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Xiaoxiao Cheng,
Vaclav Veverka,
Anand Radhakrishnan,
Lorna C Waters,
Frederick W Muskett,
Sara Morgan,
Jiandong Huo,
Chao Yu,
Edward J Evans,
Alasdair J Leslie,
Mervyn Griffiths,
Colin Stubberfield,
Robert Griffin,
Alistair J Henry,
Andreas Jansson,
John E Ladbury,
Shinji Ikemizu,
Mark D Carr, Simon J Davis
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ABSTRACT: PD-1, a receptor expressed by T-cells, B-cells and monocytes, is a potent regulator of immune responses and a promising therapeutic target. The structure and interactions of human PD-1 are, however, incompletely characterized. We present the solution nuclear magnetic resonance (NMR)-based structure of the human PD-1 extracellular region and detailed analyses of its interactions with its ligands, PD-L1 and PD-L2. PD-1 has typical immunoglobulin superfamily topology, but differs at the edge of the GFCC' sheet which is flexible and completely lacks a C″ strand. Changes in PD-1 backbone NMR signals induced by ligand binding suggest that, whilst binding is centred on the GFCC' sheet, PD-1 is engaged by its two ligands differently and in ways incompletely explained by crystal structures of mouse PD-1/ligand complexes. The affinities of these interactions and that of PD-L1 with the costimulatory protein B7-1, measured using surface plasmon resonance, are significantly weaker than expected. The three- to four-fold greater affinity of PD-L2 versus PD-L1 for human PD-1 is principally due to the three-fold smaller dissociation rate for PD-L2 binding. Isothermal titration calorimetry revealed that the PD-1/PD-L1 interaction is entropically driven, whereas PD-1/PD-L2 binding has a large enthalpic component. Mathematical simulations based on the biophysical data and quantitative expression data suggest an unexpectedly limited contribution of PD-L2 to PD-1 ligation during interactions of activated T-cells with antigen presenting cells. These findings provide a rigorous structural and biophysical framework for interpreting the important functions of PD-1, and reveal that potent inhibitory signalling can be initiated by weakly interacting receptors.
Journal of Biological Chemistry 02/2013; · 4.77 Impact Factor
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ABSTRACT: Single-particle tracking (SPT) is widely used to study processes from membrane receptor organization to the dynamics of RNAs in living cells. While single-dye labeling strategies have the benefit of being minimally invasive, this comes at the expense of data quality; typically a data set of short trajectories is obtained and analyzed by means of the mean square displacements (MSD) or the distribution of the particles' displacements in a set time interval (jump distance, JD). To evaluate the applicability of both approaches, a quantitative comparison of both methods under typically encountered experimental conditions is necessary. Here we use Monte Carlo simulations to systematically compare the accuracy of diffusion coefficients (D-values) obtained for three cases: one population of diffusing species, two populations with different D-values, and a population switching between two D-values. For the first case we find that the MSD gives more or equally accurate results than the JD analysis (relative errors of D-values <6%). If two diffusing species are present or a particle undergoes a motion change, the JD analysis successfully distinguishes both species (relative error <5%). Finally we apply the JD analysis to investigate the motion of endogenous LPS receptors in live macrophages before and after treatment with methyl-β-cyclodextrin and latrunculin B.
PLoS ONE 01/2013; 8(5):e64287. · 4.09 Impact Factor
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ABSTRACT: Determining the organization of key molecules on the surface of live cells in two dimensions and how this changes during biological processes, such as signalling, is a major challenge in cell biology and requires methods with nanoscale spatial resolution and high temporal resolution. Here, we review biophysical tools, based on scanning ion conductance microscopy and single-molecule fluorescence and the combination of both of these methods, which have recently been developed to address these issues. We then give examples of how these methods have been be applied to provide new insights into cell membrane organization and function, and discuss some of the issues that will need to be addressed to further exploit these methods in the future.
Philosophical Transactions of The Royal Society B Biological Sciences 01/2013; 368(1611):20120027. · 6.40 Impact Factor
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Nature Immunology 12/2012; 13(12):1141-2. · 26.01 Impact Factor
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Marta I. Oliveira,
Carine M. Gonçalves,
Mafalda Pinto,
Stéphanie Fabre,
Ana Mafalda Santos,
Simon F. Lee,
Mónica A. A. Castro,
Raquel J. Nunes,
Rita R. Barbosa,
Jane R. Parnes,
Chao Yu, Simon J. Davis,
Alexandra Moreira,
Georges Bismuth,
Alexandre M. Carmo
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Ricardo A Fernandes,
David A Shore,
Mai T Vuong,
Chao Yu,
Xueyong Zhu,
Selma Pereira-Lopes,
Heather Brouwer,
Janet A Fennelly,
Claire M Jessup,
Edward J Evans,
Ian A Wilson, Simon J Davis
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ABSTRACT: Native and non-native ligands of the T cell receptor (TCR), including antibodies, have been proposed to induce signaling in T cells via intra- or intersubunit conformational rearrangements within the extracellular regions of TCR complexes. We have investigated whether any signatures can be found for such postulated structural changes during TCR triggering induced by antibodies, using crystallographic and mutagenesis-based approaches. The crystal structure of murine CD3ε complexed with the mitogenic anti-CD3ε antibody 2C11 enabled the first direct structural comparisons of antibody-liganded and unliganded forms of CD3ε from a single species, which revealed that antibody binding does not induce any substantial rearrangements within CD3ε. Saturation mutagenesis of surface-exposed CD3ε residues, coupled with assays of antibody-induced signaling by the mutated complexes, suggests a new configuration for the complex within which CD3ε is highly exposed and reveals that no large new CD3ε interfaces are required to form during antibody-induced signaling. The TCR complex therefore appears to be a structure that is capable of initiating intracellular signaling in T cells without substantial structural rearrangements within or between the component subunits. Our findings raise the possibility that signaling by native ligands might also be initiated in the absence of large structural rearrangements in the receptor.
Journal of Biological Chemistry 01/2012; 287(16):13324-35. · 4.77 Impact Factor
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Frontiers in immunology. 01/2012; 3:92.
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Frontiers in endocrinology. 01/2012; 3:86.
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ABSTRACT: Modulating the activities of costimulatory molecules controlling immune responses holds considerable promise for immunotherapy. CTLA4Ig (abatacept), a soluble version of the T cell-expressed membrane receptor CTLA-4, is approved for the treatment of rheumatoid arthritis. Like natural CTLA-4 molecules, CTLA4Ig ligates B7-1 and B7-2 on antigen presenting cells, preventing CD28-mediated costimulation of T cells. However, CTLA4Ig can also prevent ligation of CTLA-4, potentially blocking vital inhibitory signals, thereby augmenting immunity. There have been no quantitative analyses of the likely effects of CTLA4Ig on costimulatory interactions at the immunological synapse. We present a mathematical model, based on rigorous biophysical and expression data, for simulating the effects of abatacept and a mutated derivative, LEA29Y, on the synaptic interactions of CD28 and CTLA-4. The simulations reveal an unexpectedly large window within which CD28, but not CTLA-4, ligation is blocked by CTLA4Ig, perhaps explaining the efficacy of abatacept at the recommended therapeutic dose (10mg/kg) and its relative safety. However, the simulations suggest that the present dosing regimen is close to the maximum theoretically safe dose. The simulations also show that, within the therapeutic window, LEA29Y enhances the interaction of CTLA-4 with the more potent of its two native ligands, B7-1. They also suggest that CTLA-4 ligation by B7-1 could, in principle, be enhanced by further decreasing the off-rate of CTLA4Ig for binding to B7-2. Our findings therefore offer molecular explanations for why LEA29Y might prove to be more effective than abatacept in a clinical setting, and suggest ways in which its therapeutic efficacy could be further optimised.
Molecular Immunology 12/2011; 49(3):527-36. · 2.90 Impact Factor
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Marta I. Oliveira,
Carine M. Gonçalves,
Mafalda Pinto,
Stéphanie Fabre,
Ana Mafalda Santos,
Simon F. Lee,
Mónica A. A. Castro,
Raquel J. Nunes,
Rita R. Barbosa,
Jane R. Parnes,
Chao Yu, Simon J. Davis,
Alexandra Moreira,
Georges Bismuth,
Alexandre M. Carmo
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ABSTRACT: The T lineage glycoprotein CD6 is generally considered to be a costimulator of T-cell activation. Here, we demonstrate that CD6 significantly reduces early and late T-cell responses upon superantigen stimulation or TCR triggering by Abs. Measuring calcium mobilization in single cells responding to superantigen, we found that human T cells expressing rat CD6 react significantly less well compared with T cells not expressing the exogenous receptor. When the cytoplasmic domain of rat CD6 was removed, calcium responses were recovered, indicating that the inhibitory properties of CD6 are attributable to its cytoplasmic domain. Calcium responses, and also late indicators of T-cell activation such as IL-2 release, were also diminished in TCR-activated Jurkat cells expressing human CD6, compared with CD6-deficient cells or cells expressing a cytoplasmic deletion mutant of human CD6. Similarly, calcium signals triggered by anti-CD3 were enhanced in human T lymphocytes following morpholino-mediated suppression of CD6 expression. Finally, the proliferation of T lymphocytes was increased when the CD6–CD166 interaction was blocked with anti-CD166 Abs, but inhibited when anti-CD6 Abs were used. Our data suggest that CD6 is a signaling attenuator whose expression alone, i.e. in the absence of ligand engagement, is sufficient to restrain signaling in T cells.
European Journal of Immunology 11/2011; 42(1):195 - 205. · 5.10 Impact Factor
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John R James,
James McColl,
Marta I Oliveira,
Paul D Dunne,
Elizabeth Huang,
Andreas Jansson,
Patric Nilsson,
David L Sleep,
Carine M Gonçalves,
Sara H Morgan,
James H Felce,
Robert Mahen,
Ricardo A Fernandes,
Alexandre M Carmo,
David Klenerman, Simon J Davis
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ABSTRACT: Understanding the component stoichiometry of the T cell antigen receptor (TCR) triggering apparatus is essential for building realistic models of signal initiation. Recent studies suggesting that the TCR and other signaling-associated proteins are preclustered on resting T cells relied on measurements of the behavior of membrane proteins at interfaces with functionalized glass surfaces. Using fluorescence recovery after photobleaching, we show that, compared with the apical surface, the mobility of TCRs is significantly reduced at Jurkat T cell/glass interfaces, in a signaling-sensitive manner. Using two biophysical approaches that mitigate these effects, bioluminescence resonance energy transfer and two-color coincidence detection microscopy, we show that, within the uncertainty of the methods, the membrane components of the TCR triggering apparatus, i.e. the TCR complex, MHC molecules, CD4/Lck and CD45, are exclusively monovalent or monomeric in human T cell lines, implying that TCR triggering depends only on the kinetics of TCR/pMHC interactions. These analyses also showed that constraining proteins to two dimensions at the cell surface greatly enhances random interactions versus those between the membrane and the cytoplasm. Simulations of TCR-pMHC complex formation based on these findings suggest how unclustered TCR triggering-associated proteins might nevertheless be capable of generating complex signaling outputs via the differential recruitment of cytosolic effectors to the cell membrane.
Journal of Biological Chemistry 07/2011; 286(37):31993-2001. · 4.77 Impact Factor
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ABSTRACT: Triggering of the T cell receptor initiates a signaling cascade resulting in the activation of the T cell. These signals are integrated alongside those resulting from the triggering of other receptors whose function is to modulate the overall response. CD5 is an immunotyrosine-based inhibition motif-bearing receptor that antagonizes the overt T cell receptor activation response by recruiting inhibitory intracellular mediators such as SHP-1, RasGAP, or Cbl. We now propose that the inhibitory effects of CD5 are also mediated by a parallel pathway that functions at the level of inhibition of Fyn, a kinase generally associated with T cell receptor-mediated activation. After CD5 ligation, phosphorylation of the negative regulatory tyrosine (Tyr(531)) of Fyn increases, and this correlates with a substantial reduction in the kinase activity of Fyn and a profound inhibition of ZAP-70 activation. The effect requires the last 23 amino acids of the cytoplasmic domain of the receptor, strongly implying the involvement of a new CD5-interacting signaling or adaptor protein. Furthermore, we show that upon CD5 ligation there is a profound shift in its distribution from the bulk fluid phase to the lipid raft environment, where it associates with Fyn, Lck, and PAG. We suggest that the relocation of CD5, which we also show is capable of forming homodimers, to the proximity of raft-resident molecules enables CD5 to inhibit membrane proximal signaling by controlling the phosphorylation and activity of Fyn, possibly by interfering with the disassembly of C-terminal Src kinase (Csk)-PAG-Fyn complexes during T cell activation.
Journal of Biological Chemistry 07/2011; 286(35):30324-36. · 4.77 Impact Factor
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ABSTRACT: Glycoproteins present problems for structural analysis since they often have to be glycosylated in order to fold correctly and because their chemical and conformational heterogeneity generally inhibits crystallization. It is shown that the α-mannosidase I inhibitor kifunensine, which has previously been used for the purpose of glycoprotein crystallization in short-term (3-5 d) cultures, is apparently stable enough to be used to produce highly endoglycosidase H-sensitive glycoprotein in long-term (3-4 week) cultures of stably transfected Chinese hamster ovary (CHO) cells. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based analysis of the extracellular region of the cytotoxic T-lymphocyte antigen 4 (CTLA-4; CD152) homodimer expressed in long-term CHO cell cultures in the presence of kifunensine revealed that the inhibitor restricted CTLA-4 glycan processing to Man9GlcNAc2 and Man5GlcNAc2 structures. Complex-type glycans were undetectable, suggesting that the inhibitor was active for the entire duration of the cultures. Endoglycosidase treatment of the homodimer yielded protein that readily formed orthorhombic crystals with unit-cell parameters a=43.9, b=51.5, c=102.9 Å and space group P2(1)2(1)2(1) that diffracted to Bragg spacings of 1.8 Å. The results indicate that kifunensine will be effective in most, if not all, transient and long-term mammalian cell-based expression systems.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 07/2011; 67(Pt 7):785-9. · 0.51 Impact Factor
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ABSTRACT: Determining the organisation of key molecules on the surface of live cells in two dimensions and how this changes during biological processes, such as signaling, is a major challenge in cell biology and requires methods with nanoscale resolution. Recent advances in fluorescence imaging both at the diffraction limit tracking single molecules and exploiting super resolution imaging have now reached a stage where they can provide fundamentally new insights. Complementary developments in scanning ion conductance microscopy also allow the cell surface to be imaged with nanoscale resolution. The challenge now is to combine the information obtained using these different methods and on different cells to obtain a coherent view of the cell surface. In the future this needs to be driven by interdisciplinary research between physical scientists and biologists.
Current opinion in chemical biology 04/2011; 15(5):696-703. · 8.30 Impact Factor
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ABSTRACT: Mutations within MHC class I-restricted epitopes have been studied in relation to T cell-mediated immune escape, but their impact on NK cells via interaction with killer Ig-like receptors (KIRs) during early HIV infection is poorly understood. In two patients acutely infected with HIV-1, we observed the appearance of a mutation within the B*57-restricted TW10 epitope (G9E) that did not facilitate strong escape from T cell recognition. The NK cell receptor KIR3DL1, carried by these patients, is known to recognize HLA-B*5703 and is associated with good control of HIV-1. Therefore, we tested whether the G9E mutation influenced the binding of HLA-B*5703 to soluble KIR3DL1 protein by surface plasmon resonance, and while the wild-type sequence and a second (T3N) variant were recognized, the G9E variant abrogated KIR3DL1 binding. We extended the study to determine the peptide sensitivity of KIR3DL1 interaction with epitopes carrying mutations near the C termini of TW10 and a second HLA-B*57-restricted epitope, IW9. Several amino acid changes interfered with KIR3DL1 binding, the most extreme of which included the G9E mutation commonly selected by HLA-B*57. Our results imply that during HIV-1 infection, some early-emerging variants could affect KIR-HLA interaction, with possible implications for immune recognition.
Journal of Virology 03/2011; 85(11):5415-22. · 5.40 Impact Factor
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Chao Yu,
Andreas F-P Sonnen,
Roger George,
Benoit H Dessailly,
Loren J Stagg,
Edward J Evans,
Christine A Orengo,
David I Stuart,
John E Ladbury,
Shinji Ikemizu,
Robert J C Gilbert, Simon J Davis
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ABSTRACT: The inhibitory T-cell surface-expressed receptor, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), which belongs to the class of cell surface proteins phosphorylated by extrinsic tyrosine kinases that also includes antigen receptors, binds the related ligands, B7-1 and B7-2, expressed on antigen-presenting cells. Conformational changes are commonly invoked to explain ligand-induced "triggering" of this class of receptors. Crystal structures of ligand-bound CTLA-4 have been reported, but not the apo form, precluding analysis of the structural changes accompanying ligand binding. The 1.8-Å resolution structure of an apo human CTLA-4 homodimer emphasizes the shared evolutionary history of the CTLA-4/CD28 subgroup of the immunoglobulin superfamily and the antigen receptors. The ligand-bound and unbound forms of both CTLA-4 and B7-1 are remarkably similar, in marked contrast to B7-2, whose binding to CTLA-4 has elements of induced fit. Isothermal titration calorimetry reveals that ligand binding by CTLA-4 is enthalpically driven and accompanied by unfavorable entropic changes. The similarity of the thermodynamic parameters determined for the interactions of CTLA-4 with B7-1 and B7-2 suggests that the binding is not highly specific, but the conformational changes observed for B7-2 binding suggest some level of selectivity. The new structure establishes that rigid-body ligand interactions are capable of triggering CTLA-4 phosphorylation by extrinsic kinase(s).
Journal of Biological Chemistry 02/2011; 286(8):6685-96. · 4.77 Impact Factor
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ABSTRACT: Exactly how ligand binding 'triggers' T cell receptor (TCR) phosphorylation is unclear. It has been proposed that ligand engagement by the TCR somehow activates the Src kinase Lck, which in turn phosphorylates the receptor. Recent data, however, suggest instead that a significant fraction of the Lck in resting T cells is already activated and that the proportion of active Lck does not change during the early stages of T cell activation. We argue that, caveats notwithstanding, these new observations offer support for the 'kinetic-segregation' model of TCR triggering, which involves spatial reorganization of signalling proteins upon ligand binding and requires a fraction of Lck to be active in resting T cells.
Trends in Immunology 01/2011; 32(1):1-5. · 10.40 Impact Factor
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ABSTRACT: N- and O-glycosylation profoundly affect the biological properties of glycoproteins, principally by influencing their structures
and cellular trafficking, and by forming the recognition sites of carbohydrate-binding ligands. For crystallographers interested
in studying the protein component of glycoproteins, the two most important aspects of glycosylation are (1) that it is often
essential for the correct folding of a given protein and for ensuring its solubility, which generally necessitates expression
of the molecule in eukaryotic cells, and (2) that there are now procedures for the efficient post-folding removal of N-linked
glycans from glycoproteins and for minimizing the effects of O-glycosylation, which will generally benefit crystallogenesis.
We provide an overview of how glycans influence glycoprotein folding and then identify the sources of structural heterogeneity
at the heart of the ‘glycosylation problem’. We then discuss the options available to structural biologists for circumventing
the problems associated with protein N- and O-glycosylation. Our emphasis is on methods for producing glycoproteins with homogeneous
and/or removable N-glycosylation in mammalian cells that can be implemented in both very high yield, stable expression systems
and in a high throughput format based on transient expression protocols. We also consider whether deglycosylation reduces
protein stability and end by emphasizing the importance of using rigorous stereochemical and biosynthetic data when building
glycosylation into partial or complete electron density.
KeywordsGlycosylation-Endoglycosidases-Mammalian expression systems-Structural biology-High throughput
12/2010: pages 127-158;
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Cell 09/2010; 142(5):668-9. · 32.40 Impact Factor
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ABSTRACT: We present the crystal structure of an immunoglobulin light-chain-like domain, CTLA-4, as a strand-swapped dimer displaying cis-trans proline isomerisation and native-like hydrogen bonding. We also show that CTLA-4 can form amyloid-like fibres and amorphous deposits explainable by the same strand swapping. Our results suggest a molecular basis for the pathological aggregation of immunoglobulin domains and why amyloid-like fibres are more often composed of homologous rather than heterologous subunits.
Journal of Molecular Biology 06/2010; 399(2):207-13. · 4.00 Impact Factor
