Detailed Comparison of Two Molecular Models of the Human CD40 Ligand with an X-ray Structure and Critical Assessment of Model-based Mutagenesis and Residue Mapping Studies
1998; Elsevier BV; Volume: 273; Issue: 38 Linguagem: Inglês
10.1074/jbc.273.38.24603
ISSN1083-351X
Autores Tópico(s)Immune Cell Function and Interaction
ResumoThe interactions between the B cell receptor CD40 and its ligand on T cells are critical for the integrity of immune responses. The human CD40 ligand gp39, a tumor necrosis factor-like protein, has been the subject of intense efforts to identify the receptor-binding site and to analyze naturally occurring mutations that compromise gp39 function in vivo. These investigations relied heavily on molecular models of gp39, built in the presence of only ∼25% sequence identity to tumor necrosis factor. The x-ray structure of gp39 has made it possible to assess modeling accuracy and to evaluate the results of model-based mutagenesis analyses. Although the models display local errors, their accuracy was sufficient to predict the CD40-binding site, to map natural mutations, and to rationalize their effects. One of five gp39 residues critical for CD40 binding was displaced in the models, and 1 of 21 point mutants was incorrectly classified. Factors most important for the reliability of the molecular models and their successful applications were valid sequence alignments and the focus of experimental studies on regions of high prediction confidence. Analysis of mutagenesis experiments correlated with anti-gp39 monoclonal antibody binding studies to assess the conformational integrity of mutant proteins. The interactions between the B cell receptor CD40 and its ligand on T cells are critical for the integrity of immune responses. The human CD40 ligand gp39, a tumor necrosis factor-like protein, has been the subject of intense efforts to identify the receptor-binding site and to analyze naturally occurring mutations that compromise gp39 function in vivo. These investigations relied heavily on molecular models of gp39, built in the presence of only ∼25% sequence identity to tumor necrosis factor. The x-ray structure of gp39 has made it possible to assess modeling accuracy and to evaluate the results of model-based mutagenesis analyses. Although the models display local errors, their accuracy was sufficient to predict the CD40-binding site, to map natural mutations, and to rationalize their effects. One of five gp39 residues critical for CD40 binding was displaced in the models, and 1 of 21 point mutants was incorrectly classified. Factors most important for the reliability of the molecular models and their successful applications were valid sequence alignments and the focus of experimental studies on regions of high prediction confidence. Analysis of mutagenesis experiments correlated with anti-gp39 monoclonal antibody binding studies to assess the conformational integrity of mutant proteins. X-linked hyper-IgM tumor necrosis factor monoclonal antibody root mean square deviation. The CD40 ligand (CD40L, gp39) is a type II transmembrane protein predominantly expressed on the surface of activated T cells (1Armitage R.J. Fanslow W.C. Strockbine L. Sato T.A. Clifford K.N. Macduff B.M. Anderson D.M. Gimpel S.D. Davis-Smith T. Maliszewski C.R. Clark E.A. Smith C.A. Grabstein K.H. Cosman D. Spriggs M.K. Nature. 1992; 357: 80-82Crossref PubMed Scopus (988) Google Scholar, 2Hollenbaugh D. Grosmaire L.S. Kullas C.D. Chalupny N.J. Braesch-Andersen S. Noelle R.J. Stamenkovic I. Ledbetter J.A. Aruffo A. EMBO J. 1992; 11: 4313-4321Crossref PubMed Scopus (499) Google Scholar). Interactions between gp39 and its receptor, CD40, a type I transmembrane protein on B cells (3Clark E.A. Ledbetter J.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4494-4498Crossref PubMed Scopus (418) Google Scholar, 4Clark E.A. Lane P.J. Annu. Rev. Immunol. 1991; 9: 97-127Crossref PubMed Scopus (138) Google Scholar), are critical for T cell-dependent B cell proliferation and the regulation of the humoral immune response (5Noelle R.J. Ledbetter J.A. Aruffo A. Immunol. Today. 1992; 13: 431-433Abstract Full Text PDF PubMed Scopus (308) Google Scholar, 6Foy T.M. Aruffo A. Bajorath J. Buhlmann J.E. Noelle R.J. Annu. Rev. Immunol. 1996; 14: 591-617Crossref PubMed Scopus (574) Google Scholar). The critical role of CD40/gp39 receptor/ligand interactions for effective antibody production and isotype switching was firmly established by the discovery of naturally occurring mutations in gp39 that cause X-linked hyper-IgM (XHIM)1 syndrome (7Aruffo A. Farrington M. Hollenbaugh D. Xu L. Milatovich A. Nonoyama S. Bajorath J. Grosmaire L.S. Stenkamp R. Neubauer M. Roberts R.L. Noelle R.J. Ledbetter J.A. Francke U. Ochs H.D. Cell. 1993; 72: 291-300Abstract Full Text PDF PubMed Scopus (752) Google Scholar). Patients with defective gp39 genes do not mount an effective humoral immune response and are severely immunocompromised (8Aruffo A. Hollenbaugh D. Ochs H.D. Curr. Opin. Hematol. 1994; 1: 12-18PubMed Google Scholar). Thus, the study of CD40/gp39 interactions has been a focal point of intense efforts to better understand T cell-dependent B cell activation and the basis of productive immune responses (6Foy T.M. Aruffo A. Bajorath J. Buhlmann J.E. Noelle R.J. Annu. Rev. Immunol. 1996; 14: 591-617Crossref PubMed Scopus (574) Google Scholar). CD40 and gp39 are members of the tumor necrosis factor (TNF) receptor (9Beutler B. van Huffel C. Science. 1994; 264: 667-668Crossref PubMed Scopus (420) Google Scholar, 10Smith C.A. Farrah T. Goodwin R.G. Cell. 1994; 76: 959-962Abstract Full Text PDF PubMed Scopus (1839) Google Scholar) and TNF (10Smith C.A. Farrah T. Goodwin R.G. Cell. 1994; 76: 959-962Abstract Full Text PDF PubMed Scopus (1839) Google Scholar) superfamilies, respectively. The extracellular region of gp39 displays ∼25% sequence identity to TNF proteins (2Hollenbaugh D. Grosmaire L.S. Kullas C.D. Chalupny N.J. Braesch-Andersen S. Noelle R.J. Stamenkovic I. Ledbetter J.A. Aruffo A. EMBO J. 1992; 11: 4313-4321Crossref PubMed Scopus (499) Google Scholar,7Aruffo A. Farrington M. Hollenbaugh D. Xu L. Milatovich A. Nonoyama S. Bajorath J. Grosmaire L.S. Stenkamp R. Neubauer M. Roberts R.L. Noelle R.J. Ledbetter J.A. Francke U. Ochs H.D. Cell. 1993; 72: 291-300Abstract Full Text PDF PubMed Scopus (752) Google Scholar). To aid in the analysis of CD40-gp39 interactions, we generated, in the absence of an experimentally determined three-dimensional structure, two molecular models of gp39 by comparative modeling (11Greer J. Proteins. 1990; 7: 317-334Crossref PubMed Scopus (391) Google Scholar,12Bajorath J. Stenkamp R. Aruffo A. Protein Sci. 1993; 2: 317-334Crossref Scopus (100) Google Scholar). These models were based on x-ray structures of TNF-α (13Eck M.J. Sprang S.R. J. Biol. Chem. 1989; 264: 17595-17605Abstract Full Text PDF PubMed Google Scholar) and TNF-β (14Eck M.J. Ultsch M. Rinderknecht E. de Vos A.M. Sprang S.R. J. Biol. Chem. 1992; 267: 2119-2122Abstract Full Text PDF PubMed Google Scholar, 15Banner D.W. D'Arcy A. Janes W. Gentz R. Schoenfeld H.-J. Broger C. Loetscher H. Lesslauer W. Cell. 1993; 73: 431-445Abstract Full Text PDF PubMed Scopus (990) Google Scholar). Initially, we constructed a TNF-α-based model of gp39 (AM) (7Aruffo A. Farrington M. Hollenbaugh D. Xu L. Milatovich A. Nonoyama S. Bajorath J. Grosmaire L.S. Stenkamp R. Neubauer M. Roberts R.L. Noelle R.J. Ledbetter J.A. Francke U. Ochs H.D. Cell. 1993; 72: 291-300Abstract Full Text PDF PubMed Scopus (752) Google Scholar), while others reported a three-dimensional model of the murine CD40 ligand (16Peitsch M.C. Jongeneel C.V. Int. Immunol. 1993; 5: 233-238Crossref PubMed Scopus (153) Google Scholar). The gp39 model was used to analyze the location of natural gp39 mutations isolated from XHIM patients (7Aruffo A. Farrington M. Hollenbaugh D. Xu L. Milatovich A. Nonoyama S. Bajorath J. Grosmaire L.S. Stenkamp R. Neubauer M. Roberts R.L. Noelle R.J. Ledbetter J.A. Francke U. Ochs H.D. Cell. 1993; 72: 291-300Abstract Full Text PDF PubMed Scopus (752) Google Scholar) and to guide an initial mutagenesis effort to identify gp39 residues important for the interaction with CD40 (17Bajorath J. Chalupny N.J. Marken J.S. Siadak A.W. Skonier J. Gordon M. Hollenbaugh D. Noelle R.J. Ochs H.D. Aruffo A. Biochemistry. 1995; 34: 1833-1844Crossref PubMed Scopus (69) Google Scholar). After an x-ray structure of the TNF receptor·TNF-β complex (15Banner D.W. D'Arcy A. Janes W. Gentz R. Schoenfeld H.-J. Broger C. Loetscher H. Lesslauer W. Cell. 1993; 73: 431-445Abstract Full Text PDF PubMed Scopus (990) Google Scholar) became available, we also generated a TNF-β-based molecular model of gp39 (BM) (18Bajorath J. Marken J.S. Chalupny N.J. Spoon T.L. Siadak A.W. Gordon M. Noelle R.J. Hollenbaugh D. Aruffo A. Biochemistry. 1995; 34: 9884-9892Crossref PubMed Scopus (55) Google Scholar). This model was used to continue the mutagenesis analysis of the CD40/gp39 interaction, to outline the receptor-binding site of gp39 (18Bajorath J. Marken J.S. Chalupny N.J. Spoon T.L. Siadak A.W. Gordon M. Noelle R.J. Hollenbaugh D. Aruffo A. Biochemistry. 1995; 34: 9884-9892Crossref PubMed Scopus (55) Google Scholar), and to carry out a three-dimensional survey of 21 naturally occurring gp39 mutations (19Bajorath J. Seyama K. Nonoyama S. Ochs H.D. Aruffo A. Protein Sci. 1996; 5: 531-534Crossref PubMed Scopus (33) Google Scholar). The more recently determined x-ray structure of gp39 (20Karpusas M. Hsu Y.M. Wang J.H. Thompson J. Lederman S. Chess L. Thomas D. Structure. 1995; 3: 1031-1039Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar) has made it possible to assess the accuracy of our comparative structure prediction and to evaluate if the models were applied in a meaningful way and if the obtained results were valid. Since a substantial body of experimental analysis was based on the gp39 models (7Aruffo A. Farrington M. Hollenbaugh D. Xu L. Milatovich A. Nonoyama S. Bajorath J. Grosmaire L.S. Stenkamp R. Neubauer M. Roberts R.L. Noelle R.J. Ledbetter J.A. Francke U. Ochs H.D. Cell. 1993; 72: 291-300Abstract Full Text PDF PubMed Scopus (752) Google Scholar, 17Bajorath J. Chalupny N.J. Marken J.S. Siadak A.W. Skonier J. Gordon M. Hollenbaugh D. Noelle R.J. Ochs H.D. Aruffo A. Biochemistry. 1995; 34: 1833-1844Crossref PubMed Scopus (69) Google Scholar, 18Bajorath J. Marken J.S. Chalupny N.J. Spoon T.L. Siadak A.W. Gordon M. Noelle R.J. Hollenbaugh D. Aruffo A. Biochemistry. 1995; 34: 9884-9892Crossref PubMed Scopus (55) Google Scholar, 19Bajorath J. Seyama K. Nonoyama S. Ochs H.D. Aruffo A. Protein Sci. 1996; 5: 531-534Crossref PubMed Scopus (33) Google Scholar), a critical assessment of these studies should be of considerable interest in addition to the prediction exercise. Therefore, a detailed comparison of the gp39 models and x-ray structure and a reevaluation of our binding site analysis and residue mapping studies were carried out. The results are presented herein. With the exception of a short β-strand at the edge of a sheet, the core regions and β-strands of gp39 were well predicted. Substantial conformational errors were observed in several loops. Modeling inaccuracies were the source of subsequent errors including the misinterpretation of 1 of 21 naturally occurring gp39 mutants and the relative displacement of a residue important for CD40 binding by 6–7 Å. However, the analysis shows that surface residues could be selected for mutagenesis with confidence and that residues important for CD40 binding were successfully predicted and experimentally confirmed. The CD40-binding site was correctly mapped to the interface between adjacent gp39 monomers; and the locations of natural gp39 mutations were well predicted, and their putative effects were rationalized in a meaningful way. Gp39 modeling, mutagenesis, and ligand binding experiments have been described in detail (7Aruffo A. Farrington M. Hollenbaugh D. Xu L. Milatovich A. Nonoyama S. Bajorath J. Grosmaire L.S. Stenkamp R. Neubauer M. Roberts R.L. Noelle R.J. Ledbetter J.A. Francke U. Ochs H.D. Cell. 1993; 72: 291-300Abstract Full Text PDF PubMed Scopus (752) Google Scholar, 17Bajorath J. Chalupny N.J. Marken J.S. Siadak A.W. Skonier J. Gordon M. Hollenbaugh D. Noelle R.J. Ochs H.D. Aruffo A. Biochemistry. 1995; 34: 1833-1844Crossref PubMed Scopus (69) Google Scholar, 18Bajorath J. Marken J.S. Chalupny N.J. Spoon T.L. Siadak A.W. Gordon M. Noelle R.J. Hollenbaugh D. Aruffo A. Biochemistry. 1995; 34: 9884-9892Crossref PubMed Scopus (55) Google Scholar) and are briefly summarized herein. Methods applied to analyze and compare the models and x-ray structure are also described. After generating structure-oriented sequence alignments of the extracellular region, gp39 models AM (7Aruffo A. Farrington M. Hollenbaugh D. Xu L. Milatovich A. Nonoyama S. Bajorath J. Grosmaire L.S. Stenkamp R. Neubauer M. Roberts R.L. Noelle R.J. Ledbetter J.A. Francke U. Ochs H.D. Cell. 1993; 72: 291-300Abstract Full Text PDF PubMed Scopus (752) Google Scholar) and BM (18Bajorath J. Marken J.S. Chalupny N.J. Spoon T.L. Siadak A.W. Gordon M. Noelle R.J. Hollenbaugh D. Aruffo A. Biochemistry. 1995; 34: 9884-9892Crossref PubMed Scopus (55) Google Scholar) were generated using TNF-α (13Eck M.J. Sprang S.R. J. Biol. Chem. 1989; 264: 17595-17605Abstract Full Text PDF PubMed Google Scholar) and TNF-β (15Banner D.W. D'Arcy A. Janes W. Gentz R. Schoenfeld H.-J. Broger C. Loetscher H. Lesslauer W. Cell. 1993; 73: 431-445Abstract Full Text PDF PubMed Scopus (990) Google Scholar) as structural templates, respectively. Regions predicted to be structurally conserved in TNF proteins and gp39 were included in the models. Nonconserved residue replacements were carried out using a side chain rotamer search procedure (21Ponder J.W. Richards F.M. J. Mol. Biol. 1987; 193: 775-791Crossref PubMed Scopus (1349) Google Scholar, 22Bajorath J. Fine R.M. Immunomethods. 1992; 1: 137-146Crossref Scopus (18) Google Scholar). Conformations of regions including insertions or deletions and nonconserved loop conformations were modeled using systematic conformational search with CONGEN (23Bruccoleri R.E. Mol. Simulat. 1993; 10: 151-174Crossref Scopus (45) Google Scholar). Loop conformations with minimum solvent-accessible surface and low potential energy were selected following a previously described protocol (24Bruccoleri R.E. Haber E. Novotny J. Nature. 1988; 335: 564-568Crossref PubMed Scopus (173) Google Scholar). The molecular contacts of the initially assembled models were refined by limited energy minimization calculations (7Aruffo A. Farrington M. Hollenbaugh D. Xu L. Milatovich A. Nonoyama S. Bajorath J. Grosmaire L.S. Stenkamp R. Neubauer M. Roberts R.L. Noelle R.J. Ledbetter J.A. Francke U. Ochs H.D. Cell. 1993; 72: 291-300Abstract Full Text PDF PubMed Scopus (752) Google Scholar, 18Bajorath J. Marken J.S. Chalupny N.J. Spoon T.L. Siadak A.W. Gordon M. Noelle R.J. Hollenbaugh D. Aruffo A. Biochemistry. 1995; 34: 9884-9892Crossref PubMed Scopus (55) Google Scholar), and the stereochemical quality of the models was confirmed using PROCHECK (25Laskowski R.A. MacArthur M.S. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1997; 26: 283-291Crossref Google Scholar). The sequence-structure compatibility of the refined models was assessed using three-dimensional profile (26Luthy R. Bowie J.U. Eisenberg D. Nature. 1992; 356: 83-85Crossref PubMed Scopus (2616) Google Scholar) or energy profile (27Sippl M.J. Proteins Struct. Funct. Genet. 1993; 17: 355-362Crossref PubMed Scopus (1792) Google Scholar) analysis. Similar procedures were applied to generate three-dimensional models of CD40 (18Bajorath J. Marken J.S. Chalupny N.J. Spoon T.L. Siadak A.W. Gordon M. Noelle R.J. Hollenbaugh D. Aruffo A. Biochemistry. 1995; 34: 9884-9892Crossref PubMed Scopus (55) Google Scholar, 28Bajorath J. Aruffo A. Proteins Struct. Funct. Genet. 1997; 27: 59-70Crossref PubMed Scopus (27) Google Scholar) based on the x-ray structure of the TNF-bound TNF receptor (15Banner D.W. D'Arcy A. Janes W. Gentz R. Schoenfeld H.-J. Broger C. Loetscher H. Lesslauer W. Cell. 1993; 73: 431-445Abstract Full Text PDF PubMed Scopus (990) Google Scholar). Residues were selected for mutagenesis based on visual inspection of the molecular models. A focal point was the interface region between adjacent gp39 monomers in the homotrimer. Point mutants were constructed by overlap extension polymerase chain reaction (17Bajorath J. Chalupny N.J. Marken J.S. Siadak A.W. Skonier J. Gordon M. Hollenbaugh D. Noelle R.J. Ochs H.D. Aruffo A. Biochemistry. 1995; 34: 1833-1844Crossref PubMed Scopus (69) Google Scholar) and expressed in soluble recombinant form as gp39-CD8 fusion proteins (17Bajorath J. Chalupny N.J. Marken J.S. Siadak A.W. Skonier J. Gordon M. Hollenbaugh D. Noelle R.J. Ochs H.D. Aruffo A. Biochemistry. 1995; 34: 1833-1844Crossref PubMed Scopus (69) Google Scholar, 18Bajorath J. Marken J.S. Chalupny N.J. Spoon T.L. Siadak A.W. Gordon M. Noelle R.J. Hollenbaugh D. Aruffo A. Biochemistry. 1995; 34: 9884-9892Crossref PubMed Scopus (55) Google Scholar). A panel of conformationally sensitive anti-gp39 monoclonal antibodies (mAbs) was generated and used to assess the gross structural integrity of mutant proteins (17Bajorath J. Chalupny N.J. Marken J.S. Siadak A.W. Skonier J. Gordon M. Hollenbaugh D. Noelle R.J. Ochs H.D. Aruffo A. Biochemistry. 1995; 34: 1833-1844Crossref PubMed Scopus (69) Google Scholar, 18Bajorath J. Marken J.S. Chalupny N.J. Spoon T.L. Siadak A.W. Gordon M. Noelle R.J. Hollenbaugh D. Aruffo A. Biochemistry. 1995; 34: 9884-9892Crossref PubMed Scopus (55) Google Scholar). Only mutants that consistently bound to these mAbs comparable to the wild type were considered structurally sound. These gp39 mutant proteins were tested for receptor binding in enzyme-linked immunosorbent assays using recombinant CD40-Ig fusion protein (17Bajorath J. Chalupny N.J. Marken J.S. Siadak A.W. Skonier J. Gordon M. Hollenbaugh D. Noelle R.J. Ochs H.D. Aruffo A. Biochemistry. 1995; 34: 1833-1844Crossref PubMed Scopus (69) Google Scholar) and also in cell binding assays (17Bajorath J. Chalupny N.J. Marken J.S. Siadak A.W. Skonier J. Gordon M. Hollenbaugh D. Noelle R.J. Ochs H.D. Aruffo A. Biochemistry. 1995; 34: 1833-1844Crossref PubMed Scopus (69) Google Scholar, 18Bajorath J. Marken J.S. Chalupny N.J. Spoon T.L. Siadak A.W. Gordon M. Noelle R.J. Hollenbaugh D. Aruffo A. Biochemistry. 1995; 34: 9884-9892Crossref PubMed Scopus (55) Google Scholar). Residues affected by naturally occurring gp39 mutations were mapped on the models using computer graphics (7Aruffo A. Farrington M. Hollenbaugh D. Xu L. Milatovich A. Nonoyama S. Bajorath J. Grosmaire L.S. Stenkamp R. Neubauer M. Roberts R.L. Noelle R.J. Ledbetter J.A. Francke U. Ochs H.D. Cell. 1993; 72: 291-300Abstract Full Text PDF PubMed Scopus (752) Google Scholar, 19Bajorath J. Seyama K. Nonoyama S. Ochs H.D. Aruffo A. Protein Sci. 1996; 5: 531-534Crossref PubMed Scopus (33) Google Scholar). These positions were then classified as described under “Results.” This classification made it possible to predict the effects of these mutations. Coordinates of the gp39 monomer (20Karpusas M. Hsu Y.M. Wang J.H. Thompson J. Lederman S. Chess L. Thomas D. Structure. 1995; 3: 1031-1039Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar) were obtained from the Brookhaven Protein Data Bank (29Bernstein F.C. Koetzle T.F. Williams G.J. Meyer Jr., E.E. Brice M.D. Rodgers J.R. Kennard O. Shimanouchi T. Tasumi M. J. Mol. Biol. 1977; 112: 535-542Crossref PubMed Scopus (8183) Google Scholar) (PDB code1ALY). The active homotrimeric form of gp39 (20Karpusas M. Hsu Y.M. Wang J.H. Thompson J. Lederman S. Chess L. Thomas D. Structure. 1995; 3: 1031-1039Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar) was generated from these coordinates by applying 3-fold symmetry around the crystallographic c axis (space group H3) using an awk script. The gp39 models and x-ray structure were compared using the Biopolymer module of InsightII (MSI, San Diego, CA) and ALIGN (30Satow Y. Cohen G.H. Padlan E.A. Davies D.R. J. Mol. Biol. 1986; 190: 593-604Crossref PubMed Scopus (532) Google Scholar). Secondary structure calculations and contact analysis were performed using PROCHECK (25Laskowski R.A. MacArthur M.S. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1997; 26: 283-291Crossref Google Scholar) and InsightII. The molecular models were compared with the x-ray structure using different superposition sets and after superposition of residues most conserved across the TNF family (10Smith C.A. Farrah T. Goodwin R.G. Cell. 1994; 76: 959-962Abstract Full Text PDF PubMed Scopus (1839) Google Scholar). Graphical representations were produced using InsightII and processed as RGB images or postscript files. A detailed comparison of the gp39 molecular models (AM and BM) and x-ray structure is presented first, followed by an analysis of model-based mutagenesis experiments, binding site prediction, and residue mapping studies. As predicted (7Aruffo A. Farrington M. Hollenbaugh D. Xu L. Milatovich A. Nonoyama S. Bajorath J. Grosmaire L.S. Stenkamp R. Neubauer M. Roberts R.L. Noelle R.J. Ledbetter J.A. Francke U. Ochs H.D. Cell. 1993; 72: 291-300Abstract Full Text PDF PubMed Scopus (752) Google Scholar), gp39 adopts the TNF fold (13Eck M.J. Sprang S.R. J. Biol. Chem. 1989; 264: 17595-17605Abstract Full Text PDF PubMed Google Scholar, 14Eck M.J. Ultsch M. Rinderknecht E. de Vos A.M. Sprang S.R. J. Biol. Chem. 1992; 267: 2119-2122Abstract Full Text PDF PubMed Google Scholar), which consists of a sandwich of two β-sheets with jelly roll topology (13Eck M.J. Sprang S.R. J. Biol. Chem. 1989; 264: 17595-17605Abstract Full Text PDF PubMed Google Scholar). Like TNF, the extracellular region of gp39 forms a 3-fold symmetric homotrimer. Fig. 1 shows the gp39 x-ray structure. After initial detection of weak sequence similarity between gp39 and TNF molecules (2Hollenbaugh D. Grosmaire L.S. Kullas C.D. Chalupny N.J. Braesch-Andersen S. Noelle R.J. Stamenkovic I. Ledbetter J.A. Aruffo A. EMBO J. 1992; 11: 4313-4321Crossref PubMed Scopus (499) Google Scholar), a detailed comparison of TNF and gp39 sequences was carried out (7Aruffo A. Farrington M. Hollenbaugh D. Xu L. Milatovich A. Nonoyama S. Bajorath J. Grosmaire L.S. Stenkamp R. Neubauer M. Roberts R.L. Noelle R.J. Ledbetter J.A. Francke U. Ochs H.D. Cell. 1993; 72: 291-300Abstract Full Text PDF PubMed Scopus (752) Google Scholar, 18Bajorath J. Marken J.S. Chalupny N.J. Spoon T.L. Siadak A.W. Gordon M. Noelle R.J. Hollenbaugh D. Aruffo A. Biochemistry. 1995; 34: 9884-9892Crossref PubMed Scopus (55) Google Scholar) in light of TNF x-ray structures. Model building of gp39 was based on the results of these structure-oriented (topological) sequence alignments. Fig. 2 summarizes the results of TNF/gp39 sequence comparisons. Although the aligned regions in TNF and gp39 show only ∼25% sequence identity, a number of key residues, which are conserved in TNF-α and TNF-β and are important for the structural integrity of theses proteins, are conserved in gp39 (Fig. 2). Mapping of conserved residues on the (then publicly available) x-ray structure of TNF-α confirmed the conservation or conservative replacement of a number of core residues in gp39 (7Aruffo A. Farrington M. Hollenbaugh D. Xu L. Milatovich A. Nonoyama S. Bajorath J. Grosmaire L.S. Stenkamp R. Neubauer M. Roberts R.L. Noelle R.J. Ledbetter J.A. Francke U. Ochs H.D. Cell. 1993; 72: 291-300Abstract Full Text PDF PubMed Scopus (752) Google Scholar). In addition, 22 of 29 residues at TNF-α dimer or trimer interfaces were conserved, at least in residue character (7Aruffo A. Farrington M. Hollenbaugh D. Xu L. Milatovich A. Nonoyama S. Bajorath J. Grosmaire L.S. Stenkamp R. Neubauer M. Roberts R.L. Noelle R.J. Ledbetter J.A. Francke U. Ochs H.D. Cell. 1993; 72: 291-300Abstract Full Text PDF PubMed Scopus (752) Google Scholar, 13Eck M.J. Sprang S.R. J. Biol. Chem. 1989; 264: 17595-17605Abstract Full Text PDF PubMed Google Scholar). On the basis of these findings, gp39 was predicted (i) to display a three-dimensional structure more similar to TNF than indicated by the relatively low level of sequence identity and (ii) to form a TNF-analogous homotrimer. Residues most conserved across the TNF family (10Smith C.A. Farrah T. Goodwin R.G. Cell. 1994; 76: 959-962Abstract Full Text PDF PubMed Scopus (1839) Google Scholar) were used as anchor points for the sequence alignments, which were equivalent for both AM versus TNF-α and BMversus TNF-β. The gp39 molecular models included residues 121–261. After optimal superposition of AM, BM, and 1ALY, the accuracy of the initial alignments was determined (Fig. 2). With the exception of the region encompassing residues 145–151, which include a topological misalignment (see below), residues in β-strands and their periodicity (exposed and buried residues) were correctly predicted. The positions of six of eight insertions/deletions in gp39 relative to TNF were also accurately predicted. The region including the topological misalignment contains an insertion and deletion in gp39 relative to TNF, the presence of which was not predicted. Topological equivalent residues in gp39 models and x-ray structure could not be assigned for two loops, residues 132–136 and 180–186, which had grossly mismodeled conformations (see below). Excluding these loop regions, the consensus alignment of gp39 versus TNF is ∼94% correct. If the loops are also considered topological misalignments, the overall alignment accuracy is ∼86%. Residues 145–151 form a short β-strand at the edge of a sheet and include residues of the adjacent loops. The absence of significant core interactions involving this edge strand in TNF was reminiscent of the C"-strand in Ig variable domains (31Bork P. Holm L. Sander C. J. Mol. Biol. 1994; 242: 309-320PubMed Google Scholar). The predicted local alignment in gp39 kept the size of the adjacent loops in TNF-α and TNF-β and thus avoided the introduction of additional insertions and deletions. However, the model/structure comparison revealed that the alignment between residues 144 and 152 was shifted by one residue. Due to this shift in register, the size of the loop preceding the edge strand in gp39 was reduced by one residue relative to TNF, and the size of the loop following this strand was increased by one residue (Fig. 2). Fig. 3shows a comparison of the predicted and experimentally determined gp39 monomer structures, obtained by superposition of the 20 most conserved residues (Fig. 2). The α-carbon positions of ∼60% of residues included in AM and BM match the corresponding positions in 1ALY within 1.5 Å Superposition of most conserved regions in proteins ensures that deviations in more variable regions are not reduced due to averaging over many residues (32Bajorath J. Harris L. Novotny J. J. Biol. Chem. 1995; 270: 22081-22084Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Table Isummarizes r.m.s.d. values obtained for model/structure comparisons. The average backbone r.m.s.d. for gp39 residues 121–261 of ∼2.6 Å was reduced to ∼1.8 Å when the six misaligned residues and the two loop regions with the largest errors were excluded from the comparison. The average backbone and all atom r.m.s.d. values for all residues in β-strands and β-turns were ∼1 and ∼1.4 Å, respectively. The smallest r.m.s.d. values were observed, as to be expected, for the subset of the 20 most conserved residues in TNF and gp39. Fig. 4 shows the comparison of a dimer interface in gp39, obtained by superposition of the trimers. The arrangement of the monomers is very similar in the models and x-ray structure.Table IOverall comparison of gp39 molecular models and x-ray structureSubsetAM b_r.m.s.d. (a_r.m.s.d.)BM b_r.m.s.d. (a_r.m.s.d.)ÅS12.49 (3.46)2.72 (3.74)S22.43 (3.27)2.64 (3.53)S31.87 (2.80)2.04 (3.07)S41.73 (2.47)1.86 (2.73)S51.02 (1.71)1.04 (1.79)S60.91 (1.53)1.00 (1.37)The TNF-α (AM)- and TNF-β (BM)-based gp39 monomer models were superimposed on the x-ray structure (PDB code 1ALY) using different residue sets (subset). Resulting backbone (b_r.m.s.d.) and all atom (a_r.m.s.d.) root mean square deviations were calculated. Subset S1, all residues (121–261) included in the models; S2, S1 without residues 145–151 (region with a topological misalignment in AM and BM); S3, S1 without two mismodeled loops (residues 132–136 and 180–186); S4, S1 without residues 132–136, 145–151, and 180–186; S5, all residues in β-strands and β-turns of 1ALY; S6, 20 residues most conserved across the TNF family (see Fig. 1). r.m.s.d. values for α-carbon superpositions were similar to b_r.m.s.d. values: for example, for S1, AM/1ALY, 2.49 Å; and BM/1ALY, 2.70 Å. Open table in a new tab Figure 4Comparison of the interface between two gp39 monomers. The stereo superposition focuses on the dimer interface and is similar to the top view in Fig. 1. The monomers are colored according to Fig. 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The TNF-α (AM)- and TNF-β (BM)-based gp39 monomer models were superimposed on the x-ray structure (PDB code 1ALY) using different residue sets (subset). Resulting backbone (b_r.m.s.d.) and all atom (a_r.m.s.d.) root mean square deviations were calculated. Subset S1, all residues (121–261) included in the models; S2, S1 without residues 145–151 (region with a topological misalignment in AM and BM); S3, S1 without two mismodeled loops (residues 132–136 and 180–186); S4, S1 without residues 132–136, 145–151, and 180–186; S5, all residues in β-strands and β-turns of 1ALY; S6, 20 residues most conserved across the TNF family (see Fig. 1). r.m.s.d. values for α-carbon superpositions were similar to b_r.m.s.d. values: for example, for S1, AM/1ALY, 2.49 Å; and BM/1ALY, 2.70 Å. Pairwise structure comparisons including TNF templates were also carried o
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