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Instruments / Technical Area	Publication Details	Published
I04-1-Macromolecular Crystallography (fixed wavelength)	Engineering a surrogate human heteromeric α/β glycine receptor orthosteric site exploiting the structural homology and stability of acetylcholine-binding protein Alice Dawson , Paul Trumper , Juliana Oliveira De Souza , Holly Parker , Mathew J. Jones , Tim G. Hales , William N. Hunter Iucrj 6 DOI: 10.1107/S205225251901114X Diamond Proposal Number(s): [19844] Open Access Show Abstract ▼ Abstract: Protein-engineering methods have been exploited to produce a surrogate system for the extracellular neurotransmitter-binding site of a heteromeric human ligand-gated ion channel, the glycine receptor. This approach circumvents two major issues: the inherent experimental difficulties in working with a membrane-bound ion channel and the complication that a heteromeric assembly is necessary to create a key, physiologically relevant binding site. Residues that form the orthosteric site in a highly stable ortholog, acetylcholine-binding protein, were selected for substitution. Recombinant proteins were prepared and characterized in stepwise fashion exploiting a range of biophysical techniques, including X-ray crystallography, married to the use of selected chemical probes. The decision making and development of the surrogate, which is termed a glycine-binding protein, are described, and comparisons are provided with wild-type and homomeric systems that establish features of molecular recognition in the binding site and the confidence that the system is suited for use in early-stage drug discovery targeting a heteromeric α/β glycine receptor.	Nov 2019
I24-Microfocus Macromolecular Crystallography	The structure of lipopolysaccharide transport protein B (LptB) from Burkholderia pseudomallei Genady Pankov , Alice Dawson , William N. Hunter Acta Crystallographica Section F Structural Biology Communications 75 DOI: 10.1107/S2053230X19001778 Diamond Proposal Number(s): [14980] Show Abstract ▼ Abstract: The thick outer membrane (OM) of Gram-negative bacteria performs an important protective role against hostile environments, supports cell integrity, and contributes to surface adhesion and in some cases also to virulence. A major component of the OM is lipopolysaccharide (LPS), a complex glycolipid attached to a core containing fatty-acyl chains. The assembly and transport of lipid A, the membrane anchor for LPS, to the OM begins when a heteromeric LptB2FG protein complex extracts lipid A from the outer leaflet of the inner membrane. This process requires energy, and upon hydrolysis of ATP one component of the heteromeric assembly, LptB, triggers a conformational change in LptFG in support of lipid A transport. A structure of LptB from the intracellular pathogen Burkholderia pseudomallei is reported here. LptB forms a dimer that displays a relatively fixed structure irrespective of whether it is in complex with LptFG or in isolation. Highly conserved sequence and structural features are discussed that allow LptB to fuel the transport of lipid A.	Apr 2019
I04-Macromolecular Crystallography	Exploiting the 2-Amino-1,3,4-thiadiazole Scaffold To Inhibit Trypanosoma brucei Pteridine Reductase in Support of Early-Stage Drug Discovery Pasquale Linciano , Alice Dawson , Ina Pöhner , David M. Costa , Monica S. Sá , Anabela Cordeiro-da-silva , Rosaria Luciani , Sheraz Gul , Gesa Witt , Bernhard Ellinger , Maria Kuzikov , Philip Gribbon , Jeanette Reinshagen , Markus Wolf , Birte Behrens , Véronique Hannaert , Paul A. M. Michels , Erika Nerini , Cecilia Pozzi , Flavio Di Pisa , Giacomo Landi , Nuno Santarem , Stefania Ferrari , Puneet Saxena , Sandra Lazzari , Giuseppe Cannazza , Lucio H. Freitas-junior , Carolina B. Moraes , Bruno S. Pascoalino , Laura M. Alcântara , Claudia P. Bertolacini , Vanessa Fontana , Ulrike Wittig , Wolfgang Müller , Rebecca C. Wade , William N. Hunter , Stefano Mangani , Luca Costantino , Maria P. Costi Acs Omega 2, 5666 - 5683 DOI: 10.1021/acsomega.7b00473 Diamond Proposal Number(s): [8574] Open Access Show Abstract ▼ Abstract: Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). We solved crystal structures of several TbPTR1-inhibitor complexes to guide the structure-based design of new thiadiazole derivatives. Subsequent synthesis and enzyme- and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compound 4m, a biphenyl-thiadiazole-2,5-diamine with IC50 = 16 μM, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC50 value. In addition, the antiparasitic activity of the combination of 4m and MTX was reversed by addition of folic acid. By adopting an efficient hit discovery platform, we demonstrate, using the 2-amino-1,3,4-thiadiazole scaffold, how a promising tool for the development of anti-T. brucei agents can be obtained.	Sep 2017
I03-Macromolecular Crystallography	An Improved Model of the Trypanosoma brucei CTP Synthetase Glutaminase Domain-Acivicin Complex Juliana Oliveira De Souza , Alice Dawson , William N. Hunter Chemmedchem 62 DOI: 10.1002/cmdc.201700118 Show Abstract ▼ Abstract: The natural product acivicin inhibits the glutaminase activity of cytidine triphosphate (CTP) synthetase and is a potent lead compound for drug discovery in the area of neglected tropical diseases, specifically trypanosomaisis. A 2.1-Å-resolution crystal structure of the acivicin adduct with the glutaminase domain from Trypanosoma brucei CTP synthetase has been deposited in the RCSB Protein Data Bank (PDB) and provides a template for structure-based approaches to design new inhibitors. However, our assessment of that data identified deficiencies in the model. We now report an improved and corrected inhibitor structure with changes to the chirality at one position, the orientation and covalent structure of the isoxazoline moiety, and the location of a chloride ion in an oxyanion binding site that is exploited during catalysis. The model is now in agreement with established chemical principles and allows an accurate description of molecular recognition of the ligand and the mode of binding in a potentially valuable drug target.	Mar 2017
I04-1-Macromolecular Crystallography (fixed wavelength)	The structure of a Trypanosoma cruzi glucose-6-phosphate dehydrogenase reveals differences from the mammalian enzyme Gustavo F. Mercaldi , Alice Dawson , Willian N. Hunter , Artur Cordeiro Febs Letters 590, 2776 - 2786 DOI: 10.1002/1873-3468.12276 Diamond Proposal Number(s): [11088] Show Abstract ▼ Abstract: The enzyme glucose-6-phosphate dehydrogenase from Trypanosoma cruzi (TcG6PDH) catalyses the first step of the pentose phosphate pathway (PPP) and is considered a promising target for the discovery of a new drug against Chagas diseases. In the present work, we describe the crystal structure of TcG6PDH obtained in a ternary complex with the substrate β-d-glucose-6-phosphate (G6P) and the reduced ‘catalytic’ cofactor NADPH, which reveals the molecular basis of substrate and cofactor recognition. A comparison with the homologous human protein sheds light on differences in the cofactor-binding site that might be explored towards the design of new NADP+ competitive inhibitors targeting the parasite enzyme.	Aug 2016
I04-Macromolecular Crystallography	Structure of Staphylococcus aureus adenylosuccinate lyase (PurB) and assessment of its potential as a target for structure-based inhibitor discovery Paul Fyfe , Alice Dawson , Marie-theres Hutchison , Scott Cameron , William Hunter Acta Crystallographica Section D Biological Crystallography 66 (8), 881-888 DOI: 10.1107/S0907444910020081 PMID: 20693687 Open Access Show Abstract ▼ Abstract: The medium-resolution structure of adenylosuccinate lyase (PurB) from the bacterial pathogen Staphylococcus aureus in complex with AMP is presented. Oxalate, which is likely to be an artifact of crystallization, has been modelled in the active site and occupies a position close to that where succinate is observed in orthologous structures. PurB catalyzes reactions that support the provision of purines and the control of AMP/fumarate levels. As such, the enzyme is predicted to be essential for the survival of S. aureus and to be a potential therapeutic target. Comparisons of this pathogen PurB with the enzyme from Escherichia coli are presented to allow discussion concerning the enzyme mechanism. Comparisons with human PurB suggest that the close similarity of the active sites would make it difficult to identify species-specific inhibitors for this enyme. However, there are differences in the way that the subunits are assembled into dimers. The distinct subunit-subunit interfaces may provide a potential area to target by exploiting the observation that creation of the enzyme active site is dependent on oligomerization.	Aug 2010
	The effect of pressure on the crystal structure of L-alanine Nicholas P. Funnell , Alice Dawson , Duncan Francis , Alistair Lennie , William G. Marshall , Stephen Moggach , John Warren , Simon Parsons Crystengcomm 12 (9), 2573-2583 DOI: 10.1039/c001296c Show Abstract ▼ Abstract: L-Alanine crystallises as a zwitterion in space group P2(1)2(1)2(1) at ambient pressure. The strongest intermolecular interactions are three N-H center dot center dot center dot O hydrogen bonds. The H-bonds link the molecules into puckered layers in the ac plane, which are then stacked along the b axis. PIXEL calculations indicate that the H-bond mediated intermolecular energies within the layers are 118 and 145 kJ mol(-1), while the stacking interactions are considerably weaker (31 kJ mol(-1)). Neutron powder diffraction data on L-alanine-d(7) to 9.87 GPa show that the effects of pressure are most prominent along a, the direction which is least well aligned with the H-bonds. The a axis decreases in length more rapidly than the c axis, and the two axis lengths are equal at ca. 2 GPa. Although the structure is metrically tetragonal at this point, the true symmetry is still orthorhombic. In fact, the structure remains in a compressed form of its starting orthorhombic phase throughout the pressure range studied, in contradiction of previous Raman and energy dispersive power diffraction studies, in which data were interpreted in terms of phase transitions at ca. 2 GPa and 9 GPa. It is likely that the spectroscopic signature of the apparent transition at 2 GPa is the result of a conformational change at the ammonium group. The molecular coordination number in L-alanine is 14, and the packing topology is a distorted form of body-centred cubic at ambient pressure. As pressure increases this topology becomes more regular, until at 9.87 GPa it is very similar to the perfect BCC topology of a structure such as tungsten. It is suggested that this behaviour can be traced to the sphericity of the alanine molecule.	Jan 2010

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