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• W2035426331 abstract "A single amino acid exchange between the major histocompatibility complex molecules HLA-B*2705 and HLA-B*2709 (Asp-116/His) is responsible for the emergence of distinct HLA-B27-restricted T cell repertoires in individuals harboring either of these two subtypes and could correlate with their differential association with the autoimmune disease ankylosing spondylitis. By using fluorescence depolarization and pKa calculations, we investigated to what extent electrostatic interactions contribute to shape antigenic differences between these HLA molecules complexed with viral, self, and non-natural peptide ligands. In addition to the established main anchor of peptides binding to HLA-B27, arginine at position 2 (pArg-2), and the secondary anchors at the peptide termini, at least two further determinants contribute to stable peptide accommodation. 1) The interaction of Asp-116 with arginine at peptide position 5, as found in pLMP2 (RRRWRRLTV; viral) and pVIPR (RRKWRRWHL; self), and with lysine in pΩ, as found in gag (KRWIILGLNK; viral), additionally stabilizes the B*2705 complexes by ∼5 and ∼27 kJ/mol, respectively, in comparison with B*2709. 2) The protonation state of the key residues Glu-45 and Glu-63 in the B-pocket, which accommodates pArg-2, affects peptide binding strength in a peptide- and subtype-dependent manner. In B*2705/pLMP2, protonation of Glu-45/Glu-63 reduces the interaction energy of pArg-2 by ∼24 kJ/mol as compared with B*2705/pVIPR. B*2705/pVIPR is stabilized by a deprotonated Glu-45/Glu-63 pair, evoked by allosteric interactions with pHis-8. The mutual electrostatic interactions of peptide and HLA molecule, including peptide- and subtype-dependent protonation of key residues, modulate complex stability and antigenic features of the respective HLA-B27 subtype. A single amino acid exchange between the major histocompatibility complex molecules HLA-B*2705 and HLA-B*2709 (Asp-116/His) is responsible for the emergence of distinct HLA-B27-restricted T cell repertoires in individuals harboring either of these two subtypes and could correlate with their differential association with the autoimmune disease ankylosing spondylitis. By using fluorescence depolarization and pKa calculations, we investigated to what extent electrostatic interactions contribute to shape antigenic differences between these HLA molecules complexed with viral, self, and non-natural peptide ligands. In addition to the established main anchor of peptides binding to HLA-B27, arginine at position 2 (pArg-2), and the secondary anchors at the peptide termini, at least two further determinants contribute to stable peptide accommodation. 1) The interaction of Asp-116 with arginine at peptide position 5, as found in pLMP2 (RRRWRRLTV; viral) and pVIPR (RRKWRRWHL; self), and with lysine in pΩ, as found in gag (KRWIILGLNK; viral), additionally stabilizes the B*2705 complexes by ∼5 and ∼27 kJ/mol, respectively, in comparison with B*2709. 2) The protonation state of the key residues Glu-45 and Glu-63 in the B-pocket, which accommodates pArg-2, affects peptide binding strength in a peptide- and subtype-dependent manner. In B*2705/pLMP2, protonation of Glu-45/Glu-63 reduces the interaction energy of pArg-2 by ∼24 kJ/mol as compared with B*2705/pVIPR. B*2705/pVIPR is stabilized by a deprotonated Glu-45/Glu-63 pair, evoked by allosteric interactions with pHis-8. The mutual electrostatic interactions of peptide and HLA molecule, including peptide- and subtype-dependent protonation of key residues, modulate complex stability and antigenic features of the respective HLA-B27 subtype. The presentation of peptides by major histocompatibility complex (MHC) 4The abbreviations used are:MHCmajor histocompatibility complexASankylosing spondylitispLMP2Epstein-Barr virus latent membrane protein 2, residues 236–244 (RRRWRRLTV)pLMP2-T8Hpeptide RRRWRRLHVpVIPRvasoactive intestinal peptide type 1 receptor, 400–408 (RRKWRRWHL)pVIPR-H8Tpeptide RRKWRRWTLpGRglucagon receptor 412–420 (RRRWHRWRL)TISepidermal growth factor-response factor, 479–487 (RRLPIFSRL)m9non-natural ligand (GRFAAAIAK)S10Rnon-natural ligand (artificial mutant of B*2707 HC 110–119) (RRLLRGHNQY)gagviral HIV-p24 gag, 263–272 (KRWIILGLNK)fluinfluenza nucleoprotein, 383–391 (SRYWAIRTR)pEBNA-3CEpstein-Barr virus EBNA3C, 258–266 (RRIYDLIEL)KIR3DL1natural killer Ig-like receptorLYIALucifer Yellow iodoacetamideHCheavy chainHIVhuman immunodeficiency virusβ2mβ2-microglobulin. class I molecules at the cell surface and their recognition by cellular ligands like T cell receptors are fundamental for immune responses. The trimeric MHC class I complex consists of a highly polymorphic transmembrane heavy chain (HC) that is noncovalently associated with a light chain, β2-microglobulin (β2m) and a peptidic fragment derived from self or non-self proteins. The human MHC class I antigen HLA-B27 is one of the best investigated MHC class I molecules, which is partly due to its strong association with the development of a variety of autoimmune diseases, including ankylosing spondylitis (AS) (1Allen R. Bowness P. McMichael A. Immunogenetics. 1999; 50: 220-227Crossref PubMed Scopus (69) Google Scholar, 2Kim T.-H. Uhm W.-S. Inman R. Curr. Opin. Rheumatol. 2005; 17: 400-405Crossref PubMed Scopus (82) Google Scholar, 3Lopez de Castro J.A. Immunol. Lett. 2007; 108: 27-33Crossref PubMed Scopus (63) Google Scholar). In addition, HLA-B27 is known to present antigenic peptides derived from major infectious agents, such as Epstein-Barr virus, influenza virus, or human immunodeficiency virus (HIV) to cytotoxic T lymphocytes (4Brooks J. Murray R. Thomas W. Kurilla M. Rickinson A. J. Exp. Med. 1993; 178: 879-887Crossref PubMed Scopus (94) Google Scholar, 5Bowness P. Moss P. Rowland-Jones S. Bell J. McMichael A. Eur. J. Immunol. 1993; 23: 1417-1421Crossref PubMed Scopus (99) Google Scholar, 6Wilson J. Ogg G. Allen R. Davis C. Shaunak S. Downie J. Dyer W. Workman C. Sullivan J. McMichael A. Rowland-Jones S. AIDS. 2000; 14: 225-233Crossref PubMed Scopus (136) Google Scholar). major histocompatibility complex ankylosing spondylitis Epstein-Barr virus latent membrane protein 2, residues 236–244 (RRRWRRLTV) peptide RRRWRRLHV vasoactive intestinal peptide type 1 receptor, 400–408 (RRKWRRWHL) peptide RRKWRRWTL glucagon receptor 412–420 (RRRWHRWRL) epidermal growth factor-response factor, 479–487 (RRLPIFSRL) non-natural ligand (GRFAAAIAK) non-natural ligand (artificial mutant of B*2707 HC 110–119) (RRLLRGHNQY) viral HIV-p24 gag, 263–272 (KRWIILGLNK) influenza nucleoprotein, 383–391 (SRYWAIRTR) Epstein-Barr virus EBNA3C, 258–266 (RRIYDLIEL) natural killer Ig-like receptor Lucifer Yellow iodoacetamide heavy chain human immunodeficiency virus β2-microglobulin. Among the HLA-B27 subtypes, HLA-B*2705 (in short, B*2705) is the most common and exhibits a clear-cut association with AS (7Khan M.A. Mathieu A. Sorrentino R. Akkoc N. Autoimmun. Rev. 2007; 6: 183-189Crossref PubMed Scopus (115) Google Scholar). There is an increasing number of comparative studies between this subtype and HLA-B*2709 (in short, B*2709), which differs from B*2705 only in one amino acid (Asp-116/His) but shows no association to AS (8D'Amato M. Fiorillo M. Carcassi C. Mathieu A. Zuccarelli A. Bitti P. Tosi R. Sorrentino R. Eur. J. Immunol. 1995; 25: 3199-3201Crossref PubMed Scopus (185) Google Scholar). A comparison of these very closely related subtypes using functional and structural studies (9Fiorillo M. Maragno M. Butler R. Dupuis M. Sorrentino R. J. Clin. Investig. 2000; 106: 47-53Crossref PubMed Scopus (159) Google Scholar, 10Hülsmeyer M. Hillig R. Volz A. Rühl M. Schröder W. Saenger W. Ziegler A. Uchanska-Ziegler B. J. Biol. Chem. 2002; 277: 47844-47853Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 11Hülsmeyer M. Fiorillo M. Bettosini F. Sorrentino R. Saenger W. Ziegler A. Uchanska-Ziegler B. J. Exp. Med. 2004; 199: 271-281Crossref PubMed Scopus (135) Google Scholar, 12Hillig R. Hülsmeyer M. Saenger W. Welfle K. Misselwitz R. Welfle H. Kozerski C. Volz A. Uchanska-Ziegler B. Ziegler A. J. Biol. Chem. 2004; 279: 652-663Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 13Hülsmeyer M. Welfle K. Pöhlmann T. Misselwitz R. Alexiev U. Welfle H. Saenger W. Uchanska-Ziegler B. Ziegler A. J. Mol. Biol. 2005; 346: 1367-1379Crossref PubMed Scopus (46) Google Scholar, 14Fiorillo M. Rückert C. Hülsmeyer M. Sorrentino R. Saenger W. Ziegler A. Uchanska-Ziegler B. J. Biol. Chem. 2005; 280: 2962-2971Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 15Rückert C. Fiorillo M. Loll B. Moretti R. Biesiadka J. Saenger W. Ziegler A. Sorrentino R. Uchanska-Ziegler B. J. Biol. Chem. 2006; 281: 2306-2316Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) as well as various biophysical methods (12Hillig R. Hülsmeyer M. Saenger W. Welfle K. Misselwitz R. Welfle H. Kozerski C. Volz A. Uchanska-Ziegler B. Ziegler A. J. Biol. Chem. 2004; 279: 652-663Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 13Hülsmeyer M. Welfle K. Pöhlmann T. Misselwitz R. Alexiev U. Welfle H. Saenger W. Uchanska-Ziegler B. Ziegler A. J. Mol. Biol. 2005; 346: 1367-1379Crossref PubMed Scopus (46) Google Scholar, 16Pöhlmann T. Böckmann R. Grubmüller H. Uchanska-Ziegler B. Ziegler A. Alexiev U. J. Biol. Chem. 2004; 279: 28197-28201Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 17Uchanska-Ziegler B. Alexiev U. Hillig R. Hülsmeyer M. Pöhlmann T. Saenger W. Volz A. Ziegler A. Hansen J.A. Immunobiology of the Human MHC: Proceedings of the 13th International Histocompatibility Workshop and Congress. 1. IHWG Press, Seattle2006: 138-147Google Scholar, 18Winkler K. Winter A. Rückert C. Uchanska-Ziegler B. Alexiev U. Biophys. J. 2007; 93: 2743-2755Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) may eventually provide clues to the pathogenic role of HLA-B27. Several theories seeking to explain the disease association have been published over the years, most of them regarding the molecule and its peptide binding properties as a key to the pathogenic role of HLA-B27 (7Khan M.A. Mathieu A. Sorrentino R. Akkoc N. Autoimmun. Rev. 2007; 6: 183-189Crossref PubMed Scopus (115) Google Scholar). This holds true for the hypotheses of molecular mimicry between foreign and self-peptides presented by HLA-B27 (14Fiorillo M. Rückert C. Hülsmeyer M. Sorrentino R. Saenger W. Ziegler A. Uchanska-Ziegler B. J. Biol. Chem. 2005; 280: 2962-2971Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 15Rückert C. Fiorillo M. Loll B. Moretti R. Biesiadka J. Saenger W. Ziegler A. Sorrentino R. Uchanska-Ziegler B. J. Biol. Chem. 2006; 281: 2306-2316Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 17Uchanska-Ziegler B. Alexiev U. Hillig R. Hülsmeyer M. Pöhlmann T. Saenger W. Volz A. Ziegler A. Hansen J.A. Immunobiology of the Human MHC: Proceedings of the 13th International Histocompatibility Workshop and Congress. 1. IHWG Press, Seattle2006: 138-147Google Scholar), but also for the involvement of misfolded or partially unfolded molecules (19Colbert R. Mol. Med. Today. 2000; 6: 224-230Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), heavy chain homodimers (20Edwards J. Bowness P. Archer J. Immunol. Today. 2000; 21: 256-260Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), or liberated β2m (21Uchanska-Ziegler B. Ziegler A. Trends Immunol. 2003; 24: 73-76Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Despite these efforts, the molecular basis of the strong linkage of HLA-B27 with spondyloarthropathies still remains elusive. Apart from its association to spondyloarthropathies, B*2705 is also connected with the long term nonprogression in chronic HIV infection through the presentation of B*2705-restricted peptides such as gag-(263–272) (KRWIILGLNK) from HIV (22Goulder P. Phillips R. Colbert R. McAdam S. Ogg G. Nowak M. Giangrande P. Luzzi G. Morgana B. Edwards A. McMichael A. Rowland-Jones S. Nat. Med. 1997; 3: 212-217Crossref PubMed Scopus (1018) Google Scholar). The recent crystal structure of B*2705 complexed with this peptide demonstrates again the pivotal role of the guanidinium group of arginine at position 2 of the peptide (pArg-2) as the main anchor for the peptide in the B-pocket of the peptide binding groove (23Stewart-Jones G. di Gleria K. Kollnberger S. McMichael A. Jones E. Bowness P. Eur. J. Immunol. 2005; 35: 341-351Crossref PubMed Scopus (90) Google Scholar); peptides with an exchange of pArg-2, as observed during HIV infections, fail to be presented by HLA-B27 antigens resulting in immune escape. Although peptide stability and HLA-B27 selectivity are mainly a consequence of the presence of pArg-2 and its binding within the B-pocket (Fig. 1), every other peptide position may in principle contribute to stable peptide binding (24López de Castro J. Alvarez I. Marcilla M. Paradela A. Ramos M. Sesma L. Vázquez M. Tissue Antigens. 2004; 63: 424-445Crossref PubMed Scopus (85) Google Scholar). The C terminal (pΩ) peptide residue is regarded as a particularly important secondary anchor, which binds within the F-pocket. In B*2705 the acidic Asp-116 at the floor of the peptide binding groove, along with two other F-pocket residues, Asp-74 and Asp-77, plays a key role in the interaction of basic pΩ peptide residues (10Hülsmeyer M. Hillig R. Volz A. Rühl M. Schröder W. Saenger W. Ziegler A. Uchanska-Ziegler B. J. Biol. Chem. 2002; 277: 47844-47853Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 23Stewart-Jones G. di Gleria K. Kollnberger S. McMichael A. Jones E. Bowness P. Eur. J. Immunol. 2005; 35: 341-351Crossref PubMed Scopus (90) Google Scholar). Peptide elution studies showed that the basic amino acids Lys and Arg are strong pΩ anchors for nonameric peptides binding to B*2705. Peptides with pΩArg or pΩLys account for 27% of the natural B*2705 ligands, whereas they are absent (pΩLys) or rare (pΩArg) in the peptide repertoire of B*2709 (24López de Castro J. Alvarez I. Marcilla M. Paradela A. Ramos M. Sesma L. Vázquez M. Tissue Antigens. 2004; 63: 424-445Crossref PubMed Scopus (85) Google Scholar, 25Ramos M. Paradela A. Vázquez M. Marina A. Vázquez J. López de Castro J. J. Biol. Chem. 2002; 277: 28749-28756Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In addition, Asp-116 has been reported to promote a noncanonical binding mode in two peptides that contain an arginine at position 5 (RRKWRRWHL from a self-protein, pVIPR; and RRRWRRLTV from a viral protein, pLMP2). In this noncanonical binding mode, pArg-5 forms a salt bridge to Asp-116 (11Hülsmeyer M. Fiorillo M. Bettosini F. Sorrentino R. Saenger W. Ziegler A. Uchanska-Ziegler B. J. Exp. Med. 2004; 199: 271-281Crossref PubMed Scopus (135) Google Scholar, 14Fiorillo M. Rückert C. Hülsmeyer M. Sorrentino R. Saenger W. Ziegler A. Uchanska-Ziegler B. J. Biol. Chem. 2005; 280: 2962-2971Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), which presumably adds to the stable accommodation of the respective peptide. In the B*2709 subtype, the exchange of Asp-116 by His does not permit this strong electrostatic interaction. As the different peptide binding properties of the subtypes also shape the antigenic features for recognition by T cell receptors or other molecules such as inhibitory receptors on natural killer cells (in case of HLA-B27, e.g. KIR3DL1) (23Stewart-Jones G. di Gleria K. Kollnberger S. McMichael A. Jones E. Bowness P. Eur. J. Immunol. 2005; 35: 341-351Crossref PubMed Scopus (90) Google Scholar), we set out to compare several different peptides (Table 1) in terms of their complex stability and electrostatic interactions between the peptide and the B*2705 and B*2709 subtypes. The results reveal that differences in complex stability as well as in peptide binding affinity between the two very closely related HLA molecules can mainly be explained by changes in the interaction energy between the peptide anchors and the binding groove, although the individual contributions to the peptide binding free energy can differ considerably, even for peptides with high sequence similarity. Furthermore, we find that peptide binding affinities are influenced not only by the expected short range but also by long range interactions between peptide residues and HC amino acids that are crucial in shaping the binding groove.TABLE 1B*2705 or B*2709 bound peptides investigated in this study The citation after each abbreviation represents the reference where the crystal structure of the given HLA-B27/peptide complex is published.SequenceOrigin/peptideAbbreviationRRLPIFSRLHuman self-peptide: epidermal growth factor response factor, residues 479–487TIS (13Hülsmeyer M. Welfle K. Pöhlmann T. Misselwitz R. Alexiev U. Welfle H. Saenger W. Uchanska-Ziegler B. Ziegler A. J. Mol. Biol. 2005; 346: 1367-1379Crossref PubMed Scopus (46) Google Scholar)RRKWRRWHLHuman self-peptide: vasoactive intestinal peptide type 1 receptor, 400–408pVIPR (11)RRKWRRWTLArtificial mutant of pVIPRpVIPR-H8TRRRWHRWRLHuman self-peptide: glucagon receptor, 412–420pGR (15)RRRWRRLTVViral: Epstein-Barr virus latent membrane protein 2, 236–244pLMP2 (14)RRRWRRLHVArtificial mutant of pLMP2pLMP2-T8HRRIYDLIELViral: Epstein-Barr virus EBNA3C, 258–266pEBNA-3C (23)RRIYDLITLArtificial mutant used for pKa calculationspEBNA-3C-E8TSRYWAIRTRViral: influenza virus nucleoprotein, 383–391flu (23Stewart-Jones G. di Gleria K. Kollnberger S. McMichael A. Jones E. Bowness P. Eur. J. Immunol. 2005; 35: 341-351Crossref PubMed Scopus (90) Google Scholar)KRWIILGLNKViral: HIV-p24 gag, 263–272gag (23Stewart-Jones G. di Gleria K. Kollnberger S. McMichael A. Jones E. Bowness P. Eur. J. Immunol. 2005; 35: 341-351Crossref PubMed Scopus (90) Google Scholar)GRFAAAIAKNon-natural ligandm9 (10Hülsmeyer M. Hillig R. Volz A. Rühl M. Schröder W. Saenger W. Ziegler A. Uchanska-Ziegler B. J. Biol. Chem. 2002; 277: 47844-47853Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar)RRLLRGHNQYNon-natural ligand (artificial mutant of B*2707 HC 110–119)S10R (12Hillig R. Hülsmeyer M. Saenger W. Welfle K. Misselwitz R. Welfle H. Kozerski C. Volz A. Uchanska-Ziegler B. Ziegler A. J. Biol. Chem. 2004; 279: 652-663Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) Open table in a new tab Protein Preparation—Complexes of the subtypes B*2705 and B*2709 with the peptides m9, pVIPR, TIS, pLMP2, gag, pGR, and the artificial peptide mutants pVIPR-H8T and pLMP2-T8H were prepared as described previously (10Hülsmeyer M. Hillig R. Volz A. Rühl M. Schröder W. Saenger W. Ziegler A. Uchanska-Ziegler B. J. Biol. Chem. 2002; 277: 47844-47853Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 11Hülsmeyer M. Fiorillo M. Bettosini F. Sorrentino R. Saenger W. Ziegler A. Uchanska-Ziegler B. J. Exp. Med. 2004; 199: 271-281Crossref PubMed Scopus (135) Google Scholar, 13Hülsmeyer M. Welfle K. Pöhlmann T. Misselwitz R. Alexiev U. Welfle H. Saenger W. Uchanska-Ziegler B. Ziegler A. J. Mol. Biol. 2005; 346: 1367-1379Crossref PubMed Scopus (46) Google Scholar, 14Fiorillo M. Rückert C. Hülsmeyer M. Sorrentino R. Saenger W. Ziegler A. Uchanska-Ziegler B. J. Biol. Chem. 2005; 280: 2962-2971Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 15Rückert C. Fiorillo M. Loll B. Moretti R. Biesiadka J. Saenger W. Ziegler A. Sorrentino R. Uchanska-Ziegler B. J. Biol. Chem. 2006; 281: 2306-2316Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 16Pöhlmann T. Böckmann R. Grubmüller H. Uchanska-Ziegler B. Ziegler A. Alexiev U. J. Biol. Chem. 2004; 279: 28197-28201Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 17Uchanska-Ziegler B. Alexiev U. Hillig R. Hülsmeyer M. Pöhlmann T. Saenger W. Volz A. Ziegler A. Hansen J.A. Immunobiology of the Human MHC: Proceedings of the 13th International Histocompatibility Workshop and Congress. 1. IHWG Press, Seattle2006: 138-147Google Scholar, 18Winkler K. Winter A. Rückert C. Uchanska-Ziegler B. Alexiev U. Biophys. J. 2007; 93: 2743-2755Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 26Garboczi D. Hung D. Wiley D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3429-3433Crossref PubMed Scopus (563) Google Scholar). The synthetic peptides as well as their fluorescent derivatives labeled with Lucifer Yellow (LY, Molecular Probes) in position C-6 and C-8, respectively, were purchased from Alta Bioscience (Birmingham, UK), Biosynthan (Berlin, Germany), or synthesized in-house. Briefly, heterotrimeric HLA-B27/peptide complexes (HC/β2m/peptide) were reconstituted from 6 m urea in the presence of the respective peptide. After size exclusion chromatography, the concentrations of highly pure MHC/peptide complexes in 10 mm phosphate buffer, pH 7.5, 150 mm NaCl were adjusted to A426 = 0.0012 OD using the absorption band of Lucifer Yellow (λmax ≈ 430 nm). Thermodynamics of Peptide Dissociation—Temperature-induced peptide dissociation from a given MHC/peptide complex was measured by detection of the change in the stationary anisotropy of LY covalently bound to the peptides in the temperature range from 5 to 85 °C (16Pöhlmann T. Böckmann R. Grubmüller H. Uchanska-Ziegler B. Ziegler A. Alexiev U. J. Biol. Chem. 2004; 279: 28197-28201Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). An average heating rate of 0.6 °C/min was applied. Lucifer Yellow was excited at λex = 428 nm, and the steady state anisotropy was determined within a time-correlated single photon counting setup (27Alexiev U. Rimke I. Pöhlmann T. J. Mol. Biol. 2003; 328: 705-719Crossref PubMed Scopus (76) Google Scholar) by collecting the integral emission intensity for λ ≥ 515 nm parallel I∥ and perpendicular I⊥ with respect to the linearly polarized excitation light. The anisotropy values were calculated according to Equation 1,r(t)=I∥(t)-I⊥(t)I∥(t)+2I⊥(t)(Eq. 1) Thermodynamic parameters, such as the enthalpy and entropy changes of the unfolding process as well as transition temperatures (Tm) for peptide dissociation, were obtained by fitting Equation 2 to the steady state anisotropy applying a least square procedure,r(T)=r0b+sbT+(r0d+sdT)exp-ΔHp0+TΔSp0RT1+exp-ΔHp0+TΔSp0RT(Eq. 2) with Equation 3Tm=ΔHp0ΔSp0(Eq. 3) This equation involves six fitting parameters, respectively; the fluorescence anisotropy of the folded state (native protein), r0b, and of the unfolded or dissociated state, r0d, the temperature dependence of the anisotropy of the peptide-bound (folded) state, Sb, and of the unfolded or dissociated state, Sd, the enthalpy change, ΔH0p, and the entropy change, ΔS0p, for the two-state dissociation reaction, the thermodynamic quantities being determined at the midpoint of the transition. HLA-B27 complex unfolding was also followed by the change in the tryptophan fluorescence intensity (16Pöhlmann T. Böckmann R. Grubmüller H. Uchanska-Ziegler B. Ziegler A. Alexiev U. J. Biol. Chem. 2004; 279: 28197-28201Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Comparison of the complex unfolding transition temperatures for HLA complexes with the natural and the LY-labeled peptide (data not shown) indicated that the modification with Lucifer Yellow does not change the thermodynamic properties of the respective complex. For comparison of the relative complex stability between B*2705 and B*2709 molecules complexed with the same peptide, we calculated the difference in free energy ΔΔGB*2709-B*2705, given by Equation 4,ΔΔGB*2709-B*2705=ΔTB*2709-B*2705·ΔS¯B*27090(Eq. 4) ΔTB*2709-B*2705 is the difference of the peptide dissociation temperatures determined for the complexes B*2705 and B*2709 and ΔS0B*2709 is the apparent entropy of the peptide dissociation reaction from B*2709 determined by applying the two-state transition model (Equation 2). According to Becktel and Shellman (28Becktel W. Schellman J. Biopolymers. 1987; 26: 1859-1877Crossref PubMed Scopus (948) Google Scholar), Equation 4 represents a method for the calculation of free energy differences of biomolecular systems showing only small perturbations, i.e. systems that differ only by small modifications, like a single point mutation or the selective binding of a ligand. Because the two HLA-B27 molecules investigated in this study are very closely related and differ only by one amino acid, the conditions for the use of Equation 4 are fulfilled. For the comparison of the relative stability between HLA-B27/pVIPR, HLA-B27/pLMP2, HLA-B27/pVIPR-H8T, and HLA-B27/pLMP2-T8H, respectively, we used the same method, now considering the difference between the sequence-related peptides pVIPR and pLMP2 or their mutants pLMP2-T8H and pVIPR-H8T as a small modification to the system. As the folded protein/peptide complex is characterized by a positive ΔG value, destabilization (stabilization) thus appears as a negative (positive) ΔΔG value (28Becktel W. Schellman J. Biopolymers. 1987; 26: 1859-1877Crossref PubMed Scopus (948) Google Scholar). pKa Analysis—Protein residue pKa calculations were performed on the crystal structures of the two B*2705 and B*2709 subtypes complexed with several different peptides (pLMP2 (14Fiorillo M. Rückert C. Hülsmeyer M. Sorrentino R. Saenger W. Ziegler A. Uchanska-Ziegler B. J. Biol. Chem. 2005; 280: 2962-2971Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), pVIPR in both canonical (conformation A) and noncanonical (conformation B) conformations (11Hülsmeyer M. Fiorillo M. Bettosini F. Sorrentino R. Saenger W. Ziegler A. Uchanska-Ziegler B. J. Exp. Med. 2004; 199: 271-281Crossref PubMed Scopus (135) Google Scholar), TIS (13Hülsmeyer M. Welfle K. Pöhlmann T. Misselwitz R. Alexiev U. Welfle H. Saenger W. Uchanska-Ziegler B. Ziegler A. J. Mol. Biol. 2005; 346: 1367-1379Crossref PubMed Scopus (46) Google Scholar), and the model peptide m9 (10Hülsmeyer M. Hillig R. Volz A. Rühl M. Schröder W. Saenger W. Ziegler A. Uchanska-Ziegler B. J. Biol. Chem. 2002; 277: 47844-47853Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar)). The pKa values were also calculated for the B*2709 subtype complexed with the S10R peptide (12Hillig R. Hülsmeyer M. Saenger W. Welfle K. Misselwitz R. Welfle H. Kozerski C. Volz A. Uchanska-Ziegler B. Ziegler A. J. Biol. Chem. 2004; 279: 652-663Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) and for the B*2705 subtype complexed with the pGR peptide presented in two different conformations (pGR A and pGR B (15Rückert C. Fiorillo M. Loll B. Moretti R. Biesiadka J. Saenger W. Ziegler A. Sorrentino R. Uchanska-Ziegler B. J. Biol. Chem. 2006; 281: 2306-2316Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar)). The peptides gag-(263–272) (gag) from HIV, EBNA3C-(258–266) (pEBNA-3C) from Epstein-Barr virus, and nucleoprotein 383–391 (flu) from influenza (23Stewart-Jones G. di Gleria K. Kollnberger S. McMichael A. Jones E. Bowness P. Eur. J. Immunol. 2005; 35: 341-351Crossref PubMed Scopus (90) Google Scholar) were also included in the study. The resolution of the used crystal structures of the complexes varies from 1.09 to 2.30 Å. The peptides are listed in Table 1. To understand how the presence or absence of a peptide and/or of β2m in the complex could influence the effective protonation state of the titratable groups, the pKa calculations were additionally performed on both HLA-B27 subtypes without peptide and with or without β2m. Peptide mutants were obtained as follows: pHis-8 of pVIPR was mutated with a threonine using the WHAT IF (29Vriend G. J. Mol. Graphics. 1990; 8: 52-56Crossref PubMed Scopus (3371) Google Scholar) package (experimental version of the function mutate (30Chinea G. Padron G. Hooft R. Sander C. Vriend G. Proteins. 1995; 23: 415-421Crossref PubMed Scopus (120) Google Scholar)). For the exchange of pThr-8 in pLMP2 with a histidine, a different strategy was adopted; after fit of the peptide backbone atoms of residues 7 to 9, the side chain of pThr-8 in pLMP2 was replaced by the side chain of the pHis-8 in pVIPR B, followed by a short energy minimization of the structure using a steepest descent algorithm (100 steps). The pKa calculations were based on the scheme proposed by Nielsen et al. (31Nielsen J.E. Vriend G. Proteins Struct. Funct. Genet. 2001; 43: 403-412Crossref PubMed Scopus (185) Google Scholar, 32Nielsen J. McCammon J. Protein Sci. 2003; 12: 313-326Crossref PubMed Scopus (105) Google Scholar, 33Nielsen J. McCammon J. Protein Sci. 2003; 12: 1894-1901Crossref PubMed Scopus (136) Google Scholar), which combines finite difference solutions to the Poisson-Boltzmann equation with a global optimization of the hydrogen bond network in all protonation states. All radii and charges of the atoms were taken from the OPLS force field (34Kaminski G. Friesner R. Tirado-Rives J. Jorgensen W. J. Phys. Chem. B. 2001; 105: 6474-6487Crossref Scopus (3194) Google Scholar). The linearized Poisson-Boltzmann equation was solved applying DELPHI II (35Rocchia W. Alexov E. Honig B. J. Phys. Chem. B. 2001; 105: 6507-6514Crossref Scopus (704) Google Scholar), with the parameters used in Ref. 32Nielsen J. McCammon J. Protein Sci. 2003; 12: 313-326Crossref PubMed Scopus (105) Google Scholar; a dielectric constant of 80 for water and 8 for the protein interior, a 65 cubed grid, a grid resolution of 3 grid points/Å for the desolvation energy and of 4 grid points/Å for the background interaction energy, a 2.0 Å ion exclusion layer, an ionic strength of 0.144 m, and a surface probe radius of 1.4 Å have been used. As suggested previously by Nielsen and Vriend (31Nielsen J.E. Vriend G. Proteins Struct. Funct. Genet. 2001; 43: 403-412Crossref PubMed Scopus (185) Google S" @default.
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• W2035426331 date "2008-08-01" @default.
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• W2035426331 title "Molecular Determinants of Major Histocompatibility Complex Class I Complex Stability" @default.
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