I04-Macromolecular Crystallography
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Open Access
Abstract: Adhesive PfEMP1 proteins are displayed on the surface of malaria-infected red blood cells. They play a critical role in the disease, tethering infected cells away from destruction by the spleen and causing many severe symptoms. A molecular understanding of how these domains maintain their binding properties while evading immune detection will be important in developing therapeutics. In malaria of pregnancy, domains from the var2csa-encoded PfEMP1 protein interact with chondroitin sulfate on the placenta surface. This causes accumulation of infected red blood cells, leading to placental inflammation and block of blood flow to the developing fetus. This is associated with maternal anemia, low birth weight, and premature delivery and can lead to the death of mother and child. Here I present the structure of the chondroitin sulfate-binding DBL3X domain from a var2csa-encoded PfEMP1 protein. The domain adopts a fold similar to malarial invasion proteins, with extensive loop insertions. One loop is flexible in the unliganded structure but observed in the presence of sulfate or disaccharide, where it completes a sulfate-binding site. This loop, and others surrounding this putative carbohydrate-binding site, are flexible and polymorphic, perhaps protecting the binding site from immune detection. This suggests a model for how the domain maintains ligand binding while evading the immune response and will guide future drug and vaccine development.
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Aug 2008
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I02-Macromolecular Crystallography
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Open Access
Abstract: Severe malaria during pregnancy is associated with accumulation of parasite-infected erythrocytes in the placenta due to interactions between VAR2CSA protein, expressed on the surface of infected-erythrocytes, and placental chondroitin sulfate proteoglycans (CSPG). VAR2CSA contains multiple CSPG-binding domains, including DBL3X and DBL6ɛ. Previous structural studies of DBL3X suggested CSPG to bind to a positively charged patch and sulfate-binding site on the concave surface of the domain. Here we present the structure of the DBL6ɛ domain from VAR2CSA. This domain displays the same overall architecture and secondary structure as that of DBL3X but differs in loop structures, disulfide bond positions and surface charge distribution. In particular, despite binding to CSPG, DBL6ɛ lacks the key features of the CSPG-binding site of DBL3X. Instead DBL6ɛ binds to CSPG through a positively charged surface on the distal side of subdomain 2 that is exposed in intact VAR2CSA on the erythrocyte surface. Finally, unlike intact VAR2CSA, both DBL3X and DBL6ɛ bind to various carbohydrates, with greatest affinity for ligands with high sulfation and negative charge. These studies provide further insight into the structure of DBL domains and suggest a model for the role of individual domains in CSPG binding by VAR2CSA in placental malaria
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Oct 2009
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I04-Macromolecular Crystallography
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Abstract: Tic22 is a component of the protein-import apparatus of the chloroplasts of plants and algae and the apicoplasts of the Apicomplexa, a large group of organisms that includes the parasites that cause malaria. Tic22 is important for protein import into these organelles and for organelle biogenesis. It lies between the two membranes of chloroplasts, making interactions with components of both the TIC and TOC complexes. In the apicoplast, it is predicted to be located between the inner two membranes and to play a similar role in import. Although Tic22 is ubiquitous, its function is as yet uncertain. Tic22 from Plasmodium falciparum was therefore overproduced, purified and crystallized. A data set extending to 2.15 Å resolution has been collected from a crystal containing selenomethionine-labelled protein and structure determination is under way.
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Mar 2012
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I04-Macromolecular Crystallography
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Open Access
Abstract: Most plastids proteins are post-translationally imported into organelles through multisubunit translocons. The TIC and TOC complexes perform this role in the two membranes of the plant chloroplast and in the inner two membranes of the apicoplasts of the apicomplexan parasites, Toxoplasma gondii and Plasmodium falciparum. Tic22 is a ubiquitous intermembrane translocon component that interacts with translocating proteins. Here, we demonstrate that T. gondii Tic22 is an apicoplast-localized protein, essential for parasite survival and protein import into the apicoplast stroma. The structure of Tic22 from P. falciparum reveals a fold conserved from cyanobacteria to plants, which displays a non-polar groove on each side of the molecule. We show that these grooves allow Tic22 to act as a chaperone. General chaperones are common components of protein translocation systems where they maintain cargo proteins in an unfolded conformation during transit. Such a chaperone had not been identified in the intermembrane space of plastids and we propose that Tic22 fulfills this role.
Apicoplast
Import
Toxoplasma
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Nov 2012
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[7495]
Abstract: African trypanosomes are protected by a densely packed surface monolayer of variant surface glycoprotein (VSG). A haptoglobin–hemoglobin receptor (HpHbR) within this VSG coat mediates heme acquisition. HpHbR is also exploited by the human host to mediate endocytosis of trypanolytic factor (TLF)1 from serum, contributing to innate immunity. Here, the crystal structure of HpHbR from Trypanosoma congolense has been solved, revealing an elongated three ?-helical bundle with a small membrane distal head. To understand the receptor in the context of the VSG layer, the dimensions of Trypanosoma brucei HpHbR and VSG have been determined by small-angle X-ray scattering, revealing the receptor to be more elongated than VSG. It is, therefore, likely that the receptor protrudes above the VSG layer and unlikely that the VSG coat can prevent immunoglobulin binding to the receptor. The HpHb-binding site has been mapped by single-residue mutagenesis and surface plasmon resonance. This site is located where it is readily accessible above the VSG layer. A single HbHpR polymorphism unique to human infective T. brucei gambiense has been shown to be sufficient to reduce binding of both HpHb and TLF1, modulating ligand affinity in a delicate balancing act that allows nutrient acquisition but avoids TLF1 uptake.
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Jan 2013
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I04-Macromolecular Crystallography
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Open Access
Abstract: Biologically active conformations of the IgG1 Fc homodimer are maintained by multiple hydrophobic interactions between the protein surface and the N-glycan. The Fc glycan modulates biological effector functions, including antibody-dependent cellular cytotoxicity (ADCC) which is mediated in part through the activatory Fc receptor, Fc?RIIIA. Consistent with previous reports, we found that site-directed mutations disrupting the protein–carbohydrate interface (F241A, F243A, V262E, and V264E) increased galactosylation and sialylation of the Fc and, concomitantly, reduced the affinity for Fc?RIIIA. We rationalized this effect by crystallographic analysis of the IgG1 Fc F241A mutant, determined here to a resolution of 1.9 Å, which revealed localized destabilization of this glycan–protein interface. Given that sialylation of Fc glycans decreases ADCC, one explanation for the effect of these mutants on Fc?RIIIA binding is their increased sialylation. However, a glycan-engineered IgG1 with hypergalactosylated and hypersialylated glycans exhibited unchanged binding affinity to Fc?RIIIA. Moreover, when we expressed these mutants as a chemically uniform (Man5GlcNAc2) glycoform, the individual effect of each mutation on Fc?RIIIA affinity was preserved. This effect was broadly recapitulated for other Fc receptors (Fc?RI, Fc?RIIA, Fc?RIIB, and Fc?RIIIB). These data indicate that destabilization of the glycan–protein interactions, rather than increased galactosylation and sialylation, modifies the Fc conformation(s) relevant for Fc?R binding. Engineering of the protein–carbohydrate interface thus provides an independent parameter in the engineering of Fc effector functions and a route to the synthesis of new classes of Fc domain with novel combinations of affinities for activatory and inhibitory Fc receptors.
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Jun 2013
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Katherine E.
Wright
,
Kathryn A.
Hjerrild
,
Jonathan
Bartlett
,
Alexander D.
Douglas
,
Jing
Jin
,
Rebecca E.
Brown
,
Joseph J.
Illingworth
,
Rebecca
Ashfield
,
Stine B.
Clemmensen
,
Willem A.
De Jongh
,
Simon J.
Draper
,
Matthew K.
Higgins
Diamond Proposal Number(s):
[9306]
Open Access
Abstract: Invasion of host erythrocytes is essential to the life cycle of Plasmodium parasites and development of the pathology of malaria. The stages of erythrocyte invasion, including initial contact, apical reorientation, junction formation, and active invagination, are directed by coordinated release of specialized apical organelles and their parasite protein contents1. Among these proteins, and central to invasion by all species, are two parasite protein families, the reticulocyte-binding protein homologue (RH) and erythrocyte-binding like proteins, which mediate hostparasite interactions2. RH5 from Plasmodium falciparum (PfRH5) is the only member of either family demonstrated to be necessary for erythrocyte invasion in all tested strains, through its interaction with the erythrocyte surface protein basigin (also known as CD147 and EMMPRIN)3, 4. Antibodies targeting PfRH5 or basigin efficiently block parasite invasion in vitro4, 5, 6, 7, 8, 9, making PfRH5 an excellent vaccine candidate. Here we present crystal structures of PfRH5 in complex with basigin and two distinct inhibitory antibodies. PfRH5 adopts a novel fold in which two three-helical bundles come together in a kite-like architecture, presenting binding sites for basigin and inhibitory antibodies at one tip. This provides the first structural insight into erythrocyte binding by the Plasmodium RH protein family and identifies novel inhibitory epitopes to guide design of a new generation of vaccines against the blood-stage parasite.
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Nov 2014
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
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Open Access
Abstract: The haptoglobin-haemoglobin receptor (HpHbR) of African trypanosomes allows acquisition of haem and provides an uptake route for trypanolytic factor-1, a mediator of innate immunity against trypanosome infection. In this study, we report the structure of Trypanosoma brucei HpHbR in complex with human haptoglobin-haemoglobin (HpHb), revealing an elongated ligand-binding site that extends along its membrane distal half. This contacts haptoglobin and the β-subunit of haemoglobin, showing how the receptor selectively binds HpHb over individual components. Lateral mobility of the glycosylphosphatidylinositol-anchored HpHbR, and a ∼50o kink in the receptor, allows two receptors to simultaneously bind one HpHb dimer. Indeed, trypanosomes take up dimeric HpHb at significantly lower concentrations than monomeric HpHb, due to increased ligand avidity that comes from bivalent binding. The structure therefore reveals the molecular basis for ligand and innate immunity factor uptake by trypanosomes and identifies adaptations that allow efficient ligand uptake in the context of the complex trypanosome cell surface. - See more at: http://elifesciences.org/content/3/e05553.full#sthash.VxBUnQfD.dpuf
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Dec 2014
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Clinton K. Y.
Lau
,
Louise
Turner
,
Jakob S.
Jespersen
,
Edward D.
Lowe
,
Bent
Petersen
,
Christian W.
Wang
,
Jens E. V.
Petersen
,
John
Lusingu
,
Thor G.
Theander
,
Thomas
Lavstsen
,
Matthew K.
Higgins
Open Access
Abstract: The PfEMP1 family of surface proteins is central for Plasmodium falciparum virulence and must retain the ability to bind to host receptors while also diversifying to aid immune evasion. The interaction between CIDRα1 domains of PfEMP1 and endothelial protein C receptor (EPCR) is associated with severe childhood malaria. We combine crystal structures of CIDRα1:EPCR complexes with analysis of 885 CIDRα1 sequences, showing that the EPCR-binding surfaces of CIDRα1 domains are conserved in shape and bonding potential, despite dramatic sequence diversity. Additionally, these domains mimic features of the natural EPCR ligand and can block this ligand interaction. Using peptides corresponding to the EPCR-binding region, antibodies can be purified from individuals in malaria-endemic regions that block EPCR binding of diverse CIDRα1 variants. This highlights the extent to which such a surface protein family can diversify while maintaining ligand-binding capacity and identifies features that should be mimicked in immunogens to prevent EPCR binding.
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Jan 2015
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9306]
Open Access
Abstract: The haptoglobin-haemoglobin receptor of the African trypanosome species, Trypanosoma brucei, is expressed when the parasite is in the bloodstream of the mammalian host, allowing it to acquire haem through the uptake of haptoglobin-haemoglobin complexes. Here we show that in Trypanosoma congolense this receptor is instead expressed in the epimastigote developmental stage that occurs in the tsetse fly, where it acts as a haemoglobin receptor. We also present the structure of the T. congolense receptor in complex with haemoglobin. This allows us to propose an evolutionary history for this receptor, charting the structural and cellular changes that took place as it adapted from a role in the insect to a new role in the mammalian host.
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Apr 2016
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