116 of the heavy chain (Asp in B*2705 and His in B*2709), but the reason for the differential disease association is not understood. Using x-ray crystallography, we show here that the viral peptide pLMP2 (RRRWRRLTV, derived from latent membrane protein 2 (residues 236–244) of Epstein-Barr virus) is presented by the B*2705 and B*2709 molecules in two drastically deviating conformations. Extensive structural similarity between pLMP2 and the self-peptide pVIPR (RRKWRRWHL, derived from vasoactive intestinal peptide type 1 receptor (residues 400–408)) is observed only when the peptides are presented by B*2705 because of a salt bridge between Arg5 of both peptides and the subtype-specific heavy chain residue Asp116. Combined with functional studies using pLMP2/pVIPR-cross-reactive cytotoxic T cell lines and clones, together with target cells presenting these peptides or a modified peptide analogue, our results reveal that a pathogen-derived peptide can exhibit major histocompatibility complex class I subtype-dependent, drastically distinct binding modes. Furthermore, the results demonstrate that molecular mimicry between pLMP2 and pVIPR in the HLA-B27 context is an allele-dependent property. Molecular mimicry is discussed as a possible mechanism that may contribute to the development of autoimmune diseases. It could also be involved in the differential association of the human major histocompatibility subtypes HLA-B*2705 and HLA-B*2709 with ankylosing spondylitis. These two subtypes differ only in residue 116 of the heavy chain (Asp in B*2705 and His in B*2709), but the reason for the differential disease association is not understood. Using x-ray crystallography, we show here that the viral peptide pLMP2 (RRRWRRLTV, derived from latent membrane protein 2 (residues 236–244) of Epstein-Barr virus) is presented by the B*2705 and B*2709 molecules in two drastically deviating conformations. Extensive structural similarity between pLMP2 and the self-peptide pVIPR (RRKWRRWHL, derived from vasoactive intestinal peptide type 1 receptor (residues 400–408)) is observed only when the peptides are presented by B*2705 because of a salt bridge between Arg5 of both peptides and the subtype-specific heavy chain residue Asp116. Combined with functional studies using pLMP2/pVIPR-cross-reactive cytotoxic T cell lines and clones, together with target cells presenting these peptides or a modified peptide analogue, our results reveal that a pathogen-derived peptide can exhibit major histocompatibility complex class I subtype-dependent, drastically distinct binding modes. Furthermore, the results demonstrate that molecular mimicry between pLMP2 and pVIPR in the HLA-B27 context is an allele-dependent property. Not all HLA-B27 subtypes are equally associated with the autoimmune disease ankylosing spondylitis (AS). 1The abbreviations used are: AS, ankylosing spondylitis; EBV, Epstein-Barr virus; HLA, human leukocyte antigen; MHC, major histocompatibility complex; HC, heavy chain; CTL, cytotoxic T lymphocyte(s); HIV, human immunodeficiency virus; TCR, T cell receptor.1The abbreviations used are: AS, ankylosing spondylitis; EBV, Epstein-Barr virus; HLA, human leukocyte antigen; MHC, major histocompatibility complex; HC, heavy chain; CTL, cytotoxic T lymphocyte(s); HIV, human immunodeficiency virus; TCR, T cell receptor. The frequent, prototypical subtype B*2705 is AS-associated, independent of ethnic origin, whereas B*2706 and B*2709, which exhibit geographically restricted distribution, are not (1Ramos M. López de Castro J.A. Tissue Antigens. 2002; 60: 191-205Crossref PubMed Scopus (97) Google Scholar). The products of the B*2705 and B*2709 alleles differ only in residue 116 of the HLA-B27 heavy chain (HC; Asp in B*2705 and His in B*2709) (2D'Amato M. Fiorillo M.T. Carcassi C. Mathieu A. Zuccarelli A. Bitti P.P. Tosi R. Sorrentino R. Eur. J. Immunol. 1995; 25: 3199-3201Crossref PubMed Scopus (184) Google Scholar). This residue is located at the floor of the peptide-binding groove, forms part of the F-pocket, and is buried upon binding of a peptide. Despite the close structural similarities, the subtypes give rise to distinct repertoires of bound peptides (3Ramos M. Paradela A. Vazquez M. Marina A. Vazquez J. López de Castro J.A. J. Biol. Chem. 2002; 277: 28749-28756Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) and cytotoxic T lymphocytes (CTL) (4Fiorillo M.T. Maragno M. Butler R. Dupuis M.L. Sorrentino R. J. Clin. Invest. 2000; 106: 47-53Crossref PubMed Scopus (158) Google Scholar). B*2705-positive but not B*2709-positive individuals possess CTL that recognize the HLA-B27-bound self-peptide pVIPR (RRK-WRRWHL). pVIPR is derived from vasoactive intestinal peptide type 1 receptor (residues 400–408) and is presented by B*2709 molecules in the common canonical conformation, whereas B*2705 presents the peptide in an unusual dual binding mode (5Hülsmeyer M. Fiorillo M.T. Bettosini F. Sorrentino R. Saenger W. Ziegler A. Uchanska-Ziegler B. J. Exp. Med. 2004; 199: 271-281Crossref PubMed Scopus (132) Google Scholar). pVIPR exhibits sequence homology with the peptide pLMP2 (RRRWRRLTV), derived from latent membrane protein 2 (residues 236–244) of Epstein-Barr virus (EBV). The existence of CTL reacting with both peptides in the context of B*2705 suggests a relationship between infection with EBV and an expansion of the pool of pLMP2/pVIPR-cross-reactive CTL (4Fiorillo M.T. Maragno M. Butler R. Dupuis M.L. Sorrentino R. J. Clin. Invest. 2000; 106: 47-53Crossref PubMed Scopus (158) Google Scholar). However, a direct correlation between EBV infection and AS pathogenesis has not been established. Molecular mimicry (6Oldstone M.B. Cell. 1987; 50: 819-820Abstract Full Text PDF PubMed Scopus (798) Google Scholar, 7Benjamin R. Parham P. Immunol. Today. 1990; 11: 137-142Abstract Full Text PDF PubMed Scopus (348) Google Scholar, 8Wucherpfennig K.W. J. Autoimmun. 2001; 16: 293-302Crossref PubMed Scopus (92) Google Scholar, 9Lang H.L. Jacobsen H. Ikemizu S. Andersson C. Harlos K. Madsen L. Hjorth P. Sondergaard L. Svejgaard A. Wucherpfennig K. Stuart D.I. Bell J.I. Jones E.Y. Fugger L. Nat. Immunol. 2002; 3: 940-943Crossref PubMed Scopus (462) Google Scholar), i.e. similarity in overall shape as well as charge distribution for an interaction surface (9Lang H.L. Jacobsen H. Ikemizu S. Andersson C. Harlos K. Madsen L. Hjorth P. Sondergaard L. Svejgaard A. Wucherpfennig K. Stuart D.I. Bell J.I. Jones E.Y. Fugger L. Nat. Immunol. 2002; 3: 940-943Crossref PubMed Scopus (462) Google Scholar), has been invoked as an explanation for the association of HLA-B27 and spondyloarthropathies (10Geczy A.F. Yap J. J. Rheumatol. 1982; 9: 97-100PubMed Google Scholar, 11Schwimmbeck P.L. Yu D.T. Oldstone M.B. J. Exp. Med. 1987; 166: 173-181Crossref PubMed Scopus (267) Google Scholar, 12Ramos M. Alvarez I. Sesma L. Logean A. Rognan D. López de Castro J.A. J. Biol. Chem. 2002; 277: 37573-37581Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), but its existence has yet to be proven (13Tiwana H. Walmsley R.S. Wilson C. Yiannakou J.Y. Ciclitira P.J. Wakefield A.J. Ebringer A. Br. J. Rheumatol. 1998; 37: 525-531Crossref PubMed Scopus (35) Google Scholar, 14Stone M.A. Payne U. Schentag C. Rahman P. Pacheco-Tena C. Inman R.D. Rheumatology. 2004; 43: 148-155Crossref PubMed Scopus (51) Google Scholar). A principal difficulty is related to the fact that a host-derived epitope is expected to share antigenic but not necessarily also extensive sequence homology with a foreign antigen (15Wucherpfennig K.W. Strominger J.L. Cell. 1995; 80: 695-705Abstract Full Text PDF PubMed Scopus (1277) Google Scholar). We have now determined the structures of both HLA-B27 subtypes in complex with pLMP2 and compare them here with the corresponding pVIPR complexes. Together with functional data, this comparison suggests that structural similarity and CTL cross-reactivity between pLMP2 and pVIPR in the context of HLA-B27 antigens are allele-dependent properties. HLA-B27-positive Donors, CTL Lines, and Clones—Seven patients with AS (with the exception of one B*2702-positive individual, all typed as B*2705) and two healthy individuals (one B*2705-positive and one B*2709-positive) were enrolled for this study (see Tables III, IV, and V). HLA-B27 typing and generation of pLMP2- and pVIPR-specific CTL lines were carried out as described (4Fiorillo M.T. Maragno M. Butler R. Dupuis M.L. Sorrentino R. J. Clin. Invest. 2000; 106: 47-53Crossref PubMed Scopus (158) Google Scholar). The CTL line MP VPAC7 was cloned by limiting dilution at 0.5–1 cell/well in 96-well U-bottom microplates in the presence of phytohemagglutinin (0.5 μg/ml), 3 × 104 allogeneic γ-irradiated peripheral blood mononuclear cells, and 20 units/ml recombinant interleukin-2 (Roche Applied Science). After 12 days, the growing cells were restimulated with pVIPR-pulsed, γ-irradiated autologous B-LCL and further expanded in the presence of recombinant interleukin-2 (20–50 units/ml).Fig. 1pLMP2 topographies, electron densities, and B factors when bound to B*2705 and B*2709. a and b, conformation of pLMP2 in B*2705 and B*2709, viewed from the side of the α2-helix together with a ribbon representation of the α1-helix and the floor (β-sheet) of the binding groove. The subtype-specific residue 116 is indicated (Asp116, red; His116, blue), with the bidentate salt bridge to Asp116 shown as dotted lines. In b, pArg5 points toward the viewer. c, superimposition of pLMP2 in B*2705 and B*2709, viewed as in a and b, showing that the peptide conformations in the two subtypes differ drastically from pTrp4 to pThr8. d and e, final 2Fo - Fc electron density contoured at the 1 σ level of pLMP2 conformations in B*2705 (d) and B*2709 (e). Water molecules are omitted for clarity from the representations in a–e. f and g, pLMP2 bound by B*2705 (f) and B*2709 (g), color-coded by isotropic B factor. A quantitative comparison is made difficult by the refinement strategies employed (anisotropic for B*2705 and isotropic for B*2709).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIIpLMP2-stimulated CTL and assessment of pLMP2/pVIPR cross-reactivityDonoraDonor CV is a healthy HLA-B*2705-positive individual. Donors MP, MA, BO, and EP are HLA-B*2705-positive patients with AS, and donor Ci is an HLA-B*2709-positive healthy individualTested on B*2705·pLMP2Tested on B*2709·pLMP2Tested on B*2705·pVIPRTested on B*2709·pVIPRCV4/4bNo. of CTL with positive reactivity/total no. of CTL tested4/40/40/4MP4/44/42/42/4MA3/33/30/30/3BO1/11/10/10/1EP4/44/40/40/4Ci9/99/91/4, 5 NTcNT, not tested2/9a Donor CV is a healthy HLA-B*2705-positive individual. Donors MP, MA, BO, and EP are HLA-B*2705-positive patients with AS, and donor Ci is an HLA-B*2709-positive healthy individualb No. of CTL with positive reactivity/total no. of CTL testedc NT, not tested Open table in a new tab Table IVpVIPR-stimulated CTL and assessment of pVIPR/pLMP2 cross-reactivity (only pVIPR-stimulated, pLMP2-cross-reactive CTL are shown)CTLaAll donors are HLA-B*2705-positive AS patientsTested on B*2705·pVIPRTested on B*2709·pVIPRTested on B*2705·pLMP2Tested on B*2709·pLMP2MP 7048bPercentage of specific lysis of T2-B*2705 or T2-B*2709 cells after background subtraction333947AS 3868633827AB 560514133MP VPAC 742456441MP VPAC 2252507862EP VIP 3767622031a All donors are HLA-B*2705-positive AS patientsb Percentage of specific lysis of T2-B*2705 or T2-B*2709 cells after background subtraction Open table in a new tab Table VpVIPR-stimulated CTL and assessment of pVIPR/pVIPR-pArg3 cross-reactivityCTLaAll donors are HLA-B*2705-positive AS patients except LV who is an HLA-B*2702-positive AS patientTested on B*2705·pVIPRTested on B*2709·pVIPRTested on B*2705·pVIPR-pArg3Tested on B*2709·pVIPR-pArg3EP 7641bPercentage of specific lysis of T2-B*2705 or T2-B*2709 cells after background subtraction4137LV 72735736PM 6554804NTcNT, not testedPM 46444897PM 45476313NTMP VPAC 757512311PM 5368632836LV 1585528NTAB 4425249NTAB 5605159NTPM 6375621077a All donors are HLA-B*2705-positive AS patients except LV who is an HLA-B*2702-positive AS patientb Percentage of specific lysis of T2-B*2705 or T2-B*2709 cells after background subtractionc NT, not tested Open table in a new tab Cytotoxicity Assays—Cytolytic activity of pLMP2-responsive (RRRWRRLTV), pVIPR-responsive (RRKWRRWHL), and pVIPR-pArg3-responsive (RRRWRRWHL) CTL lines and clones was assessed according to the standard 51Cr release procedure (4Fiorillo M.T. Maragno M. Butler R. Dupuis M.L. Sorrentino R. J. Clin. Invest. 2000; 106: 47-53Crossref PubMed Scopus (158) Google Scholar). Target cells (B*2705 or B*2709 T2 transfectants) were incubated overnight with the pLMP2 or pVIPR peptides at 70 μm or at lower concentrations (see “Results” and Fig. 4 for details) or in medium alone. One day later, the cells were labeled with sodium chromate and extensively washed before being mixed with effector T cells at 3 × 103 target cells/well. Analysis of TCR Gene Usage—Total RNA was extracted from 2 × 105 T cells, and cDNA was synthesized using oligo(dT) primer and SuperScript™ II RNase H- Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions. For the analysis of TCR α chain usage, cDNA was amplified using primers and PCR conditions as already described (16Lombardi G. Germain C. Uren J. du Fiorillo M.T. Bois R.M. Jones-Williams W. Saltini C. Sorrentino R. Lechler R. J. Immunol. 2001; 166: 3549-3555Crossref PubMed Scopus (95) Google Scholar). Additional oligonucleotides for Vα families 18–29 were designed as detailed previously (17Kalams S.A. Johnson R.P. Trocha A.K. Dynan M.J. Ngo H.S. D'Aquila R.T. Kurnick J.T. Walker B.D. J. Exp. Med. 1994; 179: 1261-1271Crossref PubMed Scopus (204) Google Scholar). PCR products were loaded on a 1.5% agarose gel stained with ethidium bromide. Specific DNA bands were cut from the gel and purified using a gel band purification kit (Amersham Biosciences). Direct sequencing was performed using an internal primer upstream of the TCR Cα reverse primer. Protein Preparation and Structure Determination—The peptide pLMP2 was purified by high pressure liquid chromatography (Alta Bioscience), and HLA-B27·peptide complexes were produced as described (5Hülsmeyer M. Fiorillo M.T. Bettosini F. Sorrentino R. Saenger W. Ziegler A. Uchanska-Ziegler B. J. Exp. Med. 2004; 199: 271-281Crossref PubMed Scopus (132) Google Scholar). Purified complexes (15–20 mg/ml in 20 mm Tris/HCl, 150 mm NaCl, 0.01% sodium azide, pH 7.5) were used for crystallization using hanging drop vapor diffusion and streak-seeding. Crystals suitable for x-ray diffraction experiments were grown in drops made of 1.5 μl of protein solution and 1.5 μl of precipitant solution (for B*2705, 15% polyethylene glycol 8000, 0.1 m Tris/HCl, pH 7.5, and for B*2709, 21% polyethylene glycol 8000, 0.1 m Tris/HCl, pH 8.5). Using glycerol as cryoprotectant, data sets were obtained from cryo-cooled (100 K) crystals at the BL2 beam line of BESSY-II. The data were processed with the HKL package (see Table I) (18Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38445) Google Scholar).Table IData collection and refinement statisticsHLA-B*2705·pLMP2HLA-B*2709·pLMP2Data collection Space groupP21P21 Unit cell (Å, Å, Å; °)51.1, 82.3, 65.9; 109.350.9, 82.6, 62.8; 104.4 Resolution (Å)aThe values in parentheses refer to the highest resolution shell40.0-1.55 (1.61-1.55)40.0-1.72 (1.78-1.72) Unique reflections74,438 (7225)53,353 (5036) Completeness (%)aThe values in parentheses refer to the highest resolution shell99.4 (97.0)99.4 (96.0) I/oaThe values in parentheses refer to the highest resolution shell21.0 (4.1)20.2 (3.7) RsymaThe values in parentheses refer to the highest resolution shell,bRsym = Σh Σi|Ih,i - 〈Ih〉|/ΣhΣiIh ,i0.051 (0.246)0.045 (0.262)Refinement Nonhydrogen atoms39923876 RcrystaThe values in parentheses refer to the highest resolution shell,cRcryst = Σh |Fo - Fc|/Σ Fo (working set, no σ cut-off applied)0.142 (0.179)0.154 (0.183) RfreeaThe values in parentheses refer to the highest resolution shell,dRfree is the same as Rcryst, but calculated on 5% of the data excluded from refinement0.177 (0.202)0.190 (0.224) Heavy chain, no. of atoms/average B factor (Å2)2301/20.02298/12.85 β2-Microglobulin, no. of atoms/average B factor (Å2)855/23.0860/17.9 Peptide, no. of atoms/average B factor (Å2)102/19.692/10.3 Water, no. of molecules/average B factor (Å2)710/38.7596/33.7 Glycerol, no. of atoms/average B factor (Å2)24/37.430/21.5 Estimated overall coordinate error (Å)eEstimated overall coordinate error based on Rfree as calculated by Refmac 5.1.1999 Root mean square deviation from ideal geometry Bond length (Å)0.0150.015 Bond angles (°)1.51.6a The values in parentheses refer to the highest resolution shellb Rsym = Σh Σi|Ih,i - 〈Ih〉|/ΣhΣiIh ,ic Rcryst = Σh |Fo - Fc|/Σ Fo (working set, no σ cut-off applied)d Rfree is the same as Rcryst, but calculated on 5% of the data excluded from refinemente Estimated overall coordinate error based on Rfree as calculated by Refmac 5.1.1999 Open table in a new tab The structure of B*2709·pLMP2 was determined by molecular replacement using peptide-stripped B*2709·m9 (Protein Data Bank code 1k5n) as a search model and the program Molrep (19Collaborative Computational Project, Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19730) Google Scholar). After rigid body refinement using Refmac (20Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16947) Google Scholar), the initial model was subjected to simulated annealing and energy minimization using CNS (21Murshudov G.N. Vagin A.A. Lebedev A. Wilson K.S. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 247-255Crossref PubMed Scopus (1007) Google Scholar) to remove model bias. Further refinement was carried out by iterative cycles of manual rebuilding using O (22Jones T.A. Kjeldgaard M. Methods Enzymol. 1997; 277: 173-208Crossref PubMed Scopus (504) Google Scholar) and restrained maximum-likelihood with Refmac comprising B-factor adjustment. Water molecules were included with ARP/wARP (23Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2563) Google Scholar). After translation, libration, screw rotation refinement (24Winn M.D. Isupov M.N. Murshudov G.N. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 122-133Crossref PubMed Scopus (1650) Google Scholar), the R factor converged at 0.154 (Rfree = 0.190). As the two HLA-B27·pLMP2 complexes crystallized isomorphously, initial phases for B*2705·pLMP2 were calculated from peptide-stripped B*2709·pLMP2 with His116 replaced by alanine. This initial model was subjected to rigid body refinement, simulated annealing, and energy minimization using CNS, improved by manual intervention using O and water molecule inclusion as described for the B*2709-data processing. Because of the higher resolution of B*2705·pLMP2, restrained maximum-likelihood refinement (Refmac) included anisotropic B-factor refinement. Evaluation of the atomic displacement parameters by Parvati (25Merritt E.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1109-1117Crossref PubMed Scopus (165) Google Scholar) provided the expected statistical distribution of 0.5 ± 0.17 for all atoms of the structure. Both structures were validated with Whatcheck (26Hooft R.W. Vriend G. Sander C. Abola E.E. Nature. 1996; 381: 272Crossref PubMed Scopus (1798) Google Scholar), and the statistics are compiled in Table I. The figures were generated using Delphi (27Nicholls A. Sharp K.A. Honig B. Proteins. 1991; 11: 281-296Crossref PubMed Scopus (5314) Google Scholar), Povray (www.povray.org), Molscript (28Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), Rastop (www.geneinfinity.org/rastop/), MSMS (29Sanner M.F. Olson A.J. Spehner J.C. Biopolymers. 1996; 38: 305-320Crossref PubMed Google Scholar), and Raster3D (30Merritt E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-527Crossref PubMed Scopus (3873) Google Scholar) together with a graphical interface (Moldraw) developed by N. Sträter (Institut für Kristallographie, Freie Universität Berlin). 2N. Sträter, unpublished program. The atomic coordinates and structure amplitudes have been deposited in the Protein Data Bank under accession codes 1uxs (B*2705·pLMP2) and 1uxw (B*2709·pLMP2). Structural Features of pLMP2 in Complex with HLA-B27 Subtypes—The B*2705·pLMP2 and B*2709·pLMP2 complexes crystallized isomorphously in space group P21 (Table I). Both show the typical MHC class I topography (31Madden D.R. Annu. Rev. Immunol. 1995; 13: 587-622Crossref PubMed Scopus (726) Google Scholar) (Fig. 1, a–c) and were refined at high resolution: 1.55 Å for B*2705·pLMP2 and 1.72 Å for B*2709·pLMP2. The HLA-B27 HC and β2-microglobulin are highly similar in the two subtypes (except for the D116H exchange) with a Cα root mean square deviation of only 0.2 Å. For each complex, the peptide could be modeled unambiguously to the electron density (Fig. 1, d and e). When complexed to B*2709, pLMP2 is bound in the conventional p4α conformation (main chain φ/Ψ torsion angles in α-helical conformation at p4), with the solvent-exposed pArg5 side chain pointing away from the binding groove (Fig. 1b) (5Hülsmeyer M. Fiorillo M.T. Bettosini F. Sorrentino R. Saenger W. Ziegler A. Uchanska-Ziegler B. J. Exp. Med. 2004; 199: 271-281Crossref PubMed Scopus (132) Google Scholar). In contrast, pLMP2 displays the drastically different p6α conformation (main chain φ/Ψ torsion angles in α-helical conformation at p6) when bound to B*2705 (Fig. 1a), with the side chain of pArg5 pointing toward the interior of the binding groove, where it forms a salt bridge with HC Asp116 (5Hülsmeyer M. Fiorillo M.T. Bettosini F. Sorrentino R. Saenger W. Ziegler A. Uchanska-Ziegler B. J. Exp. Med. 2004; 199: 271-281Crossref PubMed Scopus (132) Google Scholar). These subtype-dependent pArg5 orientations force the middle portion (residues p4–p7) of the peptide backbones and the corresponding amino acid side chains into grossly different conformations in the two subtypes (Fig. 1c). Because these regions of the two complexes do not participate in extensive crystal contacts, the observed conformational differences must be a direct consequence of the D116H polymorphism. The N-terminal pLMP2 residues pArg1, pArg2, and pArg3 occupy identical positions in both subtypes (Fig. 1c and Table II). Both subtypes exhibit also closely related interactions between HC atoms and C-terminal peptide residues pThr8 and pVal9. In B*2709, however, p8 and p9 are located slightly deeper in the binding groove than in B*2705 (Fig. 1, a–c), possibly as a consequence of altered p4–p7 conformations. To account for these changes, the binding groove residues in contact with pThr8 and pVal9 (Table II) exhibit small side chain variations.Table IIComparison of pLMP2 peptide coordination in the B*2705 and B*2709 subtypesa Intrapeptide contactb Helix α1c β–Sheet floord Helix α2 a Intrapeptide contact b Helix α1 c β–Sheet floor d Helix α2 pTrp4 is the first pLMP2 residue with substantially different positioning in the two subtypes (Fig. 1, a–e, and Table II). In p4α conformation found in complex with B*2709, the pTrp4, pArg5, and pArg6 side chains are fully solvent-exposed, with few HC contacts, whereas pLeu7 projects into the E-pocket (Fig. 1, b, c, and e, and Table II). A very different situation is found in the p6α conformation seen in B*2705 (Fig. 1, a, c, and d). Here, the pTrp4 side chain is packed against the α1-helix, and pArg5 forms a salt bridge with Asp116 that leads to deeper insertion of the middle section of the peptide into the binding groove (Fig. 1a and Table II). At pArg6, the peptide backbone bends upward (associated with the p6α conformation), so that the side chain can engage in van der Waals' contact with pTrp4 (Table II). Finally, pLeu7 is solvent-exposed in B*2705, and its side chain exhibits the highest flexibility of all pLMP2 residues in either conformation as shown by temperature (B) factors (Fig. 1, f and g, and Table I). A comparison of the B factors of the peptide reveals that pLMP2 is more flexibly bound in B*2705 despite the anchoring of its middle through the pArg5-Asp116 interaction. This differential peptide flexibility is most likely a consequence of a network of solvent molecules that is tighter in B*2709 than in B*2705, where the hydrophobic section of the pArg5 side chain prevents its formation. Structural Comparison of pLMP2 and pVIPR in Complexes with B*2705 and B*2709—The four complexes of B*2705 and B*2709 with pLMP2 and pVIPR, respectively, crystallized isomorphously (Ref. 5Hülsmeyer M. Fiorillo M.T. Bettosini F. Sorrentino R. Saenger W. Ziegler A. Uchanska-Ziegler B. J. Exp. Med. 2004; 199: 271-281Crossref PubMed Scopus (132) Google Scholar and Table I), indicating that the same crystallographic restraints (intermolecular interactions associated with crystal packing) apply to all of them. Comparison of pVIPR complexed with B*2705 and with B*2709 has already been carried out, with the dual p4α/p6α conformation found in B*2705 but not in B*2709, where only p4α occurs (5Hülsmeyer M. Fiorillo M.T. Bettosini F. Sorrentino R. Saenger W. Ziegler A. Uchanska-Ziegler B. J. Exp. Med. 2004; 199: 271-281Crossref PubMed Scopus (132) Google Scholar). Therefore, if molecular mimicry were to play a role in the context of the pLMP2/pVIPR structures, as suggested by CTL cross-reactivity (4Fiorillo M.T. Maragno M. Butler R. Dupuis M.L. Sorrentino R. J. Clin. Invest. 2000; 106: 47-53Crossref PubMed Scopus (158) Google Scholar), a comparison of side chain orientations (Fig. 2) and surface properties (Fig. 3) between the two pLMP2 complexes and the two pVIPR complexes should provide a structure-based explanation. The structures of the two peptides in B*2705 immediately reveal that pLMP2 (Fig. 3a) is much more similar to pVIPR-p6α (Cα root mean square deviation of 0.3 Å; Fig. 3c) than to pVIPR-p4α (Cα root mean square deviation of 1.6 Å; Fig. 3d). pArg1 as well as the anchor residues pArg2 and pVal9/pLeu9 occupy virtually identical positions (Fig. 2, a and b). As expected from the peptide sequences, the similarity of pVIPR and pLMP2 is most pronounced in the N-terminal half, extending to pArg6. In addition to the amino acid exchange at p7, the solvent-exposed pHis8 in pVIPR and pThr8 in pLMP2 lead to a marked topographical change near the peptide C termini (Figs. 2, a and b, and 3, a and c). The similarity between B*2705·pLMP2 (p6α binding mode) and B*2705·pVIPR-p6α extends beyond conformational (Figs. 2, a and b, and 3, a and c) to electrostatic properties of their surfaces (Fig. 3, e and g), particularly in the N-terminal halves of the peptides. In contrast, pLMP2 and pVIPR-p4α in B*2705 are less equivalent at residues p4–p8 (Figs. 2, c and d, and 3, a and d). The electrostatic surfaces of the two complexes in p4α/p6α conformation diverge considerably as well (Fig. 3, e and h). It seems therefore plausible to conclude that pLMP2/pVIPR CTL cross-reactivity is more likely to occur when pVIPR is displayed in p6α than in p4α conformation. Surprisingly, the p4α conformations of the two peptides found in B*2709 differ much more (Cα root mean square deviation of 0.9 Å; Figs. 2, e and f, and 3, b, d, f, and h) than the two p6α conformations in B*2705 (Figs. 2, a and b, and 3, a, c, e, and g). Again, residues p1, p2, p4, and p9 show negligible variations, and pLys3 (pVIPR) also occupies a similar position as pArg3 (pLMP2). However, the side chain guanidinium moieties of both pArg5 residues are solvent-exposed and point to opposite directions (Figs. 2, e and f, and 3, b and d), whereas those in p6α conformation (Figs. 2, a and b, and 3, a and c) are buried. pArg6 displays a substantial difference as well; in pLMP2, this side chain is completely solvent-accessible with only a few contacts to the α1-helix, whereas it wedges between the peptide backbone and the α1-helix in the complex with pVIPR (5Hülsmeyer M. Fiorillo M.T. Bettosini F. Sorrentino R. Saenger W. Ziegler A. Uchanska-Ziegler B. J. Exp. Med. 2004; 199: 271-281Crossref PubMed Scopus (132) Google Scholar). The Cα atoms of pArg6 deviate by 2.3 Å, and the disparity between the guanidinium groups is even larger. Although more similarly positioned, the p7-Cα atoms still differ by 1.6 Å. The side chain of pTrp7 (pVIPR) occupies the front part of the large E-pocket, an impossible location for pLeu7 (pLMP2) because of steric hindrance exerted by the large pArg3 located in the neighboring D-pocket. As a consequence, pLeu7 occupies the back part of the E-pocket, toward the peptide C terminus, is inserted deeper than pTrp7 in pVIPR, and is shifted toward the α1-helix. The peptides deviate only by 1.1 Å at Cα of p8, but the side chains display different shapes (Fig. 3, b and d)" @default.
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• W2162622550 title "Allele-dependent Similarity between Viral and Self-peptide Presentation by HLA-B27 Subtypes" @default.
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• W2162622550 doi "https://doi.org/10.1074/jbc.m410807200" @default.
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