I03-Macromolecular Crystallography
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Open Access
Abstract: The CD8 T cell response to the HLA-A2-restricted epitope LLWNGPMAV (LLW) of the non-structural protein 4b of Yellow Fever Virus (YFV) is remarkably immunodominant, highly prevalent and powerful in YFV-vaccinated humans. Here we used a combinatorial peptide library screening in the context of an A2/LLW-specific CD8 T cell clone to identify a superagonist that features a methionine to isoleucine substitution at position 7. Based on in silico modeling, the functional enhancement of this LLW-7I mutation was associated with alterations in the structural dynamics of the peptide in the major histocompatibility complex (pMHC) binding with the T cell receptor (TCR). While the TCR off-rate of LLW-7I pMHC is comparable to the wild type peptide, the rigidity of the 7I peptide seems to confer less entropy loss upon TCR binding. This LLW-7I superagonist is an example of improved functionality in human CD8 T cells associated with optimized ligand rigidity for TCR binding and not with changes in TCR:pMHC off-rate kinetics.
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Sep 2020
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Jingshan
Ren
,
Joanne E.
Nettleship
,
Gemma
Harris
,
William
Mwangi
,
Nahid
Rhaman
,
Clare
Grant
,
Abhay
Kotecha
,
Elizabeth
Fry
,
Bryan
Charleston
,
David I.
Stuart
,
John
Hammond
,
Raymond J.
Owens
Diamond Proposal Number(s):
[10627, 14744]
Open Access
Abstract: Cattle antibodies have unusually long CDR3 loops in their heavy chains (HCs), and limited light chain (LC) diversity, raising the question of whether these mask the effect of LC variation on antigen recognition. We have investigated the role of the LC in the structure and activity of two neutralizing cattle antibodies (B4 and B13) that bind the F protein of bovine respiratory syncytial virus (bRSV). Recombinant Fab fragments of B4 and B13 bound bRSV infected cells and showed similar affinities for purified bRSV F protein. Exchanging the LCs between the Fab fragments produced hybrid Fabs: B13* (B13 HC/B4 LC) and B4* (B4 HC/B13 LC). The affinity of B13* to the F protein was found to be two-fold lower than B13 whilst the binding affinity of B4* was reduced at least a hundred-fold compared to B4 such that it no longer bound to bRSV infected cells. Comparison of the structures of B4 and B13 with their LC exchanged counterparts B4* and B13* showed that paratope of the HC variable domain (VH) of B4 was disrupted on pairing with the B13 LC, consistent with the loss of binding activity. By contrast, B13 H3 adopts a similar conformation when paired with either B13 or B4 LCs. These observations confirm the expected key role of the extended H3 loop in antigen-binding by cattle antibodies but also show that the quaternary LC/HC subunit interaction can be crucial for its presentation and thus the LC variable domain (VL) is also important for antigen recognition.
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Aug 2019
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B23-Circular Dichroism
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Svetlana S.
Sakhnevych
,
Inna M.
Yasinska
,
Alison M.
Bratt
,
Ouafa
Benlaouer
,
Isabel
Gonçalves Silva
,
Rohanah
Hussain
,
Giuliano
Siligardi
,
Walter
Fiedler
,
Jasmin
Wellbrock
,
Bernhard F.
Gibbs
,
Yuri A.
Ushkaryov
,
Vadim V.
Sumbayev
Diamond Proposal Number(s):
[12578]
Abstract: The progression of acute myeloid leukemia (AML)—the most severe blood/bone marrow cancer—is determined by the ability of malignant cells to escape host immune surveillance. However, the systemic regulatory mechanisms underlying this phenomenon remain largely unknown. In this study, we discovered a fundamental systemic biochemical strategy that allows AML cells to employ physiological systems within the body to survive and escape immune attack. We found that AML cells use a crucial human adrenal cortex hormone (cortisol) to induce the expression of neuronal receptor latrophilin 1 (LPHN1), which facilitates exocytosis. This receptor interacts with the blood plasma protein fibronectin leucine rich transmembrane protein 3 (FLRT3) to cause secretion of the immune suppressor galectin-9, which impairs the anticancer activities of cytotoxic lymphoid cells.
AML is a cancer of the blood and bone marrow that originates from self-renewing malignant immature myeloid cells and rapidly progresses into a systemic, and very often fatal, malignancy.1 AML cells employ physiological systems in the body to produce factors required for proliferation/disease progression.2,3 This includes the hijacking of stem cell factor (SCF), a major hematopoietic growth factor that controls AML progression and thus can become highly oncogenic.2,3 The expression and release of SCF can be triggered by AML cells via cytokines (e.g., interleukin-1β).2 Recent evidence clearly demonstrated that AML cells are also capable of impairing the activities of cytotoxic lymphoid cells (e.g., natural killer (NK) cells and cytotoxic T cells).4 One of the biochemical mechanisms underlying this phenomenon lies in the ability of AML cells to secrete the protein galectin-9. This tandem-type galectin binds the immune receptor Tim-3 and induces a variety of intracellular and cell-to-cell signaling events leading to the inactivation of NK cells, as well as the death of cytotoxic T cells.4,5 We recently reported that the process of galectin-9 secretion in AML cells is stimulated by the unique G protein-coupled receptor LPHN1, which normally functions in neurons to facilitate exocytosis.4,6 LPHN1 is also found in hematopoietic stem cells (HSCs), but its expression disappears at the early stages of their maturation.4,7 However, upon malignant transformation, AML cells preserve their abilities to express LPHN1 and to produce high levels of galectin-9 and Tim-3, in which the latter is involved in trafficking galectin-9 during the secretion process (HSCs express neither galectin-9 nor Tim-34).
It is currently unknown which molecular mechanisms trigger elevated levels of LPHN1 expression in primary human AML cells, and in general, the mechanisms of upregulation of LPHN1 expression at the genomic level remain unclear. It is also unknown whether FLRT3, a natural LPHN1 ligand,4,8 is present in human blood plasma and in other tissues associated with AML. Unraveling these mechanisms is crucial to understanding the pathways that control the ability of AML cells to protect themselves against cytotoxic lymphoid cells and, thus, was the aim of the present study.
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Jun 2018
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9495, 7656]
Abstract: In allergic disease, mast cell activation is conventionally triggered by allergen-mediated cross-linking of receptor-bound IgE on the cell surface. In addition to its diverse range of intracellular roles in apoptosis, cell proliferation and cancer, Histamine-Releasing Factor (HRF) also activates mast cells and basophils. A subset of IgE antibodies bind HRF through their Fab regions, and two IgE binding sites on HRF have been mapped. HRF can form dimers, and a disulphide-linked dimer is critical for activity. The current model for the activity of HRF in mast cell activation involves cross-linking of receptor-bound IgE by dimeric HRF, mediated by HRF/Fab interactions. HRF crystal and solution structures have provided little insight into either the formation of disulphide-linked HRF dimers or the ability of HRF to activate mast cells. We report the first crystal structure of murine HRF (mHRF) to 4.0 Å resolution, revealing a conserved fold. We also solved the structure of human HRF (hHRF) in two new crystal forms, one at the highest resolution (1.4 Å) yet reported. The high resolution hHRF structure reveals a disulphide-linked dimer, in which the two molecules are closely associated, and provides a model for the role of both human and murine HRF in mast cell activation.
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Dec 2017
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9495]
Open Access
Abstract: The Fc region of IgG antibodies (Cγ2 and Cγ3 domains) is responsible for effector functions such as antibody-dependent cell-mediated cytotoxicity and phagocytosis, through engagement with Fcγ receptors, although the ability to elicit these functions differs between the four human IgG subclasses. A key determinant of Fcγ receptor interactions is the FG loop in the Cγ2 domain. High resolution cryogenic IgG4-Fc crystal structures have revealed a unique conformation for this loop, which could contribute to the particular biological properties of this subclass. To further explore the conformation of the IgG4 Cγ2 FG loop at near-physiological temperature, we solved a 2.7 Å resolution room temperature structure of recombinant human IgG4-Fc from crystals analysed in situ. The Cγ2 FG loop in one chain differs from the cryogenic structure, and adopts the conserved conformation found in IgG1-Fc; however, this conformation participates in extensive crystal packing interactions. On the other hand, at room temperature, and free from any crystal packing interactions, the Cγ2 FG loop in the other chain adopts the conformation previously observed in the cryogenic IgG4-Fc structures, despite both conformations being accessible. The room temperature human IgG4-Fc structure thus provides a more complete and physiologically relevant description of the conformation of this functionally critical Cγ2 FG loop.
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Dec 2016
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[7656]
Open Access
Abstract: The Fc region of IgG antibodies, important for effector functions such as antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis and complement activation, contains an oligosaccharide moiety covalently attached to each CH2 domain. The oligosaccharide not only orients the CH2 domains but plays an important role in influencing IgG effector function, and engineering the IgG-Fc oligosaccharide moiety is an important aspect in the design of therapeutic monoclonal IgG antibodies. Recently we reported the crystal structure of glycosylated IgG4-Fc, revealing structural features that could explain the anti-inflammatory biological properties of IgG4 compared with IgG1. We now report the crystal structure of enzymatically deglycosylated IgG4-Fc, derived from human serum, at 2.7 Å resolution. Intermolecular CH2-CH2 domain interactions partially bury the CH2 domain surface that would otherwise be exposed by the absence of oligosaccharide, and two Fc molecules are interlocked in a symmetric, open conformation. The conformation of the CH2 domain DE loop, to which oligosaccharide is attached, is altered in the absence of carbohydrate. Furthermore, the CH2 domain FG loop, important for Fcγ receptor and C1q binding, adopts two different conformations. One loop conformation is unique to IgG4 and would disrupt binding, consistent with IgG4's anti-inflammatory properties. The second is similar to the conserved conformation found in IgG1, suggesting that in contrast to IgG1, the IgG4 CH2 FG loop is dynamic. Finally, crystal packing reveals a hexameric arrangement of IgG4-Fc molecules, providing further clues about the interaction between C1q and IgG.
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Nov 2014
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: IgE antibodies play a central role in allergic disease. They recognize allergens via their Fab regions, whilst their effector functions are controlled through interactions of the Fc region with two principal cell surface receptors, FcɛRI and CD23. Crosslinking of FcɛRI-bound IgE on mast cells and basophils by allergen initiates an immediate inflammatory response, while the interaction of IgE with CD23 on B-cells regulates IgE production. We have determined the structures of the C-type lectin “head” domain of CD23 from seven crystal forms. The thirty-five independent structures reveal extensive conformational plasticity in two loops that are critical for IgE binding.
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Dec 2013
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I04-Macromolecular Crystallography
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Abstract: Antibodies of the human IgG4 subclass uniquely undergo a process of Fab-arm exchange in which the heavy-chains of antibodies of different specificities can dissociate and then recombine. The mechanism by which the resulting functionally monovalent but bi-specific antibodies are formed is not only key to understanding their biological role, but is also important for the design of therapeutic monoclonal antibodies. Both the hinge region and the C(H)3 domain interface are known to be involved, and of the residues that differ between human IgG1 and IgG4 in C(H)3, residue 409, the only difference at the interface itself, has been implicated. We report the high resolution (1.8 Angstrom) structure of the C(H)3 domain dimer of IgG4, and find that Arg409 in IgG4, when compared with Lys409 observed in high resolution IgG1 structures, disrupts a network of water-mediated hydrogen bonding that is conserved in IgG1. Other conformational differences were detected that are a consequence of the presence of Arg409, such as a widening of the separation between residues Asn390 in one domain and Ser 400 in the other, which opens up a groove at the edge of the interface in IgG4 compared with IgG1. The effect of all these differences on the C(H)3 interface, doubled as a result of the interface's two-fold symmetry, is weakening of the inter-domain interaction in IgG4 compared with IgG1. This suggests a mechanism by which Arg409 weakens the C(H)3 interface in IgG4, predisposing this human antibody subclass to Fab-arm exchange.
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Nov 2012
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I11-High Resolution Powder Diffraction
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Abstract: The EGF receptor is an important target of cancer immunotherapies. The 7A7 monoclonal antibody has been raised against the murine EGFR, but it cross-reacts with the human receptor. The results from experiments using immune-competent mice can therefore, in principle, be extrapolated to the corresponding scenario in humans. In this work we report the crystal structure of the 7A7 Fab at an effective resolution of 1.4 Å. The antibody binding site comprises a deep pocket, located at the interface between the light and heavy chains, with major contributions from CDR loops H1, H2, H3 and L1. Binding experiments show that 7A7 recognizes a site on the EGFR extracellular domain that is not accessible in its most stable conformations, but that becomes exposed upon treatment with a tyrosine kinase inhibitor. This suggests a recognition mechanism similar to that proposed for mAb 806.
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Jul 2011
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I04-Macromolecular Crystallography
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Open Access
Abstract: The structure of the complement-binding domain of Staphylococcus aureus protein Sbi (Sbi-IV) in complex with ligand C3d is presented. The 1.7 Å resolution structure reveals the molecular details of the recognition of thioester-containing fragment C3d of the central complement component C3, involving interactions between residues of Sbi-IV helix ?2 and the acidic concave surface of C3d. The complex provides a structural basis for the binding preference of Sbi for native C3 over C3b and explains how Sbi-IV inhibits the interaction between C3d and complement receptor 2. A second C3d binding site on Sbi-IV is identified in the crystal structure that is not observed in related S. aureus C3 inhibitors Efb-C and Ehp. This binding mode perhaps hints as to how Sbi-IV, as part of Sbi, forms a C3b–Sbi adduct and causes futile consumption of C3, an extraordinary aspect of Sbi function that is not shared by any other known Staphylococcal complement inhibitor.
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Nov 2010
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