Krios I-Titan Krios I at Diamond
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Dmitry
Ghilarov
,
Satomi
Inaba-Inoue
,
Piotr
Stepien
,
Feng
Qu
,
Elizabeth
Michalczyk
,
Zuzanna
Pakosz
,
Norimichi
Nomura
,
Satoshi
Ogasawara
,
Graham Charles
Walker
,
Sylvie
Rebuffat
,
So
Iwata
,
Jonathan
Gardiner Heddle
,
Konstantinos
Beis
Diamond Proposal Number(s):
[18659]
Open Access
Abstract: Antibiotic metabolites and antimicrobial peptides mediate competition between bacterial species. Many of them hijack inner and outer membrane proteins to enter cells. Sensitivity of enteric bacteria to multiple peptide antibiotics is controlled by the single inner membrane protein SbmA. To establish the molecular mechanism of peptide transport by SbmA and related BacA, we determined their cryo–electron microscopy structures at 3.2 and 6 Å local resolution, respectively. The structures show a previously unknown fold, defining a new class of secondary transporters named SbmA-like peptide transporters. The core domain includes conserved glutamates, which provide a pathway for proton translocation, powering transport. The structures show an outward-open conformation with a large cavity that can accommodate diverse substrates. We propose a molecular mechanism for antibacterial peptide uptake paving the way for creation of narrow-targeted therapeutics.
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Sep 2021
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I23-Long wavelength MX
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Kamel
El Omari
,
Nada
Mohamad
,
Kiran
Bountra
,
Ramona
Duman
,
Maria
Romano
,
Katja
Schlegel
,
Hok-Sau
Kwong
,
Vitaliy
Mykhaylyk
,
Claus
Olesen
,
Jesper Vuust
Moller
,
Maike
Bublitz
,
Konstantinos
Beis
,
Armin
Wagner
Open Access
Abstract: The structure determination of soluble and membrane proteins can be hindered by the crystallographic phase problem, especially in the absence of a suitable homologous structure. Experimental phasing is the method of choice for novel structures; however, it often requires heavy-atom derivatization, which can be difficult and time-consuming. Here, a novel and rapid method to obtain experimental phases for protein structure determination by vanadium phasing is reported. Vanadate is a transition-state mimic of phosphoryl-transfer reactions and it has the advantage of binding specifically to the active site of numerous enzymes catalyzing this reaction. The applicability of vanadium phasing has been validated by determining the structures of three different protein–vanadium complexes, two of which are integral membrane proteins: the rabbit sarcoplasmic reticulum Ca2+-ATPase, the antibacterial peptide ATP-binding cassette transporter McjD from Escherichia coli and the soluble enzyme RNAse A from Bos taurus. Vanadium phasing was successful even at low resolution and despite severe anisotropy in the data. This method is principally applicable to a large number of proteins, representing six of the seven Enzyme Commission classes. It relies exclusively on the specific chemistry of the protein and it does not require any modifications, making it a very powerful addition to the phasing toolkit. In addition to the phasing power of this technique, the protein–vanadium complexes also provide detailed insights into the reaction mechanisms of the studied proteins.
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Nov 2020
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I03-Macromolecular Crystallography
I23-Long wavelength MX
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[12579]
Open Access
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.
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Nov 2019
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[17221]
Open Access
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.
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Sep 2019
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B21-High Throughput SAXS
I02-Macromolecular Crystallography
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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
Open Access
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.
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Mar 2019
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I24-Microfocus Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[11641]
Open Access
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.
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May 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
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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
Diamond Proposal Number(s):
[8031]
Open Access
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.
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Sep 2017
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B23-Circular Dichroism
I24-Microfocus Macromolecular Crystallography
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Shahid
Mehmood
,
Valentina
Corradi
,
Hassan
Choudhury
,
Rohanah
Hussain
,
Patrick
Becker
,
Danny
Axford
,
Severine
Zirah
,
Sylvie
Rebuffat
,
D. Peter
Tieleman
,
Carol V.
Robinson
,
Konstantinos
Beis
Diamond Proposal Number(s):
[10136, 14484]
Open Access
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.
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Aug 2016
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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.
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Oct 2015
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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.
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Sep 2015
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