2001 |
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| 128. | Niazi, K; Chiu, M; Mendoza, R; Degano, M; Khurana, S; Moody, D; Melián, A; Wilson, I; Kronenberg, M; Porcelli, S; Modlin, R: The A' and F' pockets of human CD1b are both required for optimal presentation of lipid antigens to T cells. In: J Immunol, vol. 166, no. 4, pp. 2562–2570, 2001, ISSN: 0022-1767. (Type: Journal Article | Abstract | Links)
@article{pmid11160317,
CD1 proteins are unique in their ability to present lipid Ags to T cells. Human CD1b shares significant amino acid homology with mouse CD1d1, which contains an unusual putative Ag-binding groove formed by two large hydrophobic pockets, A' and F'. We investigated the function of the amino acid residues that line the A' and F' pockets of CD1b by engineering 36 alanine-substitution mutants and analyzing their ability to present mycobacterial glycolipid Ags. Two lipid Ags presented by CD1b were studied, a naturally occurring glucose monomycolate (GMM) isolated from mycobacteria, which contains two long alkyl chains (C54-C62 and C22-C24) and synthetic GMM (sGMM), which includes two short alkyl chains (C18 and C14). We identified eight residues in both the A' and F' pockets that were involved in the presentation of both GMM and sGMM to T cells. Interestingly, four additional residues located in the distal portion of the A' pocket were required for the optimal presentation of GMM, but not sGMM. Conversely, nine residues located between the center of the groove and the F' pocket were necessary for the optimal presentation of sGMM, but not GMM. These data indicate that both the A' and F' pockets of human CD1b are required for the presentation of lipid Ags to T cells.
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| 127. | Speir, J A; Stevens, J; Joly, E; Butcher, G W; Wilson, I A: Two different, highly exposed, bulged structures for an unusually long peptide bound to rat MHC class I RT1-Aa. In: Immunity, vol. 14, no. 1, pp. 81–92, 2001, ISSN: 1074-7613. (Type: Journal Article | Abstract | Links)
@article{pmid11163232,
The rat MHC class Ia molecule RT1-Aa has the unusual capacity to bind long peptides ending in arginine, such as MTF-E, a thirteen-residue, maternally transmitted minor histocompatibility antigen. The antigenic structure of MTF-E was unpredictable due to its extraordinary length and two arginines that could serve as potential anchor residues. The crystal structure of RT1-Aa-MTF-E at 2.55 A shows that both peptide termini are anchored, as in other class I molecules, but the central residues in two independent pMHC complexes adopt completely different bulged conformations based on local environment. The MTF-E epitope is fully exposed within the putative T cell receptor (TCR) footprint. The flexibility demonstrated by the MTF-E structures illustrates how different TCRs may be raised against chemically identical, but structurally dissimilar, pMHC complexes.
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| 126. | Beutler, E; West, C; Speir, J A; Wilson, I A; Worley, M: The hHFE gene of browsing and grazing rhinoceroses: a possible site of adaptation to a low-iron diet. In: Blood Cells Mol Dis, vol. 27, no. 1, pp. 342–350, 2001, ISSN: 1079-9796. (Type: Journal Article | Abstract | Links)
@article{pmid11358396,
When rhinoceros species that are browsers in the wild are fed in captivity they become iron overloaded. Presumably, their iron-absorptive mechanisms have evolved to become highly efficient. In humans, mutations of the HFE gene cause increased iron absorption. To determine whether the HFE gene of rhinoceroses has undergone mutation as an adaptive mechanism to improve iron absorption from iron-poor diets, we have sequenced the entire coding region of the HFE genes of four species of rhinoceros. Two of these were browsing species and two were grazing species. Although the HFE gene has been well preserved across species, numerous nucleotide differences were found between rhinoceros and human or mouse, some of which changed deduced amino acids. Of these mutations, only one found in the black rhinoceros appears to be a viable candidate mutation that might adversely affect HFE function. This mutation, S88T, is in a highly conserved region that is involved in the interaction between transferrin receptor and HFE.
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| 125. | Saphire, E O; Parren, P W; Barbas, C F; Burton, D R; Wilson, I A: Crystallization and preliminary structure determination of an intact human immunoglobulin, b12: an antibody that broadly neutralizes primary isolates of HIV-1. In: Acta Crystallogr D Biol Crystallogr, vol. 57, no. Pt 1, pp. 168–171, 2001, ISSN: 0907-4449. (Type: Journal Article | Abstract | Links)
@article{pmid11134947,
An intact human immunoglobulin with a full-length hinge has been crystallized for the first time in a form in which all of the Ig domains are ordered. The IgG1 antibody b12 is one of only three known monoclonal antibodies described that potently neutralize a broad range of HIV-1 primary isolates. It binds to an epitope overlapping the conserved CD4 binding site on the viral surface antigen gp120. Hexagonal crystals corresponding to space group R32 were grown from 0.8 M ammonium sulfate, with unit-cell parameters a = b = 271.3, c = 175.2 A and one molecule per asymmetric unit. The crystals diffract to 2.8 A and a preliminary molecular-replacement solution indicates that all 12 Ig domains of the antibody can be resolved.
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2000 |
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| 124. | Hotta, K; Lange, H; Tantillo, D J; Houk, K N; Hilvert, D; Wilson, I A: Catalysis of decarboxylation by a preorganized heterogeneous microenvironment: crystal structures of abzyme 21D8. In: J Mol Biol, vol. 302, no. 5, pp. 1213–1225, 2000, ISSN: 0022-2836. (Type: Journal Article | Abstract | Links)
@article{pmid11183784,
Antibody 21D8 catalyzes the solvent-sensitive decarboxylation of 3-carboxybenzisoxazoles. The crystal structure of chimeric Fab 21D8 with and without hapten at 1.61 A and 2.10 A, respectively, together with computational analysis, shows how a melange of polar and non-polar sites are exploited to achieve both substrate binding and acceleration of a reaction normally facilitated by purely aprotic dipolar media. The striking similarity of the decarboxylase and a series of unrelated esterase antibodies also highlights the chemical versatility of structurally conserved anion binding sites and the relatively subtle changes involved in fine-tuning the immunoglobulin pocket for recognition of different ligands and catalysis of different reactions.
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| 123. | Stratmann, T; Apostolopoulos, V; Mallet-Designe, V; Corper, A L; Scott, C A; Wilson, I A; Kang, A S; Teyton, L: The I-Ag7 MHC class II molecule linked to murine diabetes is a promiscuous peptide binder. In: J Immunol, vol. 165, no. 6, pp. 3214–3225, 2000, ISSN: 0022-1767. (Type: Journal Article | Abstract | Links)
@article{pmid10975837,
Susceptibility to insulin-dependent diabetes mellitus is linked to MHC class II genes. The only MHC class II molecule expressed by nonobese diabetic (NOD) mice, I-Ag7, shares a common alpha-chain with I-Ad but has a peculiar beta-chain. As with most beta-chain alleles linked to diabetes susceptibility, I-Ag7 contains a nonaspartic residue at position beta57. We have produced large amounts of empty I-Ag7 molecules using a fly expression system to characterize its biochemical properties and peptide binding by phage-displayed peptide libraries. The identification of a specific binding peptide derived from glutamic acid decarboxylase (GAD65) has allowed us to crystallize and obtain the three-dimensional structure of I-Ag7. Structural information was critical in evaluating the binding studies. I-Ag7, like I-Ad, appears to be very promiscuous in terms of peptide binding. Their binding motifs are degenerate and contain small and/or small hydrophobic residues at P4 and P6 of the peptide, a motif frequently found in most globular proteins. The degree of promiscuity is increased for I-Ag7 over I-Ad as a consequence of a larger P9 pocket that can specifically accommodate negatively charged residues, as well as possibly residues with bulky side chains. So, although I-Ad and I-Ag7 are structurally closely related, stable molecules and good peptide binders, they differ functionally in their ability to bind significantly different peptide repertoires that are heavily influenced by the presence or the absence of a negatively charged residue at position 57 of the beta-chain. These characteristics link I-Ag7 with autoimmune diseases, such as insulin-dependent diabetes mellitus.
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| 122. | Burdin, N; Brossay, L; Degano, M; Iijima, H; Gui, M; Wilson, I A; Kronenberg, M: Structural requirements for antigen presentation by mouse CD1. In: Proc Natl Acad Sci U S A, vol. 97, no. 18, pp. 10156–10161, 2000, ISSN: 0027-8424. (Type: Journal Article | Abstract | Links)
@article{pmid10963678,
The structural basis for the T cell response to glycolipid antigens (Ags) remains poorly understood. T lymphocytes autoreactive for mouse CD1 (mCD1.1) or reactive for the glycosphingolipid alphagalactosylceramide (alpha-GalCer) presented by mCD1.1 have been described previously. In this paper it is shown that mutations at the top of the alpha helices and in the bottom of the Ag-binding groove can disrupt both mCD1.1 autoreactivity and alpha-GalCer recognition. The locations of the positions that affect T cell responses indicate that recognition of mCD1.1 is not likely to be unconventional or superantigen-like. Furthermore, the effects of the bottom of the pocket mutation suggest that the autoreactive response could require an autologous ligand, and they indicate that alpha-GalCer binds to the groove of mCD1.1, most likely with the shorter 18-carbon hydrophobic chain in the A' pocket. Natural killer T cell hybridomas with identical T cell antigen receptor (TCR) alpha chains and different beta chains respond differently to alpha-GalCer presented by mCD1.1 mutants. This finding indicates a role for TCR beta in defining natural killer T cell specificity, despite the more restricted diversity of the alpha chains in these cells. Overall, the data are consistent with a mode of lipoglycan recognition similar to that proposed for glycopeptides, in which the TCR alpha and beta chains survey a surface composed of both mCD1.1 and the carbohydrate portion of alpha-GalCer.
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| 121. | Reyes, V M; Greasley, S E; Stura, E A; Beardsley, G P; Wilson, I A: Crystallization and preliminary crystallographic investigations of avian 5-aminoimidazole-4-carboxamide ribonucleotide transformylase-inosine monophosphate cyclohydrolase expressed in Escherichia coli. In: Acta Crystallogr D Biol Crystallogr, vol. 56, no. Pt 8, pp. 1051–1054, 2000, ISSN: 0907-4449. (Type: Journal Article | Abstract | Links)
@article{pmid10944351,
ATIC [5-aminoimidazole-4-carboxamide ribonucleotide transformylase (AICAR Tfase)-inosine monophosphate cyclohydrolase (IMPCH)] is a bifunctional enzyme that catalyzes the penultimate and final steps in the de novo purine biosynthesis pathway and thus is an attractive anticancer target. Recombinant avian ATIC has been purified from an Escherichia coli expression system and crystallized in a binary complex with methotrexate (MTX). Crystals were obtained from PEG 4000 or MPEG 5000 buffered at pH 7.0-7.2 and data were collected from a single crystal at 96 K to 2.3 A resolution at the Stanford Synchrotron Radiation Laboratory (SSRL). The crystals are monoclinic and belong to space group P2(1), with unit-cell dimensions a = 65.17, b = 105.93, c = 103.47 A, beta = 108.27 degrees. Assuming two molecules per asymmetric unit, the Matthews coefficient V(m) is 2.63 A(3) Da(-1) and the solvent volume is 52.9%.
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| 120. | Corper, A L; Stratmann, T; Apostolopoulos, V; Scott, C A; Garcia, K C; Kang, A S; Wilson, I A; Teyton, L: A structural framework for deciphering the link between I-Ag7 and autoimmune diabetes. In: Science, vol. 288, no. 5465, pp. 505–511, 2000, ISSN: 0036-8075. (Type: Journal Article | Abstract | Links)
@article{pmid10775108,
Susceptibility to murine and human insulin-dependent diabetes mellitus correlates strongly with major histocompatibility complex (MHC) class II I-A or HLA-DQ alleles that lack an aspartic acid at position beta57. I-Ag7 lacks this aspartate and is the only class II allele expressed by the nonobese diabetic mouse. The crystal structure of I-Ag7 was determined at 2.6 angstrom resolution as a complex with a high-affinity peptide from the autoantigen glutamic acid decarboxylase (GAD) 65. I-Ag7 has a substantially wider peptide-binding groove around beta57, which accounts for distinct peptide preferences compared with other MHC class II alleles. Loss of Asp(beta57) leads to an oxyanion hole in I-Ag7 that can be filled by peptide carboxyl residues or, perhaps, through interaction with the T cell receptor.
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| 119. | Karlstrom, A; Zhong, G; Rader, C; Larsen, N A; Heine, A; Fuller, R; List, B; Tanaka, F; Wilson, I A; Barbas, C F; Lerner, R A: Using antibody catalysis to study the outcome of multiple evolutionary trials of a chemical task. In: Proc Natl Acad Sci U S A, vol. 97, no. 8, pp. 3878–3883, 2000, ISSN: 0027-8424. (Type: Journal Article | Abstract | Links)
@article{pmid10760259,
Catalytic aldolase antibodies generated by immunization with two different, but structurally related, beta-diketone haptens were cloned and sequenced to study similarities and differences between independently evolved catalysts. Kinetic and sequence analysis coupled with mutagenesis, structural, and modeling studies reveal that the defining event in the evolution of these catalysts was a somatic mutation that placed a lysine residue in a deep, yet otherwise unrefined, hydrophobic pocket. We suggest that covalent chemistries may be as readily selected from the immune repertoire as the traditional noncovalent interactions that have formed the basis of immunochemistry until this time. Further, we believe that these experiments recapitulate the defining events in the evolution of nature's enzymes, particularly as they relate to chemical mechanism, catalytic promiscuity, and gene duplication.
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| 118. | Degano, M; Garcia, K C; Apostolopoulos, V; Rudolph, M G; Teyton, L; Wilson, I A: A functional hot spot for antigen recognition in a superagonist TCR/MHC complex. In: Immunity, vol. 12, no. 3, pp. 251–261, 2000, ISSN: 1074-7613. (Type: Journal Article | Abstract | Links)
@article{pmid10755612,
A longstanding question in T cell receptor signaling is how structurally similar ligands, with similar affinities, can have substantially different biological activity. The crystal structure of the 2C TCR complex of H-2Kb with superagonist peptide SIYR at 2.8 A elucidates a structural basis for TCR discrimination of altered peptide ligands. The difference in antigen potency is modulated by two cavities in the TCR combining site, formed mainly by CDRs 3alpha, 3beta, and 1beta, that complement centrally located peptide residues. This "functional hot spot" allows the TCR to finely discriminate amongst energetically similar interactions within different ligands for those in which the peptide appropriately stabilizes the TCR/pMHC complex and provides a new structural perspective for understanding differential signaling resulting from T cell cross-reactivity.
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| 117. | Luan, P; Heine, A; Zeng, K; Moyer, B; Greasely, S E; Kuhn, P; Balch, W E; Wilson, I A: A new functional domain of guanine nucleotide dissociation inhibitor (alpha-GDI) involved in Rab recycling. In: Traffic, vol. 1, no. 3, pp. 270–281, 2000, ISSN: 1398-9219. (Type: Journal Article | Abstract | Links)
@article{pmid11208110,
Guanine nucleotide dissociation inhibitor (GDI) is a 55-kDa protein that functions in vesicular membrane transport to recycle Rab GTPases. We have now determined the crystal structure of bovine alpha-GDI at ultra-high resolution (1.04 A). Refinement at this resolution highlighted a region with high mobility of its main-chain residues. This corresponded to a surface loop in the primarily alpha-helical domain II at the base of alpha-GDI containing the previously uncharacterized sequence-conserved region (SCR) 3A. Site-directed mutagenesis showed that this mobile loop plays a crucial role in binding of GDI to membranes and extraction of membrane-bound Rab. This domain, referred to as the mobile effector loop, in combination with Rab-binding residues found in the multi-sheet domain I at the apex of alpha-GDI may provide flexibility for recycling of diverse Rab GTPases. We propose that conserved residues in domains I and II synergize to form the functional face of GDI, and that domain II mediates a critical step in Rab recycling during vesicle fusion.
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| 116. | Apostolopoulos, V; Yu, M; McKenzie, I F; Wilson, I A: Structural implications for the design of molecular vaccines. In: Curr Opin Mol Ther, vol. 2, no. 1, pp. 29–36, 2000, ISSN: 1464-8431. (Type: Journal Article | Abstract)
@article{pmid11249650,
The major histocompatibility complex molecules bind and present short antigenic peptide fragments on the surface of antigen presenting cells to T-cell receptors. Recognition of peptide-MHC by cytotoxic T-cells initiates a cascade of signals to T-cells, which in turn destroy the antigen presenting cell. In the design of molecular vaccines for the treatment of diseases, an understanding of the 3-dimensional structure of MHC class I and is interaction with both peptide and T-cell receptor is an important prerequisite. In this review, we will discuss such crystal structures, as well as structures of glycopeptides and alternative T-cell antigens presented by MHC molecules.
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| 115. | Wingren, C; Crowley, M P; Degano, M; Chien, Y; Wilson, I A: Crystal structure of a gammadelta T cell receptor ligand T22: a truncated MHC-like fold. In: Science, vol. 287, no. 5451, pp. 310–314, 2000, ISSN: 0036-8075. (Type: Journal Article | Abstract | Links)
@article{pmid10634787,
Murine T10 and T22 are highly related nonclassical major histocompatibility complex (MHC) class Ib proteins that bind to certain gammadelta T cell receptors (TCRs) in the absence of other components. The crystal structure of T22b at 3.1 angstroms reveals similarities to MHC class I molecules, but one side of the normal peptide-binding groove is severely truncated, which allows direct access to the beta-sheet floor. Potential gammadelta TCR-binding sites can be inferred from functional mapping of T10 and T22 point mutants and allelic variants. Thus, T22 represents an unusual variant of the MHC-like fold and indicates that gammadelta and alphabeta TCRs interact differently with their respective MHC ligands.
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1999 |
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| 114. | Greasley, S E; Yamashita, M M; Cai, H; Benkovic, S J; Boger, D L; Wilson, I A: New insights into inhibitor design from the crystal structure and NMR studies of Escherichia coli GAR transformylase in complex with beta-GAR and 10-formyl-5,8,10-trideazafolic acid. In: Biochemistry, vol. 38, no. 51, pp. 16783–16793, 1999, ISSN: 0006-2960. (Type: Journal Article | Abstract | Links)
@article{pmid10606510,
The crystal structure of Escherichia coli GAR Tfase at 2.1 A resolution in complex with 10-formyl-5,8,10-trideazafolic acid (10-formyl-TDAF, K(i) = 260 nM), an inhibitor designed to form an enzyme-assembled multisubstrate adduct with the substrate, beta-GAR, was studied to determine the exact nature of its inhibitory properties. Rather than forming the expected covalent adduct, the folate inhibitor binds as the hydrated aldehyde (gem-diol) in the enzyme active site, in a manner that mimics the tetrahedral intermediate of the formyl transfer reaction. In this hydrated form, the inhibitor not only provides unexpected insights into the catalytic mechanism but also explains the 10-fold difference in inhibitor potency between 10-formyl-TDAF and the corresponding alcohol, and a further 10-fold difference for inhibitors that lack the alcohol. The presence of the hydrated aldehyde was confirmed in solution by (13)C-(1)H NMR spectroscopy of the ternary GAR Tfase-beta-GAR-10-formyl-TDAF complex using the (13)C-labeled 10-formyl-TDAF. This insight into the behavior of the inhibitor, which is analogous to protease or transaminase inhibitors, provides a novel and previously unrecognized basis for the design of more potent inhibitors of the folate-dependent formyl transfer enzymes of the purine biosynthetic pathway and development of anti-neoplastic agents.
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| 113. | Xu, J; Deng, Q; Chen, J; Houk, K N; Bartek, J; Hilvert, D; Wilson, I A: Evolution of shape complementarity and catalytic efficiency from a primordial antibody template. In: Science, vol. 286, no. 5448, pp. 2345–2348, 1999, ISSN: 0036-8075. (Type: Journal Article | Abstract | Links)
@article{pmid10600746,
The crystal structure of an efficient Diels-Alder antibody catalyst at 1.9 angstrom resolution reveals almost perfect shape complementarity with its transition state analog. Comparison with highly related progesterone and Diels-Alderase antibodies that arose from the same primordial germ line template shows the relatively subtle mutational steps that were able to evolve both structural complementarity and catalytic efficiency.
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| 112. | Wilson, I A: Perspectives: protein structure. Class-conscious TCR?. In: Science, vol. 286, no. 5446, pp. 1867–1868, 1999, ISSN: 0036-8075. (Type: Journal Article | Links)
@article{pmid10610577,
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| 111. | Wilson, I A; Jolliffe, L K: The structure, organization, activation and plasticity of the erythropoietin receptor. In: Curr Opin Struct Biol, vol. 9, no. 6, pp. 696–704, 1999, ISSN: 0959-440X. (Type: Journal Article | Abstract | Links)
@article{pmid10607675,
Dimerization of the erythropoietin receptor has long been accepted as the singular step in its mechanism of activation. Recent studies have revealed a regulator process for activation that is dependent on the actual configuration of the receptor-ligand dimer assembly. This aspect of the receptor subunit assembly appears to extend to the unliganded receptor, which can dimerize on the cell surface and diminish any spontaneous background signaling in the absence of ligand. This self-recognition, as well as the multiple ligand binding capabilities of the receptor binding site, is consistent with an emerging theme of plasticity in protein-protein and ligand-receptor interactions.
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| 110. | Moody, D B; Besra, G S; Wilson, I A; Porcelli, S A: The molecular basis of CD1-mediated presentation of lipid antigens. In: Immunol Rev, vol. 172, pp. 285–296, 1999, ISSN: 0105-2896. (Type: Journal Article | Abstract | Links)
@article{pmid10631954,
The CD1 family of proteins mediates a newly described pathway for presentation of lipids and glycolipids for specific recognition by T cells. All four of the known human CD1 proteins (CD1a, CD1b, CD1c and CD1d) as well as murine CD1d have now been shown to mediate T-cell recognition of lipid or glycolipid antigens. These antigens include naturally occurring foreign glycolipids from intracellular pathogens or synthetic glycolipids that are related in structure to mammalian glycolipids. The CD1b and CD1d-presented antigens differ in their fine structures but reveal a general motif in which a rigid hydrophilic cap is bound to two aliphatic hydrocarbon chains. Different T-cell populations recognize individual antigens without cross-reactivity to closely related antigen structures or CD1 isoforms, documenting the complexity and fine specificity of CD1-mediated T-cell responses. Mapping of the molecular determinants of recognition for CD1b and CD1d-presented antigens reveals that T cells discriminate the fine structure of the hydrophilic cap of the antigen, but both the length and structure of the lipid chains may be altered without loss of recognition. This pattern of lipid antigen recognition may be accounted for by a simple molecular mechanism of presentation that parallels the known mechanism for presentation of peptides, but solves the special problems related to the hydrophobic chemical nature of the lipid antigens. We propose that CD1 binds antigen by accommodating the two lipid tails within the hydrophobic groove of its two membrane distal domains, positioning the rigid hydrophilic cap of the antigen on the solvent-exposed surface of the CD1 protein, where it can directly contact the T-cell antigen receptor. This model provides a molecular basis for recognition of a new and diverse set of T-cell antigens contained within the lipid bilayers of cellular membranes.
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| 109. | Rudd, P M; Wormald, M R; Stanfield, R L; Huang, M; Mattsson, N; Speir, J A; DiGennaro, J A; Fetrow, J S; Dwek, R A; Wilson, I A: Roles for glycosylation of cell surface receptors involved in cellular immune recognition. In: J Mol Biol, vol. 293, no. 2, pp. 351–366, 1999, ISSN: 0022-2836. (Type: Journal Article | Abstract | Links)
@article{pmid10529350,
The majority of cell surface receptors involved in antigen recognition by T cells and in the orchestration of the subsequent cell signalling events are glycoproteins. The length of a typical N-linked sugar is comparable with that of an immunoglobulin domain (30 A). Thus, by virtue of their size alone, oligosaccharides may be expected to play a significant role in the functions and properties of the cell surface proteins to which they are attached. A databank of oligosaccharide structures has been constructed from NMR and crystallographic data to aid in the interpretation of crystal structures of glycoproteins. As unambiguous electron density can usually only be assigned to the glycan cores, the remainder of the sugar is then modelled into the crystal lattice by superimposing the appropriate oligosaccharide from the database. This approach provides insights into the roles that glycosylation might play in cell surface receptors, by providing models that delineate potential close packing interactions on the cell surface. It has been proposed that the specific recognition of antigen by T cells results in the formation of an immunological synapse between the T cell and the antigen-presenting cell. The cell adhesion glycoproteins, such as CD2 and CD48, help to form a cell junction, providing a molecular spacer between opposing cells. The oligosaccharides located on the membrane proximal domains of CD2 and CD48 provide a scaffold to orient the binding faces, which leads to increased affinity. In the next step, recruitment of the peptide major histocompatibility complex (pMHC) by the T-cell receptors (TCRs) requires mobility on the membrane surface. The TCR sugars are located such that they could prevent non-specific aggregation. Importantly, the sugars limit the possible geometry and spacing of TCR/MHC clusters which precede cell signalling. We postulate that, in the final stage, the sugars could play a general role in controlling the assembly and stabilisation of the complexes in the synapse and in protecting them from proteolysis during prolonged T-cell engagement.
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| 108. | Gruber, K; Zhou, B; Houk, K N; Lerner, R A; Shevlin, C G; Wilson, I A: Structural basis for antibody catalysis of a disfavored ring closure reaction. In: Biochemistry, vol. 38, no. 22, pp. 7062–7074, 1999, ISSN: 0006-2960. (Type: Journal Article | Abstract | Links)
@article{pmid10353817,
The catalysis of disfavored chemical reactions, especially those with no known natural enzyme counterparts, is one of the most promising achievements of catalytic antibody research. Antibodies 5C8, 14B9, 17F6, and 26D9, elicited by two different transition-state analogues, catalyze disfavored endo-tet cyclization reactions of trans-epoxy alcohols, in formal violation of Baldwin's rules for ring closure. Thus far, neither chemical nor enzyme catalysis has been capable of emulating the extraordinary activity and specificity of these antibodies. X-ray structures of two complexes of Fab 5C8 with the original hapten and with an inhibitor have been determined to 2.0 A resolution. The Fab structure has an active site that contains a putative catalytic diad, consisting of AspH95 and HisL89, capable of general acid/base catalysis. The stabilization of a positive charge that develops along the reaction coordinate appears to be an important factor for rate enhancement and for directing the reaction along the otherwise disfavored pathway. Sequence analysis of the four catalytic antibodies, as well as four inactive antibodies that strongly bind the transition-state analogues, suggests a conserved catalytic mechanism. The occurrence of the putative base HisL89 in all active antibodies, its absence in three out of the four analyzed inactive antibodies, and the rarity of a histidine at this position in immunoglobulins support an important catalytic role for this residue.
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| 107. | Middleton, S A; Barbone, F P; Johnson, D L; Thurmond, R L; You, Y; McMahon, F J; Jin, R; Livnah, O; Tullai, J; Farrell, F X; Goldsmith, M A; Wilson, I A; Jolliffe, L K: Shared and unique determinants of the erythropoietin (EPO) receptor are important for binding EPO and EPO mimetic peptide. In: J Biol Chem, vol. 274, no. 20, pp. 14163–14169, 1999, ISSN: 0021-9258. (Type: Journal Article | Abstract | Links)
@article{pmid10318834,
We have shown previously that Phe93 in the extracellular domain of the erythropoietin (EPO) receptor (EPOR) is crucial for binding EPO. Substitution of Phe93 with alanine resulted in a dramatic decrease in EPO binding to the Escherichia coli-expressed extracellular domain of the EPOR (EPO-binding protein or EBP) and no detectable binding to full-length mutant receptor expressed in COS cells. Remarkably, Phe93 forms extensive contacts with a peptide ligand in the crystal structure of the EBP bound to an EPO-mimetic peptide (EMP1), suggesting that Phe93 is also important for EMP1 binding. We used alanine substitution of EBP residues that contact EMP1 in the crystal structure to investigate the function of these residues in both EMP1 and EPO binding. The three largest hydrophobic contacts at Phe93, Met150, and Phe205 and a hydrogen bonding interaction at Thr151 were examined. Our results indicate that Phe93 and Phe205 are important for both EPO and EMP1 binding, Met150 is not important for EPO binding but is critical for EMP1 binding, and Thr151 is not important for binding either ligand. Thus, Phe93 and Phe205 are important binding determinants for both EPO and EMP1, even though these ligands share no sequence or structural homology, suggesting that these residues may represent a minimum epitope on the EPOR for productive ligand binding.
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| 106. | Stanfield, R; Cabezas, E; Satterthwait, A; Stura, E; Profy, A; Wilson, I: Dual conformations for the HIV-1 gp120 V3 loop in complexes with different neutralizing fabs. In: Structure, vol. 7, no. 2, pp. 131–142, 1999, ISSN: 0969-2126. (Type: Journal Article | Abstract | Links)
@article{pmid10368281,
BACKGROUND: The third hypervariable (V3) loop of HIV-1 gp120 has been termed the principal neutralizing determinant (PND) of the virus and is involved in many aspects of virus infectivity. The V3 loop is required for viral entry into the cell via membrane fusion and is believed to interact with cell surface chemokine receptors on T cells and macrophages. Sequence changes in V3 can affect chemokine receptor usage, and can, therefore, modulate which types of cells are infected. Antibodies raised against peptides with V3 sequences can neutralize laboratory-adapted strains of the virus and inhibit syncytia formation. Fab fragments of these neutralizing antibodies in complex with V3 loop peptides have been studied by X-ray crystallography to determine the conformation of the V3 loop.nnRESULTS: We have determined three crystal structures of Fab 58.2, a broadly neutralizing antibody, in complex with one linear and two cyclic peptides the amino acid sequence of which comes from the MN isolate of the gp120 V3 loop. Although the peptide conformations are very similar for the linear and cyclic forms, they differ from that seen for the identical peptide bound to a different broadly neutralizing antibody, Fab 59.1, and for a similar peptide bound to the MN-specific Fab 50.1. The conformational difference in the peptide is localized around residues Gly-Pro-Gly-Arg, which are highly conserved in different HIV-1 isolates and are predicted to adopt a type II beta turn.nnCONCLUSIONS: The V3 loop can adopt at least two different conformations for the highly conserved Gly-Pro-Gly-Arg sequence at the tip of the loop. Thus, the HIV-1 V3 loop has some inherent conformational flexibility that may relate to its biological function.
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| 105. | Remy, I; Wilson, I A; Michnick, S W: Erythropoietin receptor activation by a ligand-induced conformation change. In: Science, vol. 283, no. 5404, pp. 990–993, 1999, ISSN: 0036-8075. (Type: Journal Article | Abstract | Links)
@article{pmid9974393,
Erythropoietin and other cytokine receptors are thought to be activated through hormone-induced dimerization and autophosphorylation of JAK kinases associated with the receptor intracellular domains. An in vivo protein fragment complementation assay was used to obtain evidence for an alternative mechanism in which unliganded erythropoietin receptor dimers exist in a conformation that prevents activation of JAK2 but then undergo a ligand-induced conformation change that allows JAK2 to be activated. These results are consistent with crystallographic evidence of distinct dimeric configurations for unliganded and ligand-bound forms of the erythropoietin receptor.
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| 104. | Livnah, O; Stura, E A; Middleton, S A; Johnson, D L; Jolliffe, L K; Wilson, I A: Crystallographic evidence for preformed dimers of erythropoietin receptor before ligand activation. In: Science, vol. 283, no. 5404, pp. 987–990, 1999, ISSN: 0036-8075. (Type: Journal Article | Abstract | Links)
@article{pmid9974392,
Erythropoietin receptor (EPOR) is thought to be activated by ligand-induced homodimerization. However, structures of agonist and antagonist peptide complexes of EPOR, as well as an EPO-EPOR complex, have shown that the actual dimer configuration is critical for the biological response and signal efficiency. The crystal structure of the extracellular domain of EPOR in its unliganded form at 2.4 angstrom resolution has revealed a dimer in which the individual membrane-spanning and intracellular domains would be too far apart to permit phosphorylation by JAK2. This unliganded EPOR dimer is formed from self-association of the same key binding site residues that interact with EPO-mimetic peptide and EPO ligands. This model for a preformed dimer on the cell surface provides insights into the organization, activation, and plasticity of recognition of hematopoietic cell surface receptors.
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| 103. | Garcia, K C; Teyton, L; Wilson, I A: Structural basis of T cell recognition. In: Annu Rev Immunol, vol. 17, pp. 369–397, 1999, ISSN: 0732-0582. (Type: Journal Article | Abstract | Links)
@article{pmid10358763,
Exciting breakthroughs in the last two years have begun to elucidate the structural basis of cellular immune recognition. Crystal structures have been determined for full-length and truncated forms of alpha beta T cell receptor (TCR) heterodimers, both alone and in complex with their peptide-MHC (pMHC) ligands or with anti-TCR antibodies. In addition, a truncated CD8 coreceptor has been visualized with a pMHC. Aided in large part by the substantial body of knowledge accumulated over the last 25 years on antibody structure, a number of general conclusions about TCR structure and its recognition of antigen can already be derived from the relatively few TCR structures that have been determined. Small, but important, variations between TCR and antibody structures bear on their functional differences as well as on their specific antigen recognition requirements. As observed in antibodies, canonical CDR loop structures are already emerging for some of the TCR CDR loops. Highly similar docking orientations of the TCR V alpha domains in the TCR/pMHC complex appear to play a primary role in dictating orientation, but the V beta positions diverge widely. Similar TCR contact positions, but whose exact amino acid content can vary, coupled with relatively poor interface shape complementarity, may explain the flexibility and short half-lives of many TCR interactions with pMHC. Here we summarize the current state of this field, and suggest that the knowledge gap between the three-dimensional structure and the signaling function of the TCR can be bridged through a synthesis of molecular biological and biophysical techniques.
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| 102. | Speir, J A; Abdel-Motal, U M; Jondal, M; Wilson, I A: Crystal structure of an MHC class I presented glycopeptide that generates carbohydrate-specific CTL. In: Immunity, vol. 10, no. 1, pp. 51–61, 1999, ISSN: 1074-7613. (Type: Journal Article | Abstract | Links)
@article{pmid10023770,
T cell receptor (TCR) recognition of nonpeptidic and modified peptide antigens has been recently uncovered but is still poorly understood. Immunization with an H-2Kb-restricted glycopeptide RGY8-6H-Gal2 generates a population of cytotoxic T cells that express both alpha/beta TCR, specific for glycopeptide, and gamma/delta TCR, specific for the disaccharide, even on glycolipids. The crystal structure of Kb/RGY8-6H-Gal2 now demonstrates that the peptide and H-2Kb structures are unaffected by the peptide glycosylation, but the central region of the putative TCR binding site is dominated by the extensive exposure of the tethered carbohydrate. These features of the Kb/RGY8-6H-Gal2 structure are consistent with the individual ligand binding preferences identified for the alpha/beta and gamma/delta TCRs and thus explain the generation of a carbohydrate-specific T cell response.
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| 101. | Garcia, K C; Degano, M; Speir, J A; Wilson, I A: Emerging principles for T cell receptor recognition of antigen in cellular immunity. In: Rev Immunogenet, vol. 1, no. 1, pp. 75–90, 1999, ISSN: 1398-1714. (Type: Journal Article | Abstract)
@article{pmid11256574,
The structural basis of antigen recognition in cellular immunity has been elucidated through the determination of crystal structures of major histocompatibility complex (MHC) molecules bound to antigenic peptides, T cell receptors (TCR), CD8 and CD4 co-receptors and, most recently, TCRs in complex with peptide-MHC (pMHC). The mechanisms that generate the diversity of the immune response to invading microorganisms were first realized at a genetic level and are necessary in order to cope with the enormous number of potential antigens. This diversity is manifested in the protein products of the genes which code for the components of the TCR signalling complex. The structure of the TCR reveals both striking similarities with and fundamental differences from its functional counterpart, the antibody, in the humoral immune system. The conserved manner in which the TCR recognizes and interacts with its peptide-MHC ligand allows the TCR great latitude in its potential to form productive interactions with antigen-presenting cells that bear numerous ligands to which the TCR has not been previously exposed. This phenomenon of cross-, or alloreactivity arises from a combination of conserved structural features across all MHC molecules, both self and foreign, and some degree of molecular mimicry. Non-classical MHC ligands presenting either modified or specialized peptides, lipids, carbohydrates, or no ligand at all, are now thought to play increasingly important roles in cellular immunity. We review some of the recent structural results and our current state of knowledge about TCR structure, and how this relates to its function.
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| 100. | Grant, E P; Degano, M; Rosat, J P; Stenger, S; Modlin, R L; Wilson, I A; Porcelli, S A; Brenner, M B: Molecular recognition of lipid antigens by T cell receptors. In: J Exp Med, vol. 189, no. 1, pp. 195–205, 1999, ISSN: 0022-1007. (Type: Journal Article | Abstract | Links)
@article{pmid9874576,
The T cell antigen receptor (TCR) mediates recognition of peptide antigens bound in the groove of major histocompatibility complex (MHC) molecules. This dual recognition is mediated by the complementarity-determining residue (CDR) loops of the alpha and beta chains of a single TCR which contact exposed residues of the peptide antigen and amino acids along the MHC alpha helices. The recent description of T cells that recognize hydrophobic microbial lipid antigens has challenged immunologists to explain, in molecular terms, the nature of this interaction. Structural studies on the murine CD1d1 molecule revealed an electrostatically neutral putative antigen-binding groove beneath the CD1 alpha helices. Here, we demonstrate that alpha/beta TCRs, when transferred into TCR-deficient recipient cells, confer specificity for both the foreign lipid antigen and CD1 isoform. Sequence analysis of a panel of CD1-restricted, lipid-specific TCRs reveals the incorporation of template-independent N nucleotides that encode diverse sequences and frequent charged basic residues at the V(D)J junctions. These sequences permit a model for recognition in which the TCR CDR3 loops containing charged residues project between the CD1 alpha helices, contacting the lipid antigen hydrophilic head moieties as well as adjacent CD1 residues in a manner that explains antigen specificity and CD1 restriction.
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1998 |
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| 99. | Livnah, O; Johnson, D L; Stura, E A; Farrell, F X; Barbone, F P; You, Y; Liu, K D; Goldsmith, M A; He, W; Krause, C D; Pestka, S; Jolliffe, L K; Wilson, I A: An antagonist peptide-EPO receptor complex suggests that receptor dimerization is not sufficient for activation. In: Nat Struct Biol, vol. 5, no. 11, pp. 993–1004, 1998, ISSN: 1072-8368. (Type: Journal Article | Abstract | Links)
@article{pmid9808045,
Dimerization of the erythropoietin (EPO) receptor (EPOR), in the presence of either natural (EPO) or synthetic (EPO-mimetic peptides, EMPs) ligands is the principal extracellular event that leads to receptor activation. The crystal structure of the extracellular domain of EPOR bound to an inactive (antagonist) peptide at 2.7 A resolution has unexpectedly revealed that dimerization still occurs, but the orientation between receptor molecules is altered relative to active (agonist) peptide complexes. Comparison of the biological properties of agonist and antagonist EMPs with EPO suggests that the extracellular domain orientation is tightly coupled to the cytoplasmic signaling events and, hence, provides valuable new insights into the design of synthetic ligands for EPOR and other cytokine receptors.
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| 98. | Cai, Z; Brunmark, A B; Luxembourg, A T; Garcia, K C; Degano, M; Teyton, L; Wilson, I; Peterson, P A; Sprent, J; Jackson, M R: Probing the activation requirements for naive CD8+ T cells with Drosophila cell transfectants as antigen presenting cells. In: Immunol Rev, vol. 165, pp. 249–265, 1998, ISSN: 0105-2896. (Type: Journal Article | Abstract | Links)
@article{pmid9850865,
Activation of T cells involves multiple receptor-ligand interactions between T cells and antigen presenting cells (APC). At least two signals are required for T-cell activation: Signal 1 results from recognition of MHC/peptide complexes on the APC by cell surface T-cell receptors (TCR), whereas Signal 2 is induced by the interactions of co-stimulatory molecules on APC with their complementary receptors on T cells. This review focuses on our attempts to understand these various signals in a model system involving the 2C TCR. The structural basis of Signal 1 was investigated by determining the crystal structure of 2C TCR alone and in complex with MHC/peptide. Analysis of these structures has provided some basic rules for how TCR and MHC/peptide interact; however, the critical question of how this interaction transduces Signal 1 to T cells remains unclear. The effects of Signal 1 and Signal 2 on T-cell activation were examined with naive T cells from the 2C TCR transgenic mice, defined peptides as antigen and transfected Drosophila cells as APC. The results suggest that, except under extreme conditions, Signal 1 alone is unable to activate naive CD8 T cells despite the induction of marked TCR downregulation. Either B7 or intercellular adhesion molecule (ICAM)-1 can provide the second signal for CD8 T-cell activation. However, especially at low MHC/peptide densities, optimal activation and differentiation of CD8 T cells required interaction with both B7 and ICAM-1 on the same APC. Thus, the data suggest that at least two qualitatively different co-stimulation signals are required for full activation of CD8 T cells under physiological conditions.
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| 97. | Su, Y; Yamashita, M M; Greasley, S E; Mullen, C A; Shim, J H; Jennings, P A; Benkovic, S J; Wilson, I A: A pH-dependent stabilization of an active site loop observed from low and high pH crystal structures of mutant monomeric glycinamide ribonucleotide transformylase at 1.8 to 1.9 A. In: J Mol Biol, vol. 281, no. 3, pp. 485–499, 1998, ISSN: 0022-2836. (Type: Journal Article | Abstract | Links)
@article{pmid9698564,
A mutation in the dimer interface of Escherichia coli glycinamide ribonucleotide transformylase (GarTfase) disrupts the observed pH-dependent association of the wild-type enzyme, but has no observable effect on the enzyme activity. Here, we assess whether a pH effect on the enzyme's conformation is sufficient by itself to explain the pH-dependence of the GarTfase reaction. A pH-dependent conformational change is observed between two high-resolution crystal structures of the Glu70Ala mutant GarTfase at pH 3.5 (1.8 A) and 7.5 (1.9 A). Residues 110 to 131 in GarTfase undergo a transformation from a disordered loop at pH 3.5, where the enzyme is inactive, to an ordered loop-helix structure at pH 7.5, where the enzyme is active. The ordering of this flexible loop-helix has a direct effect on catalytic residues in the active site, binding of the folate cofactor and shielding of the active site from solvent. A main-chain carbonyl oxygen atom from Tyr115 in the ordered loop forms a hydrogen bond with His108, and thereby provides electronic and structural stabilization of this key active site residue. Kinetic data indicate that the pKa of His108 is in fact raised to 9. 2. The loop movement can be correlated with elevation of the His pKa, but with further stabilization, probably from Asp144, after the binding of folate cofactor. Leu118, also in the loop, becomes positioned near the p-amino benzoic acid binding site, providing additional hydrophobic interactions with the cofactor 10-formyl tetrahydrofolate. Thus, the pH-dependence of the enzyme activity appears to arise from local active site rearrangements and not from differences due to monomer-dimer association.
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| 96. | Porcelli, S A; Segelke, B W; Sugita, M; Wilson, I A; Brenner, M B: The CD1 family of lipid antigen-presenting molecules. In: Immunol Today, vol. 19, no. 8, pp. 362–368, 1998, ISSN: 0167-5699. (Type: Journal Article | Links)
@article{pmid9709504,
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| 95. | Speir, J A; Garcia, K C; Brunmark, A; Degano, M; Peterson, P A; Teyton, L; Wilson, I A: Structural basis of 2C TCR allorecognition of H-2Ld peptide complexes. In: Immunity, vol. 8, no. 5, pp. 553–562, 1998, ISSN: 1074-7613. (Type: Journal Article | Abstract | Links)
@article{pmid9620676,
MHC class I H-2Ld complexed with peptide QL9 (or p2Ca) is a high-affinity alloantigen for the 2C TCR. We used the crystal structure of H-2Ld with a mixture of bound peptides at 3.1 A to construct a model of the allogeneic 2C-Ld/QL9 complex for comparison with the syngeneic 2C-Kb/dEV8 structure. A prominent ridge on the floor of the Ld peptide-binding groove, not present in Kb, creates a C-terminal bulge in Ld peptides that greatly increases interactions with the 2C beta-chain. Furthermore, weak electrostatic complementarity between Asp77 on the alpha1 helix of Kb and 2C is enhanced in the allogeneic complex by closer proximity of QL9 peptide residue AspP8 to the 2C HV4 loop.
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| 94. | Manning, T C; Schlueter, C J; Brodnicki, T C; Parke, E A; Speir, J A; Garcia, K C; Teyton, L; Wilson, I A; Kranz, D M: Alanine scanning mutagenesis of an alphabeta T cell receptor: mapping the energy of antigen recognition. In: Immunity, vol. 8, no. 4, pp. 413–425, 1998, ISSN: 1074-7613. (Type: Journal Article | Abstract | Links)
@article{pmid9586632,
The T cell receptor (TCR) from the alloreactive T lymphocyte 2C recognizes a nonamer peptide QL9 complexed with the MHC class I molecule H2-Ld. Forty-two single-site alanine substitutions of the 2C TCR were analyzed for binding to QL9/Ld and anti-TCR antibodies. The results provided a detailed energy map of T cell antigen recognition and indicated that the pMHC and clonotypic antibody epitopes on the TCR were similar. Although residues in each Valpha and Vbeta CDR are important in binding pMHC, the most significant energy for the TCR/QL9/Ld interaction was contributed by CDRs 1 and 2 of both alpha and beta chains. The extent to which the individual energy contributions are directed at class I helices or peptide was also assessed.
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| 93. | Heine, A; Stura, E A; Yli-Kauhaluoma, J T; Gao, C; Deng, Q; Beno, B R; Houk, K N; Janda, K D; Wilson, I A: An antibody exo Diels-Alderase inhibitor complex at 1.95 angstrom resolution. In: Science, vol. 279, no. 5358, pp. 1934–1940, 1998, ISSN: 0036-8075. (Type: Journal Article | Abstract | Links)
@article{pmid9506943,
A highly specific Diels-Alder protein catalyst was made by manipulating the antibody repertoire of the immune system. The catalytic antibody 13G5 catalyzes a disfavored exo Diels-Alder transformation in a reaction for which there is no natural enzyme counterpart and that yields a single regioisomer in high enantiomeric excess. The crystal structure of the antibody Fab in complex with a ferrocenyl inhibitor containing the essential haptenic core that elicited 13G5 was determined at 1.95 angstrom resolution. Three key antibody residues appear to be responsible for the observed catalysis and product control. Tyrosine-L36 acts as a Lewis acid activating the dienophile for nucleophilic attack, and asparagine-L91 and aspartic acid-H50 form hydrogen bonds to the carboxylate side chain that substitutes for the carbamate diene substrate. This hydrogen-bonding scheme leads to rate acceleration and also pronounced stereoselectivity. Docking experiments with the four possible ortho transition states of the reaction explain the specific exo effect and suggest that the (3R,4R)-exo stereoisomer is the preferred product.
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| 92. | Johnson, D L; Farrell, F X; Barbone, F P; McMahon, F J; Tullai, J; Hoey, K; Livnah, O; Wrighton, N C; Middleton, S A; Loughney, D A; Stura, E A; Dower, W J; Mulcahy, L S; Wilson, I A; Jolliffe, L K: Identification of a 13 amino acid peptide mimetic of erythropoietin and description of amino acids critical for the mimetic activity of EMP1. In: Biochemistry, vol. 37, no. 11, pp. 3699–3710, 1998, ISSN: 0006-2960. (Type: Journal Article | Abstract | Links)
@article{pmid9521688,
To obtain information about the functional importance of amino acids required for effective erythropoietin (EPO) mimetic action, the conserved residues of a peptide mimetic of EPO, recently discovered by phage display, were subjected to an alanine replacement strategy. Further, to identify a minimal mimetic peptide sequence, a series of truncation peptides has been generated. One EPO mimetic peptide sequence, EMP1, was targeted and more than 25 derivatives of this sequence were evaluated for their ability to compete with [125I]EPO for receptor binding and for their ability to support the proliferation of two EPO-responsive cell lines. Two hydrophobic amino acids, Tyr4 and Trp13, appear essential for mimetic action, and aromatic residues appear to be important at these sites. These findings are consistent with the previously reported X-ray crystal structure of EMP1 complexed with the extracellular domain of the EPO receptor (EPO binding protein; EBP). In our efforts to define the structural elements required for EPO mimetic action, a 13 amino acid peptide was identified which possesses mimetic properties and contains a minimal agonist epitope. The ability of this peptide to effectively serve as a mimetic capable of the induction of EPO-responsive cell proliferation appears to reside within a single residue, equivalent to position Tyr4 of EMP1, when present in a sequence that includes the cyclic core peptide structure. Although these peptides are less potent than EPO, they should serve as an excellent starting point for the design of compounds with EPO mimetic activity.
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| 91. | Scott, C A; Peterson, P A; Teyton, L; Wilson, I A: Crystal structures of two I-Ad-peptide complexes reveal that high affinity can be achieved without large anchor residues. In: Immunity, vol. 8, no. 3, pp. 319–329, 1998, ISSN: 1074-7613. (Type: Journal Article | Abstract | Links)
@article{pmid9529149,
We have determined the structures of I-Ad covalently linked to an ovalbumin peptide (OVA323-339) and to an influenza virus hemagglutinin peptide (HA126-138). The floor of the peptide-binding groove contains an unusual beta bulge, not seen in I-E and DR structures, that affects numerous interactions between the alpha and beta chains and bound peptide. Unlike other MHC-peptide complexes, the peptides do not insert any large anchor residues into the binding pockets of the shallow I-Ad binding groove. The previously identified six-residue "core" binding motif of I-Ad occupies only the P4 to P9 pockets, implying that specificity of T cell receptor recognition of I-Ad-peptide complexes can be accomplished by peptides that only partially fill the MHC groove.
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| 90. | Garcia, K C; Degano, M; Pease, L R; Huang, M; Peterson, P A; Teyton, L; Wilson, I A: Structural basis of plasticity in T cell receptor recognition of a self peptide-MHC antigen. In: Science, vol. 279, no. 5354, pp. 1166–1172, 1998, ISSN: 0036-8075. (Type: Journal Article | Abstract | Links)
@article{pmid9469799,
The T cell receptor (TCR) inherently has dual specificity. T cells must recognize self-antigens in the thymus during maturation and then discriminate between foreign pathogens in the periphery. A molecular basis for this cross-reactivity is elucidated by the crystal structure of the alloreactive 2C TCR bound to self peptide-major histocompatibility complex (pMHC) antigen H-2Kb-dEV8 refined against anisotropic 3.0 angstrom resolution x-ray data. The interface between peptide and TCR exhibits extremely poor shape complementarity, and the TCR beta chain complementarity-determining region 3 (CDR3) has minimal interaction with the dEV8 peptide. Large conformational changes in three of the TCR CDR loops are induced upon binding, providing a mechanism of structural plasticity to accommodate a variety of different peptide antigens. Extensive TCR interaction with the pMHC alpha helices suggests a generalized orientation that is mediated by the Valpha domain of the TCR and rationalizes how TCRs can effectively "scan" different peptides bound within a large, low-affinity MHC structural framework for those that provide the slight additional kinetic stabilization required for signaling.
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| 89. | Huang, M; Syed, R; Stura, E A; Stone, M J; Stefanko, R S; Ruf, W; Edgington, T S; Wilson, I A: The mechanism of an inhibitory antibody on TF-initiated blood coagulation revealed by the crystal structures of human tissue factor, Fab 5G9 and TF.G9 complex. In: J Mol Biol, vol. 275, no. 5, pp. 873–894, 1998, ISSN: 0022-2836. (Type: Journal Article | Abstract | Links)
@article{pmid9480775,
The tissue factor (TF)-initiated blood coagulation protease cascade can be greatly inhibited in vivo by a potent anti-human-TF monoclonal antibody, 5G9. This antibody binds the carboxyl module of the extracellular domain of TF with a nanomolar binding constant and inhibits the formation of the TF.VIIa.X ternary initiation complex. We have determined the crystal structures of the extra-cellular modules of human TF, Fab 5G9, and their complex (TF.5G9) to 2.4 A, 2. 5 A, and 3.0 A, respectively, and measured the apparent inhibition constants of 5G9 on a panel of TF mutants. In our unliganded TF structure, a 7 degrees change in the relative orientation between the D1 and D2 modules was observed when compared with other published TF structures. Comparison of the free and bound Fab 5G9 indicates that small segmental and side chain variation of the antibody complementarity determining regions occurred on complexation with TF. The antibody-antigen recognition involves 18 TF antigen residues and 19 Fab residues from six CDR with one of the largest buried surface areas seen to date. A combination of structural and mutagenesis data indicate that Tyr156, Lys169, Arg200, and Lys201 play the major role in the antibody recognition. The TF. 5G9 structure provides insights into the mechanism by which the antibody 5G9 inhibits formation of the TF.VIIa.X ternary complex.
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| 88. | Wilson, I A; Bjorkman, P J: Unusual MHC-like molecules: CD1, Fc receptor, the hemochromatosis gene product, and viral homologs. In: Curr Opin Immunol, vol. 10, no. 1, pp. 67–73, 1998, ISSN: 0952-7915. (Type: Journal Article | Abstract | Links)
@article{pmid9523114,
The MHC fold, with its well-characterized peptide-binding groove, can perform other functions in addition to presentation of antigenic peptides to T cells. Homologs of MHC molecules have diverse roles that include presentation of lipid antigens (by CD1), transport of immunoglobulins (by the neonatal Fc receptor), regulation of iron metabolism (by the hemochromatosis gene product, HFE), and deception of the host immune system (by viral homologs). Recent crystal structures of two of these non-standard MHC-like molecules have allowed comparison of the recognition properties of classical. MHC molecules with those of their unusual homologs.
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| 87. | Scott, C A; Garcia, K C; Stura, E A; Peterson, P A; Wilson, I A; Teyton, L: Engineering protein for X-ray crystallography: the murine Major Histocompatibility Complex class II molecule I-Ad. In: Protein Sci, vol. 7, no. 2, pp. 413–418, 1998, ISSN: 0961-8368. (Type: Journal Article | Abstract | Links)
@article{pmid9521118,
Class II Major Histocompatibility (MHC) molecules are cell surface heterodimeric glycoproteins that play a central role in the immune response by presenting peptide antigens for surveillance by T cells. Due to the inherent instability of the class II MHC heterodimer, and its dependence on bound peptide for proper assembly, the production of electrophoretically pure samples of class II MHC proteins in complex with specific peptides has been problematic. A soluble form of the murine class II MHC molecule, I-Ad, with a leucine zipper tail added to each chain to enhance dimer assembly and secretion, has been produced in Drosophila melanogaster SC2 cells. To facilitate peptide loading, a high affinity ovalbumin peptide was covalently engineered to be attached by a six-residue linker to the amino terminus of the I-Adbeta chain. This modified I-Ad molecule was purified using preparative IEF and one fraction, after removal of the leucine zipper tails, produced crystals suitable for X-ray crystallographic analysis. The protein engineering and purification methods described here should be of general value for the expression of I-A and other class II MHC-peptide complexes.
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1997 |
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| 86. | Barbas, C F; Heine, A; Zhong, G; Hoffmann, T; Gramatikova, S; Björnestedt, R; List, B; Anderson, J; Stura, E A; Wilson, I A; Lerner, R A: Immune versus natural selection: antibody aldolases with enzymic rates but broader scope. In: Science, vol. 278, no. 5346, pp. 2085–2092, 1997, ISSN: 0036-8075. (Type: Journal Article | Abstract | Links)
@article{pmid9405338,
Structural and mechanistic studies show that when the selection criteria of the immune system are changed, catalytic antibodies that have the efficiency of natural enzymes evolve, but the catalytic antibodies are much more accepting of a wide range of substrates. The catalytic antibodies were prepared by reactive immunization, a process whereby the selection criteria of the immune system are changed from simple binding to chemical reactivity. This process yielded aldolase catalytic antibodies that approximated the rate acceleration of the natural enzyme used in glycolysis. Unlike the natural enzyme, however, the antibody aldolases catalyzed a variety of aldol reactions and decarboxylations. The crystal structure of one of these antibodies identified the reactive lysine residue that was selected in the immunization process. This lysine is deeply buried in a hydrophobic pocket at the base of the binding site, thereby accounting for its perturbed pKa.
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| 85. | Garcia, K C; Tallquist, M D; Pease, L R; Brunmark, A; Scott, C A; Degano, M; Stura, E A; Peterson, P A; Wilson, I A; Teyton, L: Alphabeta T cell receptor interactions with syngeneic and allogeneic ligands: affinity measurements and crystallization. In: Proc Natl Acad Sci U S A, vol. 94, no. 25, pp. 13838–13843, 1997, ISSN: 0027-8424. (Type: Journal Article | Abstract | Links)
@article{pmid9391114,
Cellular immunity is mediated by the interaction of an alphabeta T cell receptor (TCR) with a peptide presented within the context of a major histocompatibility complex (MHC) molecule. Alloreactive T cells have alphabeta TCRs that can recognize both self- and foreign peptide-MHC (pMHC) complexes, implying that the TCR has significant complementarity with different pMHC. To characterize the molecular basis for alloreactive TCR recognition of pMHC, we have produced a soluble, recombinant form of an alloreactive alphabeta T cell receptor in Drosophila melanogaster cells. This recombinant TCR, 2C, is expressed as a correctly paired alphabeta heterodimer, with the chains covalently connected via a disulfide bond in the C-terminal region. The native conformation of the 2C TCR was probed by surface plasmon resonance (SPR) analysis by using conformation-specific monoclonal antibodies, as well as syngeneic and allogeneic pMHC ligands. The 2C interaction with H-2Kb-dEV8, H-2Kbm3-dEV8, H-2Kb-SIYR, and H-2Ld-p2Ca spans a range of affinities from Kd = 10(-4) to 10(-6)M for the syngeneic (H-2Kb) and allogeneic (H-2Kbm3, H-2Ld) ligands. In general, the syngeneic ligands bind with weaker affinities than the allogeneic ligands, consistent with current threshold models of thymic selection and T cell activation. Crystallization of the 2C TCR required proteolytic trimming of the C-terminal residues of the alpha and beta chains. X-ray quality crystals of complexes of 2C with H-2Kb-dEV8, H-2Kbm3-dEV8 and H-2Kb-SIYR have been grown.
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| 84. | Wilson, I A; Garcia, K C: T-cell receptor structure and TCR complexes. In: Curr Opin Struct Biol, vol. 7, no. 6, pp. 839–848, 1997, ISSN: 0959-440X. (Type: Journal Article | Abstract | Links)
@article{pmid9434905,
The first crystal structures of intact T-cell receptors (TCRs) and their complexes with MHC peptide antigens (pMHC) were reported during the past year, along with those of a single-chain TCR Fv fragment and a beta-chain complexed with two different bacterial superantigens. These structures have shown the similarities and differences in the architecture of the antigen-binding regions of TCRs and antibodies, and how the TCR interacts with pMHC ligands as well as with superantigens.
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| 83. | Zeng, Z; Castaño, A R; Segelke, B W; Stura, E A; Peterson, P A; Wilson, I A: Crystal structure of mouse CD1: An MHC-like fold with a large hydrophobic binding groove. In: Science, vol. 277, no. 5324, pp. 339–345, 1997, ISSN: 0036-8075. (Type: Journal Article | Abstract | Links)
@article{pmid9219685,
CD1 represents a third lineage of antigen-presenting molecules that are distantly related to major histocompatibility complex (MHC) molecules in the immune system. The crystal structure of mouse CD1d1, corresponding to human CD1d, at 2.8 resolution shows that CD1 adopts an MHC fold that is more closely related to that of MHC class I than to that of MHC class II. The binding groove, although significantly narrower, is substantially larger because of increased depth and it has only two major pockets that are almost completely hydrophobic. The extreme hydrophobicity and shape of the binding site are consistent with observations that human CD1b and CD1c can present mycobacterial cell wall antigens, such as mycolic acid and lipoarabinomannans. However, mouse CD1d1 can present very hydrophobic peptides, but must do so in a very different way from MHC class Ia and class II molecules.
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| 82. | He, M; Gani, M; Livnah, O; Stura, E A; Beale, D; Coley, J; Wilson, I A; Taussig, M J: Sequence, specificity and crystallization of an oestrone-3-glucuronide antibody (3910). In: Immunology, vol. 90, no. 4, pp. 632–639, 1997, ISSN: 0019-2805. (Type: Journal Article | Abstract | Links)
@article{pmid9176119,
We describe the specificity profile and V region sequences of a high-affinity monoclonal antibody (mAb), 3910, directed against oestrone-3-glucuronide (E3G). Inhibition studies show that the D-ring is critical for steroid specificity, while the glucuronic acid attached to the A ring is required for high binding affinity, suggesting that both 'ends' of the E3G ligand are recognized. The VH domain is encoded by a gene from the VH7183 family, while VL appears to be encoded by the Vk5.1 gene (kappa II subgroup) with a deletion of six residues from complementarity-determining region-1 (CDR1). The VH CDR3 is 10 amino acid residues in length, of which D/N contributes five residues. Comparison of VH CDR of 3910 with those of mAb against progesterone (DB3) and digoxin (26-10, 40-50), for which crystal structures have been determined, suggests that aromatic side chains are important for E3G binding and that tyrosine residues H50, H97 and H100 may interact with the ligand. The Fab fragment of 3910 has been crystallized in its native and steroid (E3G and oestriol-3-glucuronide) complexed forms. An X-ray diffraction data set to 3 A resolution has been collected for the native Fab.
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| 81. | Ghiara, J B; Ferguson, D C; Satterthwait, A C; Dyson, H J; Wilson, I A: Structure-based design of a constrained peptide mimic of the HIV-1 V3 loop neutralization site. In: J Mol Biol, vol. 266, no. 1, pp. 31–39, 1997, ISSN: 0022-2836. (Type: Journal Article | Abstract | Links)
@article{pmid9054968,
Antigenic variation among different HIV-1 isolates has been a major problem in the development of an effective vaccine against AIDS. Peptide vaccines incorporating structural elements common to groups of viral isolates, such as the clade subtypes of HIV-1, hold promise; however, the design of such immunogens has been hampered by the lack of specific structural information on the viral proteins to be targeted. As part of a structure-based approach to this problem, we report the design and characterization of a conformationally restricted peptide analog (Aib142) of a highly conserved HIV-1 clade-B sequence from the third variable loop of the membrane glycoprotein gp120. The design strategy incorporates peptide conformational data derived from crystal structure analysis of an MN-isolate peptide (RP142) in complex with the Fab fragment (Fab59.1) of a broadly neutralizing antibody. The synthetic peptide (Aib142) replaces an alanine residue within the V3 loop epitope sequence GPGRAF by the conformationally restricted helicogenic alpha-aminoisobutyryl residue. As expected, the crystal structure of the Fab 59.1-Aib142 complex at 2.8 A resolution shows that the peptide interacts very similarly with the neutralizing antibody. Proton nuclear magnetic resonance (NMR) studies indicate that the free Aib142 peptide is indeed more ordered in solution with a conformational preference that corresponds to the X-ray structure of its Fab-bound form. Aib142 thus represents the first step in the design of conformationally constrained peptide analogs built to mimic biologically relevant structural forms of HIV-1 neutralization sites.
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| 80. | Desmet, J; Wilson, I A; Joniau, M; Maeyer, M De; Lasters, I: Computation of the binding of fully flexible peptides to proteins with flexible side chains. In: FASEB J, vol. 11, no. 2, pp. 164–172, 1997, ISSN: 0892-6638. (Type: Journal Article | Abstract | Links)
@article{pmid9039959,
Docking algorithms play an important role in the process of rational drug design and in understanding the mechanism of molecular recognition. An important determinant for successful docking is the extent to which the configurational space (including conformational changes) of the ligand/receptor system is searched. Here we describe a new, combinatorial method for flexible docking of peptides to proteins that allows full rotation around all single bonds of the peptide ligand and around those of a large set of receptor side chains. We have simulated the binding of several viral peptides to murine major histocompatibility complex class I H-2Kb. In addition, we have explored the limits of our method by simulating a complex between calmodulin and an 18-residue long helical peptide from calmodulin-dependent protein kinase IIalpha. The calculated peptide conformations generally matched well with the X-ray structures. Essential information about local flexibility and about residues that are responsible for strong binding was obtained. We have frequently observed considerable side-chain flexibility during the simulations, showing the need for a flexible treatment of the receptor. Our method may also be useful whenever the receptor side-chain conformation is not available or uncertain, as illustrated by the docking of an H-2Kb binding nonapeptide to the receptor structure taken from an octapeptide/H-2Kb complex.
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1996 |
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| 79. | Garcia, K C; Scott, C A; Brunmark, A; Carbone, F R; Peterson, P A; Wilson, I A; Teyton, L: CD8 enhances formation of stable T-cell receptor/MHC class I molecule complexes. In: Nature, vol. 384, no. 6609, pp. 577–581, 1996, ISSN: 0028-0836. (Type: Journal Article | Abstract | Links)
@article{pmid8955273,
T-cell antigen receptors (TCR) generally interact with moderate affinity with the complex formed by major histocompatibility complex (MHC) molecules and foreign peptides. MHC/TCR recognition is followed by the generation of a signal to the T cell through a monomorphic multicomponent system that includes the CD3 complex and accessory molecules such as CD4 and CD8. The interaction between the extracellular domains of MHC and TCR molecules, and the interaction of MHC and CD4/CD8 molecules, have been considered to occur independently of one another. We report here that the affinity of CD8 dimers for MHC class I molecules is independent of haplotype and peptide content, and that the affinity of the TCR for its specific ligand is enhanced through a reduced 'off' rate in the presence of either CD8alpha alpha homo- or CD8alpha beta heterodimers. Moreover, CD8 seems to help recognition of the specific MHC-peptide complex either by guiding an energetically favourable docking of TCR onto MHC, or by inducing conformational changes in the MHC complex that can augment the TCR/MHC-peptide interaction. CD8 should therefore be considered as an active participant in the T-cell recognition complex, rather than simply as an accessory molecule.
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