FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2098086095> ?p ?o ?g. }

• W2098086095 endingPage "10625" @default.
• W2098086095 startingPage "10618" @default.
• W2098086095 abstract "Classic major histocompatibility complex (MHC) proteins associate with antigen- and self-derived peptides in an allele-specific manner. Herein we present the crystal structure of the MHC class I protein H-2Kd (Kd) expressed by BALB/c mice in complex with an antigenic peptide derived from influenza A/PR/8/34 nucleoprotein (Flu, residues 147-155, TYQRTRALV). Analysis of our structure in conjunction with the sequences of naturally processed epitopes provides a comprehensive understanding of the dominant Kd peptide-binding motif. We find that Flu residues TyrP2, ThrP5, and ValP9 are sequestered into the B, C, and F pockets of the Kd groove, respectively. The shape and chemistry of the polymorphic B pocket make it an optimal binding site for the side chain of TyrP2 as the dominant anchoring residue of nonameric peptides. The non-polar F pocket limits the amino acid repertoire at P9 to hydrophobic residues such as Ile, Leu, or Val, whereas the C pocket restricts the size of the P5-anchoring side chain. We also show that Flu is accommodated in the complex through an unfavorable kink in the otherwise extended peptide backbone due to the presence of a prominent ridge in the Kd groove. Surprisingly, this backbone conformation is strikingly similar to Db-presented peptides despite the fact that these proteins employ distinct motif-anchoring strategies. The results presented in this study provide a solid foundation for the understanding of Kd-restricted antigen presentation and recognition events. Classic major histocompatibility complex (MHC) proteins associate with antigen- and self-derived peptides in an allele-specific manner. Herein we present the crystal structure of the MHC class I protein H-2Kd (Kd) expressed by BALB/c mice in complex with an antigenic peptide derived from influenza A/PR/8/34 nucleoprotein (Flu, residues 147-155, TYQRTRALV). Analysis of our structure in conjunction with the sequences of naturally processed epitopes provides a comprehensive understanding of the dominant Kd peptide-binding motif. We find that Flu residues TyrP2, ThrP5, and ValP9 are sequestered into the B, C, and F pockets of the Kd groove, respectively. The shape and chemistry of the polymorphic B pocket make it an optimal binding site for the side chain of TyrP2 as the dominant anchoring residue of nonameric peptides. The non-polar F pocket limits the amino acid repertoire at P9 to hydrophobic residues such as Ile, Leu, or Val, whereas the C pocket restricts the size of the P5-anchoring side chain. We also show that Flu is accommodated in the complex through an unfavorable kink in the otherwise extended peptide backbone due to the presence of a prominent ridge in the Kd groove. Surprisingly, this backbone conformation is strikingly similar to Db-presented peptides despite the fact that these proteins employ distinct motif-anchoring strategies. The results presented in this study provide a solid foundation for the understanding of Kd-restricted antigen presentation and recognition events. Class I major histocompatibility complex (MHC) 2The abbreviations used are: MHC, major histocompatibility complex; Flu, antigenic peptide derived from Influenza A/PR/8/34 nucleoprotein residues 147-155; TCR, T cell receptor; mβ2m, murine β2-microglobulin; CTL, cytotoxic T lymphocyte; r.m.s.d., root mean square deviation; ERK, extracellular signal-regulated kinase. proteins serve a critical role in the adaptive immune response by binding short peptide fragments intracellularly and presenting them at the cell surface for surveillance by cytotoxic T lymphocytes (1Townsend A. Bodmer H. Annu. Rev. Immunol. 1989; 7: 601-624Crossref PubMed Scopus (1108) Google Scholar, 2Kourilsky P. Claverie J.M. Adv. Immunol. 1989; 45: 107-193Crossref PubMed Scopus (86) Google Scholar, 3Bjorkman P.J. Parham P. Annu. Rev. Biochem. 1990; 59: 253-288Crossref PubMed Scopus (621) Google Scholar, 4Tsomides T.J. Eisen H.N. J. Biol. Chem. 1991; 266: 3357-3360Abstract Full Text PDF PubMed Google Scholar). Structural studies of human and murine MHC class I proteins in complex with a variety of peptides have revealed conservative structural elements that promote efficient binding and presentation of peptide epitopes (5Jones E.Y. Curr. Opin. Immunol. 1997; 9: 75-79Crossref PubMed Scopus (69) Google Scholar, 6Madden D.R. Annu. Rev. Immunol. 1995; 13: 587-622Crossref PubMed Scopus (732) Google Scholar, 7Wilson I.A. Fremont D.H. Semin. Immunol. 1993; 5: 75-80Crossref PubMed Scopus (39) Google Scholar, 8Natarajan K. Li H. Mariuzza R.A. Margulies D.H. Rev. Immunogenet. 1999; 1: 32-46PubMed Google Scholar, 9Young A.C. Nathenson S.G. Sacchettini J.C. FASEB J. 1995; 9: 26-36Crossref PubMed Scopus (52) Google Scholar). Peptides of 8-10 residues are bound in a predominantly extended conformation within a narrow groove formed by two antiparallel α-helices positioned above an eight-strand β-sheet platform. Conservative hydrogen bonding networks are established in the binding groove with peptide main-chain and terminal atoms that enable largely sequence-independent ligation. Although low affinity, kinetically short-lived peptide-MHC complexes can be established by highly diverse epitope sequences, stable association requires the anchoring of peptide side chains into specific pockets in the MHC groove. MHC polymorphisms are clustered in these pockets (3Bjorkman P.J. Parham P. Annu. Rev. Biochem. 1990; 59: 253-288Crossref PubMed Scopus (621) Google Scholar, 10Garrett T.P. Saper M.A. Bjorkman P.J. Strominger J.L. Wiley D.C. Nature. 1989; 342: 692-696Crossref PubMed Scopus (596) Google Scholar, 11Abastado J.P. Casrouge A. Kourilsky P. J. Immunol. 1993; 151: 3569-3575PubMed Google Scholar), and their shape and chemistry impose constraints that are reflected by allele-specific motifs found in the sequences of naturally processed peptides (12Falk K. Rotzschke O. Stevanovic S. Jung G. Rammensee H.G. Nature. 1991; 351: 290-296Crossref PubMed Scopus (2132) Google Scholar, 13Rammensee H.G. Falk K. Rotzschke O. Annu. Rev. Immunol. 1993; 11: 213-244Crossref PubMed Scopus (716) Google Scholar). For example, H-2d cell lines (P815) and H-2b cell lines (EL4) infected with the same influenza virus present different antigenic peptides for CTL recognition (14Rotzschke O. Falk K. Deres K. Schild H. Norda M. Metzger J. Jung G. Rammensee H.G. Nature. 1990; 348: 252-254Crossref PubMed Scopus (640) Google Scholar). Thus, outbred populations that express varied MHC proteins can survey diverse peptide fragments from a given pathogen despite specificity constraints imposed by each individual allele. The sequences of a number of naturally processed peptides that are presented by Kd have been identified, including self-peptides (12Falk K. Rotzschke O. Stevanovic S. Jung G. Rammensee H.G. Nature. 1991; 351: 290-296Crossref PubMed Scopus (2132) Google Scholar, 15Ikeda H. Ohta N. Furukawa K. Miyazaki H. Wang L. Kuribayashi K. Old L.J. Shiku H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6375-6379Crossref PubMed Scopus (93) Google Scholar, 16Nagata Y. Furugen R. Hiasa A. Ikeda H. Ohta N. Furukawa K. Nakamura H. Kanematsu T. Shiku H. J. Immunol. 1997; 159: 1336-1343PubMed Google Scholar, 17Matsui K. O'Mara L.A. Allen P.M. Int. Immunol. 2003; 15: 797-805Crossref PubMed Scopus (27) Google Scholar, 18Wallny H.J. Deres K. Faath S. Jung G. Van Pel A. Boon T. Rammensee H.G. Int. Immunol. 1992; 4: 1085-1090Crossref PubMed Scopus (49) Google Scholar, 19Sibille C. Chomez P. Wildmann C. Van Pel A. De Plaen E. Maryanski J.L. de Bergeyck V. Boon T. J. Exp. Med. 1990; 172: 35-45Crossref PubMed Scopus (134) Google Scholar, 20Wong F.S. Moustakas A.K. Wen L. Papadopoulos G.K. Janeway Jr., C.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5551-5556Crossref PubMed Scopus (54) Google Scholar, 21Harpur A.G. Zimiecki A. Wilks A.F. Falk K. Rotzschke O. Rammensee H.G. Immunol. Lett. 1993; 35: 235-237Crossref PubMed Scopus (11) Google Scholar, 60Suri A. Walters J.J. Levisetti M.G. Gross M.L. Unanue E.R. Eur. J. Immunol. 2006; 36: 544-557Crossref PubMed Scopus (23) Google Scholar) and those encoded by viruses (14Rotzschke O. Falk K. Deres K. Schild H. Norda M. Metzger J. Jung G. Rammensee H.G. Nature. 1990; 348: 252-254Crossref PubMed Scopus (640) Google Scholar, 22Spaulding A.C. Kurane I. Ennis F.A. Rothman A.L. J. Virol. 1999; 73: 398-403Crossref PubMed Google Scholar, 23Tamura M. Kuwano K. Kurane I. Ennis F.A. J. Virol. 1998; 72: 9404-9406Crossref PubMed Google Scholar), parasites (24Weiss W.R. Mellouk S. Houghten R.A. Sedegah M. Kumar S. Good M.F. Berzofsky J.A. Miller L.H. Hoffman S.L. J. Exp. Med. 1990; 171: 763-773Crossref PubMed Scopus (175) Google Scholar, 25Romero P. Maryanski J.L. Corradin G. Nussenzweig R.S. Nussenzweig V. Zavala F. Nature. 1989; 341: 323-326Crossref PubMed Scopus (438) Google Scholar), and bacteria (26Pamer E.G. Harty J.T. Bevan M.J. Nature. 1991; 353: 852-855Crossref PubMed Scopus (382) Google Scholar). The first virally encoded T-cell epitope ever described was in fact a Kd-binding peptide derived from influenza A/PR/8/34 nucleo-protein (27Taylor P.M. Davey J. Howland K. Rothbard J.B. Askonas B.A. Immunogenetics. 1987; 26: 267-272Crossref PubMed Scopus (74) Google Scholar). Although early studies using synthetic peptides suggested that an 11-residue peptide is presented (28Bodmer H.C. Pemberton R.M. Rothbard J.B. Askonas B.A. Cell. 1988; 52: 253-258Abstract Full Text PDF PubMed Scopus (122) Google Scholar), sequencing of the naturally processed peptide from virally infected cells revealed that the nucleo-protein epitope is only nine residues long (residues 147-155, TYQRTRALV) (14Rotzschke O. Falk K. Deres K. Schild H. Norda M. Metzger J. Jung G. Rammensee H.G. Nature. 1990; 348: 252-254Crossref PubMed Scopus (640) Google Scholar). In fact, the vast majority of Kd-binding peptides are nine residues in length, which nearly invariantly contain Tyr at the second position (P2) (29Maryanski J.L. Romero P. Van Pel A. Boon T. Salemme F.R. Cerottini J.C. Corradin G. Int. Immunol. 1991; 3: 1035-1042Crossref PubMed Scopus (39) Google Scholar, 30Romero P. Corradin G. Luescher I.F. Maryanski J.L. J. Exp. Med. 1991; 174: 603-612Crossref PubMed Scopus (141) Google Scholar, 31Quesnel A. Casrouge A. Kourilsky P. Abastado J.P. Trudelle Y. Pept. Res. 1995; 8: 44-51PubMed Google Scholar). To resolve the structural underpinnings of the dominant Kd-binding motif we have undertaken crystallographic studies of Kd in complex with the antigenic peptide from influenza virus nucleoprotein (Flu). The 2.6-Å resolution structure of Kd-Flu provided an excellent framework to delineate the role of polymorphic anchoring pockets in determining Kd-specific peptide binding. To extend our understanding of the overall binding motif to a broad population of Kd epitopes, we analyzed 95 naturally processed Kd peptides in conjunction with our structural data. Comparisons of Kd-Flu to other class I peptide-MHC complexes reveal that the conformation of Flu in the Kd groove is similar to that of peptides associated with Db despite differences in anchoring strategies between the two MHC proteins. Lastly, our structural studies provide a detailed framework for understanding the role of individual peptide residues in T-cell recognition events. Expression and Purification—The extracellular domains of Kd (heavy chain, residues 1-283; murine β2-microglobulin (mβ2m), residues 1-99, with signal peptides omitted) were expressed separately in the bacterial strain BL21CodonPlus® (DE3)RIL (Stratagene) as insoluble inclusion bodies. LB media (8 liters, 37 °C) was inoculated from a single colony, and protein expression was induced at A595 of 0.8 with 0.5 mm isopropyl 1-thio-β-D-galactopyranoside. Cells were harvested and suspended in 200 ml of buffer containing 50 mm Tris, pH 8.0, 25% (w:v) sucrose, 1 mm EDTA, 10 mm dithiothreitol, and 0.01% (w:v) NaN3. Lysozyme (0.4 mg/ml), DNase I (40 μg/ml), and MgCl2 (10 mm) were added to the suspension, and the cells were lysed by the addition of 200 ml of buffer containing 50 mm Tris, pH 8.0, 1% (v:v) Triton X-100, 1% (w:v) sodium deoxycholate, 100 mm NaCl, 10 mm dithiothreitol, and 0.01% NaN3. After lysis, EDTA was added (12.5 mm) and insoluble protein was pelleted by centrifugation. The inclusion bodies were washed three times with buffer containing 50 mm Tris, pH 8.0, 0.5% Triton X-100, 100 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, and 0.01% NaN3. To remove detergent the inclusion bodes were washed twice with buffer as described above but without Triton X-100. Protein purity was confirmed by SDS-PAGE. The purified, detergent-free, inclusion bodies were solubilized overnight in 6 m Gdn·HCl, 10 mm Tris, pH 8.0, and 10 mm β-mercaptoethanol. To form the Kd-Flu complex, murine β2m, and heavy chain were refolded under oxidative conditions in the presence of 10 molar excess of Flu (influenza A/PR8/34 nucleoprotein residues 147-155, TYQRTRALV). Refolding was performed at 4 °C using a rapid dilution method. Briefly, Flu was diluted to 15 μm in 500 ml of refolding buffer (100 mm Tris, pH 8.0, 400 mm l-Arg, 2.0 mm EDTA, 0.5 mm GSSG, 5.0 mm GSH, and protease inhibitors). Murine β2m was injected into the refolding reaction to concentration of 4.5 μm. Following 30 min of incubation the heavy chain (final concentration 1.5 μm) was injected in three separate batches spaced over a 24-h period. The final concentration of Gdn·HCl in the refolding reaction did not exceed 100 mm. After an overnight incubation the refolding reaction was concentrated to 4 ml using an Amicon ultrafiltration device (Millipore, Billerica, MA). The Kd-Flu complex was purified from protein aggregates and other impurities on a Superdex75 (GE Healthcare, Piscataway, NJ) size exclusion column using a running buffer containing 20 mm HEPES, pH 7.5, 150 mm NaCl, and 0.01% NaN3. The fractions containing Kd-Flu were pooled, diluted 3-fold in buffer containing 20 mm Tris, pH 8.5, loaded on an anion exchange Mono Q column (GE Healthcare), and eluted with a NaCl gradient (0 mm to 400 mm NaCl over 30 ml). Prior to crystallization the pure Kd-Flu complex was exchanged in buffer containing 20 mm HEPES, pH 7.5, and 150 mm NaCl. Typically 400 μg of purified complex were obtained from a 500-ml refolding reaction. Crystallization and Data Collection—Diffraction-quality crystals of Kd-Flu were obtained by hanging-drop vapor diffusion method. Protein at 6-8 mg/ml was equilibrated at 20 °C against 12% (w:v) polyethylene glycol 2000, 5% (w:v) 2-methyl-2,4-pentanediol, and 100 mm HEPES, pH 6.9. Small crystals obtained in these drops were used to microseed protein hanging drops equilibrated against similar conditions with marginally lower concentration of polyethylene glycol 2000. Larger crystals appeared overnight and grew over 3-4 weeks. As the crystallization conditions were cryoprotective, no additional compounds were added for liquid nitrogen flash cooling. X-ray diffraction data for Kd-Flu were collected on a Raxis IV detector (Rigaku/MCS, The Woodlands, TX) to 2.6-Å resolution. A total of 249 frames was collected each representing a 1.5° oscillation range. These data were indexed and integrated using DENZO (HKL Suite, HKL Research, Inc., Charlottesville, VA) in the primitive orthorhombic lattice with cell dimensions a = 117.2 Å, b = 85.3 Å, c = 42.6 Å and scaled and merged using SCALEPACK (HKL Suite, HKL Research, Inc., Charlottesville VA). Wilson scaling was applied to the final output structure factor amplitudes (Collaborative Computing Project 4 (CCP4), Daresbury Laboratory, Warrington UK (32Number Collaborative Computational Project Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar)). Structure Determination and Refinement—The structure of Kd-Flu was determined by molecular replacement using AMoRe (CCP4). The coordinates of Kb-Ova (PDB 1VAC) with the Ova peptide and water molecules omitted were used as a search model. The rotation search yielded a single, distinct solution, and translation searches were run in all possible orthorhombic symmetry groups. The highest signal (correlation coefficient = 33.1% and R value = 49.4% for all 15-4.0 Å data) was obtained in the P212121 space group, consistent with the systematic absences in the data. Rigid body refinement of the three domains of the search model (α1α2, α3, and murine β2m) against the Kd data yielded an R value of 41.8% for the 20- to 2.6-Å resolution data. Extensive model building was performed with the macromolecular modeling program O (O version 6.22, Uppsala Software Factory, Sweden) using 2Fo - Fc, Fo - Fc, and 2Fo - Fc composite omit maps (CNS, Yale University, New Haven CT (33Brunger 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. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar)). Atomic refinement was done employing simulated annealing, energy minimization, and restrained B-factor refinement protocols as implemented in CNS. The final model includes a total of 383 residues (residues A1 to A275 for the heavy chain, B1 to B99 for murine β2m, and P1 to P9 for Flu) and 114 water molecules. Atomic coordinates were not assigned to residues 276-283 from the heavy chain and the N-terminal methionine of β2m as no interpretable electron density was seen for these regions of Kd-Flu. Refinement of this final model against the 20- to 2.6-Å resolution data converged to an R value of 21.6% with an Rfree of 26.9% (4.3% test set) with good geometry (Table 1).TABLE 1Summary of data collection and refinement statisticsCrystal space groupP212121Unit cell (Å)a = 117.2; b = 85.3; c = 42.6Data processingObservations to 2.6 Å/unique72,826/12,404Completeness (%)89.1 (92.6)aValues in parentheses are for data in the highest resolution shell for data processing (2.72-2.60) and refinement (2.76-2.60).RsymbStatistics as defined in SCALEPACK.0.129 (0.597)I/σ12.8 (2.17)RefinementData range (Å)20.0-2.6Reflections (F>0)12,244Completeness (%)85.0 (81.6)Reflections in Rfree set592 (86)Non-hydrogen atoms3,092Solvent molecules114r.m.s. Δ bond lengths (Å)cStatistics as defined in CNS.0.008r.m.s. Δ bond angles (°)1.4r.m.s. Δ dihedral angles (°)25.0r.m.s. Δ improper angles (°)0.92Average B factor for H-2Kd53Average B factor for Flu peptide49Average B factor for water molecules43Est. coord. error from Luzzati plot (Å)0.34Rcryst (%)cStatistics as defined in CNS.21.8 (34.3)Rfree (%)cStatistics as defined in CNS.26.9 (36.4)Ramachandran plotMost favored/additional (%)86.5/12.6Generously allowed/disallowed (%)0.9/0.0a Values in parentheses are for data in the highest resolution shell for data processing (2.72-2.60) and refinement (2.76-2.60).b Statistics as defined in SCALEPACK.c Statistics as defined in CNS. Open table in a new tab Computational Analysis—Graphical structure representations were primarily created using Ribbons (34Carson M. J. Mol. Graphics. 1987; 5: 103-106Crossref Scopus (514) Google Scholar). Molecular surfaces of the peptide-binding groove (Figs. 4A and 5) were generated using InsightII (Biosym Technologies, San Diego CA). r.m.s.d. values between the different MHC proteins were calculated using an incremental combinatorial extension algorithm (35Shindyalov I.N. Bourne P.E. Protein Eng. 1998; 11: 739-747Crossref PubMed Scopus (1702) Google Scholar). r.m.s.d. values between the different MHC peptides were calculated using Lsqkab (CCP4). HBPLUS (36McDonald I.K. Thornton J.M. J. Mol. Biol. 1994; 238: 777-793Crossref PubMed Scopus (1885) Google Scholar) was used to catalogue contacting atoms and putative hydrogen bonds. Shape complementarity scores (37Lawrence M.C. Colman P.M. J. Mol. Biol. 1993; 234: 946-950Crossref PubMed Scopus (1107) Google Scholar) were calculated using CCP4. Atomic accessible surfaces were calculated using the program NACCESS. 3S. J. Hubbard and J. M. Thornton, Dept. of Chemistry and Molecular Biology, University College London. FIGURE 5The peptide-binding motif of Kd and characterized TCR interactions. The solvent-accessible surface of the peptide-binding groove is displayed as a blue dotted surface. Aligned under the rendering are the sequences of Flu (boxed) and six other antigenic peptides representative of the Kd peptide-binding motif as well as three antigenic peptides for which TCR contact residues (denoted by the asterisk) have been determined. Pocket B and the P2 residue are highlighted in red while the secondary anchors P5 and P9 and their respective pockets C and F are highlighted in green. Percent values in parentheses reflect the frequencies at which each amino acid is found at the indicated position; only the most prevalent amino acids are denoted. The shorthand above each aligned residue denotes the orientation of each residue of Flu in the complex. The anchor symbol denotes anchoring residues that point down toward the peptide binding platform; ↑ denotes residues pointing away from the peptide binding platform and toward solvent; → denotes residues pointing toward the α1 helix; and ← denotes residues pointing toward the α2 helix (see Fig. 4A).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Structure Determination—The extracellular domains of Kd (heavy chain, residues 1-283 and murine β2m, residues 1-99 plus an N-terminal methionine) were expressed separately in Escherichia coli as insoluble inclusion bodies. The Kd-Flu complex was formed in vitro under oxidative refolding conditions in the presence of excess peptide and was purified using size exclusion and anion exchange chromatographies. Electrospray mass spectral analysis of the complex confirmed the presence of abundant peaks at 32,865.69 Da and 11,817.81 Da corresponding to the predicted molecular weights of the heavy chain and murine β2m, respectively. Further inspection of the mass spectrum over lower mass ranges revealed a monoisotopic, singly charged peak at 1,106.7 Da corresponding to Flu. The Kd-Flu complex crystallized in the orthorhombic space group P212121 with one complex per asymmetric unit. Initial phase estimates were obtained by molecular replacement. After initial refinement, easily interpretable electron density was seen for the bound peptide that improved upon further building and refinement cycles. Diffraction data to 2.6-Å resolution were used for refinement of the final atomic model, which has an R factor of 21.6% (Rfree = 26.9%) with good angle and bond geometry (Table 1). The electron density maps for the whole complex were of good quality (Fig. 1A). No ambiguities were seen for the main chain and the side chains of the bound peptide except for a small break in the electron density between the Cβ and Cδ carbons of the exposed ArgP6 side chain (Fig. 1A). Overall Structural Features of the Complex—Kd-Flu is very similar to the structures of other MHC class I proteins (Fig. 1B). Minor differences were observed for the conformations of solvent-exposed loops and the terminal regions of the complex. Flu is bound in the Kd groove between the α1 and α2 helices and on top of the β-sheet platform (Fig. 1) in canonical manner (7Wilson I.A. Fremont D.H. Semin. Immunol. 1993; 5: 75-80Crossref PubMed Scopus (39) Google Scholar). Structural alignment of Kd-Flu to the structures of other murine MHC class I complexes yielded overall pairwise r.m.s.d. values of 1.14 Å (Dd, 80.4% sequence identity) to 2.15 Å (Db, 80.7% sequence identity). An alignment of the α1α2 domains alone yielded pairwise r.m.s.d. values of 0.63 Å (Kb, 80.4% sequence identity) to 1.01 Å (Ld, 81.1% sequence identity) reflecting the high degree of general similarity among these proteins. Backbone Conformation of Kd-presented Flu—Like most MHC-bound nonameric peptides, the backbone of Flu assumes a predominantly extended conformation with a bulge at residues P6 and P7. Surprisingly, the main-chain kink adopted by Flu results from an infrequently observed, unfavorable conformation of ArgP6 that is well supported by our experimental data (Fig. 1A). The P6-P7 bulge is associated with a hydrophobic ridge formed by polymorphic residues Tyr156 and Trp73 in the Kd groove. While favorable Ramachandran angles would be observed if the P6-P7 peptide bond were flipped, this conformation would preclude several favorable interactions and engender steric clashes with Kd (see Fig. 4A). We compared the conformation of Flu to that of nine-residue peptides bound to Kb (38Fremont D.H. Stura E.A. Matsumura M. Peterson P.A. Wilson I.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2479-2483Crossref PubMed Scopus (241) Google Scholar, 39Fremont D.H. Matsumura M. Stura E.A. Peterson P.A. Wilson I.A. Science. 1992; 257: 919-927Crossref PubMed Scopus (825) Google Scholar), Ld (40Balendiran G.K. Solheim J.C. Young A.C. Hansen T.H. Nathenson S.G. Sacchettini J.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6880-6885Crossref PubMed Scopus (73) Google Scholar), and Db (41Glithero A. Tormo J. Haurum J.S. Arsequell G. Valencia G. Edwards J. Springer S. Townsend A. Pao Y.L. Wormald M. Dwek R.A. Jones E.Y. Elliott T. Immunity. 1999; 10: 63-74Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 42Tissot A.C. Ciatto C. Mittl P.R. Grutter M.G. Pluckthun A. J. Mol. Biol. 2000; 302: 873-885Crossref PubMed Scopus (39) Google Scholar, 43Ciatto C. Tissot A.C. Tschopp M. Capitani G. Pecorari F. Pluckthun A. Grutter M.G. J. Mol. Biol. 2001; 312: 1059-1071Crossref PubMed Scopus (16) Google Scholar, 44Ostrov D.A. Roden M.M. Shi W. Palmieri E. Christianson G.J. Mendoza L. Villaflor G. Tilley D. Shastri N. Grey H. Almo S.C. Roopenian D. Nathenson S.G. J. Immunol. 2002; 168: 283-289Crossref PubMed Scopus (38) Google Scholar, 45Young A.C. Zhang W. Sacchettini J.C. Nathenson S.G. Cell. 1994; 76: 39-50Abstract Full Text PDF PubMed Scopus (241) Google Scholar). The main-chain conformations of Kb-presented peptides vary significantly from that of Flu with overall r.m.s.d. values ranging from 1.43 to 1.47 Å. The greatest differences were observed in the region between P5 and P7 where the r.m.s.d. values for the Cα atoms ranged from 1.22 to 3.66 Å (Fig. 2A). The conformation of the Ld peptide resembles more closely that of Flu with r.m.s.d. 0.95 Å, but nevertheless differs significantly between P4 and P6 (Fig. 2A). Comparison of Flu with Db-presented peptides reveals that they adopt nearly identical main-chain conformations all the way from P1 to P9 with r.m.s.d. values ranging from 0.62 to 0.81 Å (Fig. 2A). Interestingly, Db has a similar hydrophobic ridge as Kd located beneath the P6-P7 kink (43Ciatto C. Tissot A.C. Tschopp M. Capitani G. Pecorari F. Pluckthun A. Grutter M.G. J. Mol. Biol. 2001; 312: 1059-1071Crossref PubMed Scopus (16) Google Scholar). We also compared the dihedral angles of the aligned peptides (Fig. 2B). This analysis revealed that the backbones of the Db peptides adopt a P6-P7 bulge associated with unfavorable dihedral angles for their P6 residues similar to the one in Flu (Fig. 2B). This bulging at P6-P7 was absent in the Ld peptide (Fig. 2B) despite the presence of a similar hydrophobic ridge in the same region of the Ld groove (40Balendiran G.K. Solheim J.C. Young A.C. Hansen T.H. Nathenson S.G. Sacchettini J.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6880-6885Crossref PubMed Scopus (73) Google Scholar). Hydrogen Bonding to the Flu Backbone—The Flu main chain has 19 nitrogen and oxygen atoms, 14 of which hydrogen bond with Kd either directly or through water-mediated networks. Of the 17 MHC amino acids that participate in hydrogen bonding 8 are invariant among MHC class I proteins. These residues anchor the N- and C-terminal regions of Flu through highly conservative hydrogen bonding networks at each end of the binding groove (Fig. 3). Eight main-chain nitrogen and oxygen atoms between GlnP3 and ValP8 mediate hydrogen bonds with polymorphic groove residues (Fig. 3). Of particular note are the hydrogen bonds to the main-chain oxygen of ArgP6 and the main-chain oxygen and nitrogen of AlaP7. The carbonyl oxygen atoms of both residues participate in a bifurcate hydrogen bonding network with the Nϵ1 nitrogen of Trp73, whereas on the opposite side of the Flu backbone the amide nitrogen atom of P7 hydrogen bonds with Asp152 (Fig. 3). This hydrogen bonding arrangement can only form as a result of the unfavorable turn in the Flu main chain at ArgP6. Binding Pockets in the Kd Groove—Specificity of peptide-MHC association is imparted through a myriad of interactions with peptide anchor side chains, which are sequestered in distinct pockets of the MHC groove. To visualize these pockets in Kd we calculated a solvent-accessible surface (46Connolly M.L. Science. 1983; 221: 709-713Crossref PubMed Scopus (2453) Google Scholar) for a spherical probe with a radius of 1.4 Å for the α1α2 domain. Five distinct pockets are clearly apparent in the Kd groove, which correspond to pockets A, B, C, D, and F according to the nomen-clature of Matsumura et al. (47Matsumura M. Fremont D.H. Peterson P.A. Wilson I.A. Science. 1992; 257: 927-934Crossref PubMed Scopus (653) Google Scholar) (Fig. 4A). Pocket E, which is the most variable between the different MHC proteins, is absent in our structure. Instead, the polymorphic residues Trp73 and Tyr156 fill the E pocket location creating the hydrophobic ridge across the Kd groove that accommodates the P6-P7 turn in Flu (Fig. 4A). Interestingly, this ridge is absent in Kb, which preferentially binds 8-residue peptides. To systematically identify the MHC residues that make up the Kd pockets, we calculated solvent-accessible surfaces of the α1α2 domain" @default.
• W2098086095 created "2016-06-24" @default.
• W2098086095 creator A5061136971 @default.
• W2098086095 creator A5065780658 @default.
• W2098086095 date "2006-04-01" @default.
• W2098086095 modified "2023-10-18" @default.
• W2098086095 title "Structural Definition of the H-2Kd Peptide-binding Motif" @default.
• W2098086095 cites W1529539669 @default.
• W2098086095 cites W1537166599 @default.
• W2098086095 cites W1595191255 @default.
• W2098086095 cites W1679981289 @default.
• W2098086095 cites W1771845532 @default.
• W2098086095 cites W1832913110 @default.
• W2098086095 cites W1893184572 @default.
• W2098086095 cites W1957876511 @default.
• W2098086095 cites W1967582433 @default.
• W2098086095 cites W1969857935 @default.
• W2098086095 cites W1971302107 @default.
• W2098086095 cites W1971837823 @default.
• W2098086095 cites W1978720797 @default.
• W2098086095 cites W1979804467 @default.
• W2098086095 cites W1990395260 @default.
• W2098086095 cites W1990806106 @default.
• W2098086095 cites W1991022642 @default.
• W2098086095 cites W1994048581 @default.
• W2098086095 cites W1995017064 @default.
• W2098086095 cites W1995506240 @default.
• W2098086095 cites W2001641653 @default.
• W2098086095 cites W2009912771 @default.
• W2098086095 cites W2014916785 @default.
• W2098086095 cites W2019993505 @default.
• W2098086095 cites W2027262407 @default.
• W2098086095 cites W2029955341 @default.
• W2098086095 cites W2031101268 @default.
• W2098086095 cites W2043120431 @default.
• W2098086095 cites W2049151427 @default.
• W2098086095 cites W2051628563 @default.
• W2098086095 cites W2051959131 @default.
• W2098086095 cites W2054965969 @default.
• W2098086095 cites W2055415050 @default.
• W2098086095 cites W2056034415 @default.
• W2098086095 cites W2063841466 @default.
• W2098086095 cites W2073883777 @default.
• W2098086095 cites W2076558278 @default.
• W2098086095 cites W2083424266 @default.
• W2098086095 cites W2091257232 @default.
• W2098086095 cites W2099533131 @default.
• W2098086095 cites W2104011738 @default.
• W2098086095 cites W2107971340 @default.
• W2098086095 cites W2108508060 @default.
• W2098086095 cites W2115603980 @default.
• W2098086095 cites W2115610174 @default.
• W2098086095 cites W2118599971 @default.
• W2098086095 cites W2119013789 @default.
• W2098086095 cites W2131609927 @default.
• W2098086095 cites W2134674240 @default.
• W2098086095 cites W2141488911 @default.
• W2098086095 cites W2149911790 @default.
• W2098086095 cites W2150383532 @default.
• W2098086095 cites W2152326664 @default.
• W2098086095 cites W2153918116 @default.
• W2098086095 cites W2159404298 @default.
• W2098086095 cites W2162424523 @default.
• W2098086095 cites W2167449756 @default.
• W2098086095 cites W2917210380 @default.
• W2098086095 doi "https://doi.org/10.1074/jbc.m510511200" @default.
• W2098086095 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16473882" @default.
• W2098086095 hasPublicationYear "2006" @default.
• W2098086095 type Work @default.
• W2098086095 sameAs 2098086095 @default.
• W2098086095 citedByCount "27" @default.
• W2098086095 countsByYear W20980860952012 @default.
• W2098086095 countsByYear W20980860952013 @default.
• W2098086095 countsByYear W20980860952014 @default.
• W2098086095 countsByYear W20980860952016 @default.
• W2098086095 countsByYear W20980860952017 @default.
• W2098086095 countsByYear W20980860952018 @default.
• W2098086095 countsByYear W20980860952019 @default.
• W2098086095 countsByYear W20980860952020 @default.
• W2098086095 countsByYear W20980860952021 @default.
• W2098086095 countsByYear W20980860952022 @default.
• W2098086095 crossrefType "journal-article" @default.
• W2098086095 hasAuthorship W2098086095A5061136971 @default.
• W2098086095 hasAuthorship W2098086095A5065780658 @default.
• W2098086095 hasBestOaLocation W20980860951 @default.
• W2098086095 hasConcept C107038049 @default.
• W2098086095 hasConcept C138885662 @default.
• W2098086095 hasConcept C185592680 @default.
• W2098086095 hasConcept C2779281246 @default.
• W2098086095 hasConcept C32276052 @default.
• W2098086095 hasConcept C55493867 @default.
• W2098086095 hasConcept C70721500 @default.
• W2098086095 hasConcept C86803240 @default.
• W2098086095 hasConceptScore W2098086095C107038049 @default.
• W2098086095 hasConceptScore W2098086095C138885662 @default.
• W2098086095 hasConceptScore W2098086095C185592680 @default.
• W2098086095 hasConceptScore W2098086095C2779281246 @default.
• W2098086095 hasConceptScore W2098086095C32276052 @default.

• next