52 results found
Peter MF, Bountra K, Beis K, et al., 2021, PELDOR/DEER: An electron paramagnetic resonance method to study membrane proteins in lipid bilayers., Biophysics of Membrane Proteins, Editors: Postis, Goldman, Pages: 313-333
Every membrane protein is involved in close interactions with the lipid environment of cellular membranes. The annular lipids, that are in direct contact with the polypeptide, can in principle be seen as an integral part of its structure, akin to the first hydration shell of soluble proteins. It is therefore desirable to investigate the structure of membrane proteins and especially their conformational flexibility under conditions that are as close as possible to their native state. This can be achieved by reconstituting the protein into proteoliposomes, nanodiscs, or bicelles. In recent years, PELDOR/DEER spectroscopy has proved to be a very useful method to study the structure and function of membrane proteins in such artificial membrane environments. The technique complements both X-ray crystallography and cryo-EM and can be used in combination with virtually any artificial membrane environment and under certain circumstances even in native membranes. Of the above-mentioned membrane mimics, bicelles are currently the least often used for PELDOR studies, although they offer some advantages, especially their ease of use. Here, we provide a step-by-step protocol for studying a bicelle reconstituted membrane protein with PELDOR/DEER spectroscopy.
El Omari K, Mohamad N, Bountra K, et al., 2020, Experimental phasing with vanadium and application to nucleotide-binding membrane proteins, IUCRJ, Vol: 7, Pages: 1092-1101, ISSN: 2052-2525
Thomas C, Aller SG, Beis K, et al., 2020, Structural and functional diversity calls for a new classification of ABC transporters, FEBS LETTERS, Vol: 594, Pages: 3767-3775, ISSN: 0014-5793
Smits SHJ, Schmitt L, Beis K, 2020, Self-immunity to antibacterial peptides by ABC transporters, FEBS LETTERS, Vol: 594, Pages: 3920-3942, ISSN: 0014-5793
Qu F, ElOmari K, Wagner A, et al., 2019, Desolvation of the substrate binding protein TauA dictates ligand specificity for the alkanesulfonate ABC importer TauABC, Biochemical Journal, Vol: 476, Pages: 3649-3660, ISSN: 0264-6021
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 characterised 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 characterised 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 in 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.
Beis K, Rebuffat S, 2019, Multifaceted ABC transporters associated to microcin and bacteriocin export, RESEARCH IN MICROBIOLOGY, Vol: 170, Pages: 399-406, ISSN: 0923-2508
Wong JLC, Romano M, Kerry L, et al., 2019, OmpK36-mediated Carbapenem resistance attenuates ST258 Klebsiella pneumoniae in vivo, Nature Communications, Vol: 10, ISSN: 2041-1723
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 suggest 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.
Pang SL, Ho KL, Waterman J, et al., 2019, Crystal structure and epitope analysis of house dust mite allergen Der f 21, SCIENTIFIC REPORTS, Vol: 9, ISSN: 2045-2322
Wang L, Bateman B, Zanetti-Domingues LC, et al., 2019, Solid immersion microscopy images cells under cryogenic conditions with 12 nm resolution, Communications Biology, Vol: 2, ISSN: 2399-3642
Super-resolution fluorescence microscopy plays a crucial role in our understanding of cell structure and function by reporting cellular ultrastructure with 20–30 nm resolution. However, this resolution is insufficient to image macro-molecular machinery at work. A path to improve resolution is to image under cryogenic conditions. This substantially increases the brightness of most fluorophores and preserves native ultrastructure much better than chemical fixation. Cryogenic conditions are, however, underutilised because of the lack of compatible high numerical aperture objectives. Here, using a low-cost super-hemispherical solid immersion lens (superSIL) and a basic set-up we achieve 12 nm resolution under cryogenic conditions, to our knowledge the best yet attained in cells using simple set-ups and/or commercial systems. By also allowing multicolour imaging, and by paving the way to total-internal-reflection fluorescence imaging of mammalian cells under cryogenic conditions, superSIL microscopy opens a straightforward route to achieve unmatched resolution on bacterial and mammalian cell samples.
Ford RC, Beis K, 2019, Learning the ABCs one at a time: structure and mechanism of ABC transporters, Biochemical Society Transactions, Pages: BST20180147-BST20180147, ISSN: 0300-5127
Husada F, Bountra K, Tasis K, et al., 2018, Conformational dynamics of the ABC transporter McjD seen by single-molecule FRET, EMBO Journal, Vol: 37, ISSN: 0261-4189
ABC transporters utilize ATP for export processes to provide cellular resistance against toxins, antibiotics, and harmful metabolites in eukaryotes and prokaryotes. Based on static structure snapshots, it is believed that they use an alternating access mechanism, which couples conformational changes to ATP binding (outward‐open conformation) and hydrolysis (inward‐open) for unidirectional transport driven by ATP. Here, we analyzed the conformational states and dynamics of the antibacterial peptide exporter McjD from Escherichia coli using single‐molecule Förster resonance energy transfer (smFRET). For the first time, we established smFRET for an ABC exporter in a native‐like lipid environment and directly monitor conformational dynamics in both the transmembrane‐ (TMD) and nucleotide‐binding domains (NBD). With this, we unravel the ligand dependences that drive conformational changes in both domains. Furthermore, we observe intrinsic conformational dynamics in the absence of ATP and ligand in the NBDs. ATP binding and hydrolysis on the other hand can be observed via NBD conformational dynamics. We believe that the progress made here in combination with future studies will facilitate full understanding of ABC transport cycles.
Romano M, Fusco G, Choudhury H, et al., 2018, Structural basis for natural product selection and export by bacterial ABC transporters, ACS Chemical Biology, Vol: 13, Pages: 1598-1609, ISSN: 1554-8929
Bacteria under stress produce ribosomally synthesized and post-translationally modified peptides (RiPPs) to target closely related species, such as the lasso peptide microcin J25 (MccJ25). These peptides are also toxic to the producing organisms that utilize dedicated ABC transporters to achieve self-immunity. MccJ25 is exported by the Escherichia coli ABC transporter McjD through a complex mechanism of recognition that has remained elusive. Here, we used biomolecular NMR to study this interaction and identified a region of the toxic peptide that is crucial to its recognition by the ABC transporter. Our study provides evidence that McjD is highly specific to MccJ25 and not to other RiPPs or antibiotics, unlike multidrug ABC transporters. Additionally, we show that MccJ25 is not exported by another natural product ABC transporter. Therefore, we propose that specific interactions between natural product ABC transporters and their substrate provides them with their high degree of specificity. Taken together, these findings suggest that ABC transporters might have acquired structural elements in their binding cavity to recognize and allow promiscuous export of a larger variety of compounds.
Wahlgren WY, Dunevall E, North RA, et al., 2018, Substrate-bound outward-open structure of a Na+-coupled sialic acid symporter reveals a new Na+ site, Nature Communications, Vol: 9, ISSN: 2041-1723
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.
Bountra K, Hagelueken G, Choudhury HG, et al., 2017, Structural basis for antibacterial peptide self-immunity by the bacterial ABC transporter McjD, The EMBO Journal, Vol: 36, Pages: 3062-3079, ISSN: 0261-4189
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.
Mehmood S, Corradi V, Choudhury HG, et al., 2016, Structural and functional basis for lipid synergy on the activity of the antibacterial peptide ABC transporter McjD, Journal of Biological Chemistry, Vol: 291, Pages: 21656-21668, ISSN: 1083-351X
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.
Beis K, 2015, Structural basis for the mechanism of ABC transporters, BIOCHEMICAL SOCIETY TRANSACTIONS, Vol: 43, Pages: 889-893, ISSN: 0300-5127
Gu R-X, Corradi V, Singh G, et al., 2015, Conformational Changes of the Antibacterial Peptide ATP Binding Cassette Transporter McjD Revealed by Molecular Dynamics Simulations, BIOCHEMISTRY, Vol: 54, Pages: 5989-5998, ISSN: 0006-2960
Axford D, Foadi J, Hu N-J, et al., 2015, Structure determination of an integral membrane protein at room temperature from crystals in situ, ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY, Vol: 71, Pages: 1228-1237, ISSN: 2059-7983
Gu R-X, Corradi V, Singh G, et al., 2015, Conformational Changes of the ABC Transporter McjD Revealed by Molecular Dynamics Simulations, BIOPHYSICAL JOURNAL, Vol: 108, Pages: 89A-89A, ISSN: 0006-3495
Lee C, Kang HJ, Hjelm A, et al., 2014, MemStar: A one-shot Escherichia coli-based approach for high-level bacterial membrane protein production, FEBS LETTERS, Vol: 588, Pages: 3761-3769, ISSN: 0014-5793
Choudhury HG, Tong Z, Mathavan I, et al., 2014, Structure of an antibacterial peptide ATP-binding cassette transporter in a novel outward occluded state, Proceedings of the National Academy of Sciences of the United States of America, Vol: 111, Pages: 9145-9150, ISSN: 0027-8424
Enterobacteriaceae produce antimicrobial peptides for survival under nutrient starvation. Microcin J25 (MccJ25) is an antimicrobial peptide with a unique lasso topology. It is secreted by the ATP-binding cassette (ABC) exporter McjD, which ensures self-immunity of the producing strain through efficient export of the toxic mature peptide from the cell. Here we have determined the crystal structure of McjD from Escherichia coli at 2.7-Å resolution, which is to the authors’ knowledge the first structure of an antibacterial peptide ABC transporter. Our functional and biochemical analyses demonstrate McjD-dependent immunity to MccJ25 through efflux of the peptide. McjD can directly bind MccJ25 and displays a basal ATPase activity that is stimulated by MccJ25 in both detergent solution and proteoliposomes. McjD adopts a new conformation, termed nucleotide-bound outward occluded. The new conformation defines a clear cavity; mutagenesis and ligand binding studies of the cavity have identified Phe86, Asn134, and Asn302 as important for recognition of MccJ25. Comparisons with the inward-open MsbA and outward-open Sav1866 structures show that McjD has structural similarities with both states without the intertwining of transmembrane (TM) helices. The occluded state is formed by rotation of TMs 1 and 2 toward the equivalent TMs of the opposite monomer, unlike Sav1866 where they intertwine with TMs 3–6 of the opposite monomer. Cysteine cross-linking studies on the McjD dimer in inside-out membrane vesicles of E. coli confirmed the presence of the occluded state. We therefore propose that the outward-occluded state represents a transition intermediate between the outward-open and inward-open conformation of ABC exporters.
Mathavan I, Zirah S, Mehmood S, et al., 2014, Structural basis for hijacking siderophore receptors by antimicrobial lasso peptides, Nature Chemical Biology, Vol: 10, Pages: 340-342, ISSN: 1552-4450
The lasso peptide microcin J25 is known to hijack the siderophore receptor FhuA for initiating internalization. Here, we provide what is to our knowledge the first structural evidence on the recognition mechanism, and our biochemical data show that another closely related lasso peptide cannot interact with FhuA. Our work provides an explanation on the narrow activity spectrum of lasso peptides and opens the path to the development of new antibacterials.
Runti G, Ruiz MDCL, Stoilova T, et al., 2013, Functional Characterization of SbmA, a Bacterial Inner Membrane Transporter Required for Importing the Antimicrobial Peptide Bac7(1-35), JOURNAL OF BACTERIOLOGY, Vol: 195, Pages: 5343-5351, ISSN: 0021-9193
Corbalan N, Runti G, Adler C, et al., 2013, Functional and Structural Study of the Dimeric Inner Membrane Protein SbmA, JOURNAL OF BACTERIOLOGY, Vol: 195, Pages: 5352-5361, ISSN: 0021-9193
Choudhury HG, Beis K, 2013, The dimeric form of the unphosphorylated response regulator BaeR, PROTEIN SCIENCE, Vol: 22, Pages: 1287-1293, ISSN: 0961-8368
Choudhury HG, Beis K, 2013, Tellurite-Detoxifying Protein TehB from Escherichia coli, Encyclopedia of Metalloproteins, Editors: Kretsinger, Uversky, Permyakov, Publisher: Springer, ISBN: 978-1-4614-1532-9
Choudhury HG, Beis K, 2013, Tellurite-Resistance Protein TehA from Escherichia coli, Encyclopedia of Metalloproteins, Editors: Kretsinger, Uversky, Permyakov, Publisher: Springer, ISBN: 978-1-4614-1532-9
Mathavan I, Beis K, 2012, The role of bacterial membrane proteins in the internalization of microcin MccJ25 and MccB17, Biochemical Society Transactions, Vol: 40, Pages: 1539-1543
Lou H, Chen M, Black SS, et al., 2011, Altered Antibiotic Transport in OmpC Mutants Isolated from a Series of Clinical Strains of Multi-Drug Resistant E. coli, PLOS ONE, Vol: 6, ISSN: 1932-6203
Choudhury HG, Cameron AD, Iwata S, et al., 2011, Escherichia coli detoxification of chalcolgens by TehAB, 36th FEBS Congress of the Biochemistry for Tomorrows Medicine, Publisher: WILEY-BLACKWELL, Pages: 101-101, ISSN: 1742-464X
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