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22 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography I23-Long wavelength MX I24-Microfocus Macromolecular Crystallography	Desolvation of the substrate binding protein TauA dictates ligand specificity for the alkanesulfonate ABC importer TauABC Feng Qu , Kamel El Omari , Armin Wagner , Alfonso De Simone , Konstantinos Beis Biochemical Journal DOI: 10.1042/BCJ20190779 Diamond Proposal Number(s): [12579] Open Access Show Abstract ▼ Abstract: Under limiting sulfur availability, bacteria can assimilate sulfur from alkanesulfonates. Bacteria utilize ATP-binding cassette (ABC) transporters to internalise them for further processing to release sulfur. In gram-negative bacteria the TauABC and SsuABC ensure internalization, although, these two systems have common substrates, the former has been characterized as a taurine specific system. TauA and SsuA are substrate-binding proteins (SBPs) that bind and bring the alkanesulfonates to the ABC importer for transport. Here, we have determined the crystal structure of TauA and have characterized its thermodynamic binding parameters by isothermal titration calorimetry in complex with taurine and different alkanesulfonates. Our structures revealed that the coordination of the alkanesulfonates is conserved, with the exception of Asp205 that is absent from SsuA, but the thermodynamic parameters revealed a very high enthalpic penalty cost for binding of the other alkanesulfonates relative to taurine. Our molecular dynamic simulations indicated that the different levels of hydration of the binding site contributed to the selectivity for taurine over the other alkanesulfonates. Such selectivity mechanism is very likely to be employed by other SBPs of ABC transporters.	Nov 2019
I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	OmpK36-mediated Carbapenem resistance attenuates ST258 Klebsiella pneumoniae in vivo Joshua L. C. Wong , Maria Romano , Louise E. Kerry , Hok-sau Kwong , Wen-wen Low , Stephen J. Brett , Abigail Clements , Konstantinos Beis , Gad Frankel Nature Communications 10 DOI: 10.1038/s41467-019-11756-y Diamond Proposal Number(s): [17221] Open Access Show Abstract ▼ Abstract: Carbapenem-resistance in Klebsiella pneumoniae (KP) sequence type ST258 is mediated by carbapenemases (e.g. KPC-2) and loss or modification of the major non-selective porins OmpK35 and OmpK36. However, the mechanism underpinning OmpK36-mediated resistance and consequences of these changes on pathogenicity remain unknown. By solving the crystal structure of a clinical ST258 OmpK36 variant we provide direct structural evidence of pore constriction, mediated by a di-amino acid (Gly115-Asp116) insertion into loop 3, restricting diffusion of both nutrients (e.g. lactose) and Carbapenems. In the presence of KPC-2 this results in a 16-fold increase in MIC to Meropenem. Additionally, the Gly-Asp insertion impairs bacterial growth in lactose-containing medium and confers a significant in vivo fitness cost in a murine model of ventilator-associated pneumonia. Our data suggests that the continuous selective pressure imposed by widespread Carbapenem utilisation in hospital settings drives the expansion of KP expressing Gly-Asp insertion mutants, despite an associated fitness cost.	Sep 2019
B21-High Throughput SAXS I02-Macromolecular Crystallography	Crystal structure and epitope analysis of house dust mite allergen Der f 21 Sze Lei Pang , Kok Lian Ho , Jitka Waterman , Robert Paul Rambo , Aik-hong Teh , Indran Mathavan , Gemma Harris , Konstantinos Beis , Yee-how Say , Matta Sri Anusha , Yang Yie Sio , Fook Tim Chew , Chyan Leong Ng Scientific Reports 9 DOI: 10.1038/s41598-019-40879-x Open Access Show Abstract ▼ Abstract: Group 21 and 5 allergens are homologous house dust mite proteins known as mid-tier allergens. To reveal the biological function of group 21 allergens and to understand better the allergenicity of the rDer f 21 allergen, we determined the 1.5 Å crystal structure of rDer f 21 allergen from Dermatophagoides farinae. The rDer f 21 protein consists of a three helical bundle, similar to available structures of group 21 and homologous group 5 allergens. The rDer f 21 dimer forms a hydrophobic binding pocket similar to the one in the Der p 5 allergen, which indicates that both of the homologous groups could share a similar function. By performing structure-guided mutagenesis, we mutated all 38 surface-exposed polar residues of the rDer f 21 allergen and carried out immuno-dot blot assays using 24 atopic sera. Six residues, K10, K26, K42, E43, K46, and K48, which are located in the region between the N-terminus and the loop 1 of rDer f 21 were identified as the major IgE epitopes of rDer f 21. Epitope mapping of all potential IgE epitopes on the surface of the rDer f 21 crystal structure revealed heterogeneity in the sIgE recognition of the allergen epitopes in atopic individuals. The higher the allergen-sIgE level of an individual, the higher the number of epitope residues that are found in the allergen. The results illustrate the clear correlation between the number of specific major epitope residues in an allergen and the sIgE level of the atopic population.	Mar 2019
I24-Microfocus Macromolecular Crystallography	Substrate-bound outward-open structure of a Na+-coupled sialic acid symporter reveals a new Na+ site Weixiao Yuan Wahlgren , Elin Dunevall , Rachel A. North , Aviv Paz , Mariafrancesca Scalise , Paola Bisignano , Johan Bengtsson-palme , Parveen Goyal , Elin Claesson , Rhawnie Caing-carlsson , Rebecka Andersson , Konstantinos Beis , Ulf J. Nilsson , Anne Farewell , Lorena Pochini , Cesare Indiveri , Michael Grabe , Renwick C. J. Dobson , Jeff Abramson , Sneha Ramaswamy , Rosmarie Friemann Nature Communications 9 DOI: 10.1038/s41467-018-04045-7 Diamond Proposal Number(s): [11641] Open Access Show Abstract ▼ Abstract: Many pathogenic bacteria utilise sialic acids as an energy source or use them as an external coating to evade immune detection. As such, bacteria that colonise sialylated environments deploy specific transporters to mediate import of scavenged sialic acids. Here, we report a substrate-bound 1.95 Å resolution structure and subsequent characterisation of SiaT, a sialic acid transporter from Proteus mirabilis. SiaT is a secondary active transporter of the sodium solute symporter (SSS) family, which use Na+ gradients to drive the uptake of extracellular substrates. SiaT adopts the LeuT-fold and is in an outward-open conformation in complex with the sialic acid N-acetylneuraminic acid and two Na+ ions. One Na+ binds to the conserved Na2 site, while the second Na+ binds to a new position, termed Na3, which is conserved in many SSS family members. Functional and molecular dynamics studies validate the substrate-binding site and demonstrate that both Na+ sites regulate N-acetylneuraminic acid transport.	May 2018
I04-1-Macromolecular Crystallography (fixed wavelength)	Structural basis for antibacterial peptide self‐immunity by the bacterial ABC transporter McjD Kiran Bountra , Gregor Hagelueken , Hassanul Choudhury , Valentina Corradi , Kamel El Omari , Armin Wagner , Indran Mathavan , Séverine Zirah , Weixiao Yuan Wahlgren , D. Peter Tieleman , Olav Schiemann , Sylvie Rebuffat , Konstantinos Beis The Embo Journal DOI: 10.15252/embj.201797278 Diamond Proposal Number(s): [8031] Open Access Show Abstract ▼ Abstract: Certain pathogenic bacteria produce and release toxic peptides to ensure either nutrient availability or evasion from the immune system. These peptides are also toxic to the producing bacteria that utilize dedicated ABC transporters to provide self‐immunity. The ABC transporter McjD exports the antibacterial peptide MccJ25 in Escherichia coli. Our previously determined McjD structure provided some mechanistic insights into antibacterial peptide efflux. In this study, we have determined its structure in a novel conformation, apo inward‐occluded and a new nucleotide‐bound state, high‐energy outward‐occluded intermediate state, with a defined ligand binding cavity. Predictive cysteine cross‐linking in E. coli membranes and PELDOR measurements along the transport cycle indicate that McjD does not undergo major conformational changes as previously proposed for multi‐drug ABC exporters. Combined with transport assays and molecular dynamics simulations, we propose a novel mechanism for toxic peptide ABC exporters that only requires the transient opening of the cavity for release of the peptide. We propose that shielding of the cavity ensures that the transporter is available to export the newly synthesized peptides, preventing toxic‐level build‐up.	Sep 2017
B23-Circular Dichroism I24-Microfocus Macromolecular Crystallography	Structural and functional basis for lipid synergy on the activity of the antibacterial peptide ABC transporter McjD Shahid Mehmood , Valentina Corradi , Hassan Choudhury , Rohanah Hussain , Patrick Becker , Danny Axford , Severine Zirah , Sylvie Rebuffat , D. Peter Tieleman , Carol V. Robinson , Konstantinos Beis Journal Of Biological Chemistry DOI: 10.1074/jbc.M116.732107 Diamond Proposal Number(s): [10136, 14484] Show Abstract ▼ Abstract: The lipid bilayer is a dynamic environment that consists of a mixture of lipids with different properties that regulate the function of membrane proteins; these lipids are either annular, masking the protein hydrophobic surface, or specific lipids, essential for protein function. In this study, using tandem mass spectrometry, we have identified specific lipids associated with the Escherichia coli ABC transporter McjD, which translocates the antibacterial peptide MccJ25. Using non-denaturing mass spectrometry, we show that McjD in complex with MccJ25 survives the gas-phase. Partial delipidation of McjD resulted in reduced ATPase activity and thermostability as shown by Circular Dichroism, both of which could be restored upon addition of defined E. coli lipids. We have resolved a phosphatidylglycerol lipid associated with McjD at 3.4 Å resolution, while molecular dynamic simulations carried out in different lipid environments assessed the binding of specific lipids to McjD. Combined, our data show a synergistic effect of zwitterionic and negatively charged lipids on the activity of McjD; the zwitterionic lipids provide structural stability to McjD whereas the negatively charged lipids are essential for its function.	Aug 2016
	Structural basis for the mechanism of ABC transporters K. Beis Biochemical Society Transactions 43 (5), 889 - 893 DOI: 10.1042/BST20150047 PMID: 26517899 Show Abstract ▼ Abstract: The ATP-binding cassette (ABC) transporters are primary transporters that couple the energy stored in adenosine triphosphate (ATP) to the movement of molecules across the membrane. ABC transporters can be divided into exporters and importers; importers mediate the uptake of essential nutrients into cells and are found predominantly in prokaryotes whereas exporters transport molecules out of cells or into organelles and are found in all organisms. ABC exporters have been linked with multi-drug resistance in both bacterial and eukaryotic cells. ABC transporters are powered by the hydrolysis of ATP and transport their substrate via the alternating access mechanism, whereby the protein alternates between a conformation in which the substrate-binding site is accessible from the outside of the membrane, outward-facing and one in which it is inward-facing. In this mini-review, the structures of different ABC transporter types in different conformations are presented within the context of the alternating access mechanism and how they have shaped our current understanding of the mechanism of ABC transporters.	Oct 2015
	Conformational Changes of the Antibacterial Peptide ATP Binding Cassette Transporter McjD Revealed by Molecular Dynamics Simulations Ruo-xu Gu , Valentina Corradi , Gurpreet Singh , Hassanul G. Choudhury , Konstantinos Beis , D. Peter Tieleman Biochemistry 54 (38), 5989 - 5998 DOI: 10.1021/acs.biochem.5b00753 PMID: 26334959 Show Abstract ▼ Abstract: : The ATP binding cassette (ABC) transporters form one of the largest protein superfamilies. They use the energy of ATP hydrolysis to transport chemically diverse ligands across membranes. An alternating access mechanism in which the transporter switches between inward- and outwardfacing conformations has been proposed to describe the translocation process. One of the main open questions in this process is the degree of opening of the transporter at different stages of the transport cycle, as crystal structures and biochemical data have suggested a wide range of distances between nucleotide binding domains. Recently, the crystal structure of McjD, an antibacterial peptide ABC transporter from Escherichia coli, revealed a new occluded intermediate state of the transport cycle. The transmembrane domain is closed on both sides of the membrane, forming a cavity that can accommodate its ligand, MccJ25, a lasso peptide of 21 amino acids. In this work, we investigate the degree of opening of the transmembrane cavity equired for ligand translocation. By means of steered molecular dynamics simulations, the ligand was pulled from the internal cavity to the extracellular side. This resulted in an outward-facing state. Comparison with existing outward-facing crystal structures shows a smaller degree of opening in the simulations, suggesting that the large conformational changes in some crystal structures may not be necessary even for a large substrate like MccJ25.	Sep 2015
I24-Microfocus Macromolecular Crystallography	Structure determination of an integral membrane protein at room temperature from crystals in situ Danny Axford , James Foadi , Nien-jen Hu , Hassan Choudhury , So Iwata , Konstantinos Beis , Gwyndaf Evans , Yilmaz Alguel Acta Crystallographica Section D Biological Crystallography 71 DOI: 10.1107/S139900471500423X PMID: 26057664 Open Access Show Abstract ▼ Abstract: The structure determination of an integral membrane protein using synchrotron X-ray diffraction data collected at room temperature directly in vapour diffusion crystallization plates (in situ) is demonstrated. Exposing the crystals in situ eliminates manual sample handling and, since it is performed at room temperature, removes the complication of cryoprotection and potential structural anomalies induced by sample cryocooling. Essential to the method is the ability to limit radiation damage by recording a small amount of data per sample from many samples and subsequently assembling the resulting data sets using specialized software. The validity of this procedure is established by the structure determination of Haemophilus influenza TehA at 2.3 A˚ resolution. The method presented offers an effective protocol for the fast and efficient determination of membrane-protein structures at room temperature using third generation synchrotron beamlines.	Jun 2015
	MemStar: A one-shot Escherichia coli-based approach for high-level bacterial membrane protein production Chiara Lee , Haejoo Kang , Anna Hjelm , Abdul Aziz Qureshi , Emmanuel Nji , Hassanul Choudhury , Konstantinos Beis , Jan-willam De Gier , David Drew Febs Letters 588 (20), 3761 - 3769 DOI: 10.1016/j.febslet.2014.08.025 PMID: 25176409 Show Abstract ▼ Abstract: Optimising membrane protein production yields in Escherichiacoli can be time- and resource-consuming. Here, we present a simple and effective Membrane protein Single shot amplification recipe: MemStar. This one-shot amplification recipe is based on the E. coli strain Lemo21(DE3), the PASM-5052 auto-induction medium and, contradictorily, an IPTG induction step. Using MemStar, production yields for most bacterial membrane proteins tested were improved to reach an average of 5 mg L−1 per OD600 unit, which is significantly higher than yields obtained with other common production strategies. With MemStar, we have been able to obtain new structural information for several transporters, including the sodium/proton antiporter NapA.	Oct 2014

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