I24-Microfocus Macromolecular Crystallography
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Meriem
El Ghachi
,
Nicole
Howe
,
Chia-ying
Huang
,
Vincent
Olieric
,
Rangana
Warshamanage
,
Thierry
Touzé
,
Dietmar
Weichert
,
Phillip J.
Stansfeld
,
Meitian
Wang
,
Fred
Kerff
,
Martin
Caffrey
Diamond Proposal Number(s):
[12710]
Open Access
Abstract: As a protective envelope surrounding the bacterial cell, the peptidoglycan sacculus is a site of vulnerability and an antibiotic target. Peptidoglycan components, assembled in the cytoplasm, are shuttled across the membrane in a cycle that uses undecaprenyl-phosphate. A product of peptidoglycan synthesis, undecaprenyl-pyrophosphate, is converted to undecaprenyl-phosphate for reuse in the cycle by the membrane integral pyrophosphatase, BacA. To understand how BacA functions, we determine its crystal structure at 2.6 Å resolution. The enzyme is open to the periplasm and to the periplasmic leaflet via a pocket that extends into the membrane. Conserved residues map to the pocket where pyrophosphorolysis occurs. BacA incorporates an interdigitated inverted topology repeat, a topology type thus far only reported in transporters and channels. This unique topology raises issues regarding the ancestry of BacA, the possibility that BacA has alternate active sites on either side of the membrane and its possible function as a flippase.
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Mar 2018
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I24-Microfocus Macromolecular Crystallography
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Joanne L.
Parker
,
Chenghan
Li
,
Allete
Brinth
,
Zhi
Wang
,
Lutz
Vogeley
,
Nicolae
Solcan
,
Gregory
Ledderboge-vucinic
,
Jessica M. J.
Swanson
,
Martin
Caffrey
,
Gregory A.
Voth
,
Simon
Newstead
Diamond Proposal Number(s):
[9406]
Open Access
Abstract: POT transporters represent an evolutionarily well-conserved family of proton-coupled transport systems in biology. An unusual feature of the family is their ability to couple the transport of chemically diverse ligands to an inwardly directed proton electrochemical gradient. For example, in mammals, fungi, and bacteria they are predominantly peptide transporters, whereas in plants the family has diverged to recognize nitrate, plant defense compounds, and hormones. Although recent structural and biochemical studies have identified conserved sites of proton binding, the mechanism through which transport is coupled to proton movement remains enigmatic. Here we show that different POT transporters operate through distinct proton-coupled mechanisms through changes in the extracellular gate. A high-resolution crystal structure reveals the presence of ordered water molecules within the peptide binding site. Multiscale molecular dynamics simulations confirm proton transport occurs through these waters via Grotthuss shuttling and reveal that proton binding to the extracellular side of the transporter facilitates a reorientation from an inward- to outward-facing state. Together these results demonstrate that within the POT family multiple mechanisms of proton coupling have likely evolved in conjunction with variation of the extracellular gate.
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Nov 2017
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[11890, 10357, 9406]
Abstract: The lipid cubic phase (in meso) method is an important approach for generating crystals and high-resolution X-ray structures of integral membrane proteins. However, as a consequence of instability, it can be impossible—using traditional methods—to concentrate certain membrane proteins and complexes to values suitable for in meso crystallization and structure determination. The cubicon method described here exploits the amphiphilic nature of membrane proteins and their natural tendency to partition preferentially into lipid bilayers from aqueous solution. Using several rounds of reconstitution, the protein concentration in the bilayer of the cubic mesophase can be ramped up stepwise from less than a milligram per milliliter to tens of milligrams per milliliter for crystallogenesis. The general applicability of the method is demonstrated with five integral membrane proteins: the β2-adrenergic G protein-coupled receptor (β2AR), the peptide transporter (PepTSt), diacylglycerol kinase (DgkA), the alginate transporter (AlgE) and the cystic fibrosis transmembrane conductance regulator (CFTR). In the cases of β2AR, PepTSt, DgkA and AlgE, an effective 20- to 45-fold concentration was realized, resulting in a protein-laden mesophase that allowed the formation of crystals using the in meso method and structure determination to resolutions ranging from 2.4 Å to 3.2 Å. In addition to opening up in meso crystallization to a broader range of integral membrane protein targets, the cubicon method should find application in situations that require membrane protein reconstitution in a lipid bilayer at high concentrations. These applications include functional and biophysical characterization studies for ligand screening, drug delivery, antibody production and protein complex formation. A typical cubicon experiment can be completed in 3–5 h.
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Aug 2017
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I24-Microfocus Macromolecular Crystallography
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Maciej
Wiktor
,
Dietmar
Weichert
,
Nicole
Howe
,
Chia-ying
Huang
,
Vincent
Olieric
,
Coilín
Boland
,
Jonathan
Bailey
,
Lutz
Vogeley
,
Phillip J.
Stansfeld
,
Nienke
Buddelmeijer
,
Meitian
Wang
,
Martin
Caffrey
Diamond Proposal Number(s):
[11890, 12710]
Open Access
Abstract: Lipoproteins serve essential roles in the bacterial cell envelope. The posttranslational modification pathway leading to lipoprotein synthesis involves three enzymes. All are potential targets for the development of new antibiotics. Here we report the crystal structure of the last enzyme in the pathway, apolipoprotein N-acyltransferase, Lnt, responsible for adding a third acyl chain to the lipoprotein’s invariant diacylated N-terminal cysteine. Structures of Lnt from Pseudomonas aeruginosa and Escherichia coli have been solved; they are remarkably similar. Both consist of a membrane domain on which sits a globular periplasmic domain. The active site resides above the membrane interface where the domains meet facing into the periplasm. The structures are consistent with the proposed ping-pong reaction mechanism and suggest plausible routes by which substrates and products enter and leave the active site. While Lnt may present challenges for antibiotic development, the structures described should facilitate design of therapeutics with reduced off-target effects.
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Jul 2017
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I24-Microfocus Macromolecular Crystallography
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Abstract: With functions that range from cell envelope structure to signal transduction and transport, lipoproteins constitute 2 to 3% of bacterial genomes and play critical roles in bacterial physiology, pathogenicity, and antibiotic resistance. Lipoproteins are synthesized with a signal peptide securing them to the cytoplasmic membrane with the lipoprotein domain in the periplasm or outside the cell. Posttranslational processing requires a signal peptidase II (LspA) that removes the signal peptide. Here, we report the crystal structure of LspA from Pseudomonas aeruginosa complexed with the antimicrobial globomycin at 2.8 angstrom resolution. Mutagenesis studies identify LspA as an aspartyl peptidase. In an example of molecular mimicry, globomycin appears to inhibit by acting as a noncleavable peptide that sterically blocks the active site. This structure should inform rational antibiotic drug discovery.
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Feb 2016
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I24-Microfocus Macromolecular Crystallography
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Dianfan
Li
,
Phillip J.
Stansfeld
,
Mark S. P.
Sansom
,
Aaron
Keogh
,
Lutz
Vogeley
,
Nicole
Howe
,
Joseph
Lyons
,
David
Aragao
,
Petra
Fromme
,
Raimund
Fromme
,
Shibom
Basu
,
Ingo
Grotjohann
,
Christopher
Kupitz
,
Kimberley
Rendek
,
Uwe
Weierstall
,
Nadia A.
Zatsepin
,
Vadim
Cherezov
,
Wei
Liu
,
Sateesh
Bandaru
,
Niall J.
English
,
Cornelius
Gati
,
Anton
Barty
,
Oleksandr
Yefanov
,
Henry N.
Chapman
,
Kay
Diederichs
,
Marc
Messerschmidt
,
Sébastien
Boutet
,
Garth J.
Williams
,
M.
Marvin Seibert
,
Martin
Caffrey
Open Access
Abstract: Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.
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Dec 2015
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I24-Microfocus Macromolecular Crystallography
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Philip w
Fowler
,
Marcella
Orwick-rydmark
,
Sebastian
Radestock
,
Nicolae
Solcan
,
Patricia m
Dijkman
,
Joseph
Lyons
,
Jane
Kwok
,
Martin
Caffrey
,
Anthony
Watts
,
Lucy r
Forrest
,
Simon
Newstead
Diamond Proposal Number(s):
[8224, 8518]
Open Access
Abstract: Proton-coupled oligopeptide transporters belong to the major facilitator superfamily (MFS) of membrane transporters. Recent crystal structures suggest the MFS fold facilitates transport through rearrangement of their two six-helix bundles around a central ligand binding site; how this is achieved, however, is poorly understood. Using modeling, molecular dynamics, crystallography, functional assays, and site-directed spin labeling combined with double electron-electron resonance (DEER) spectroscopy, we present a detailed study of the transport dynamics of two bacterial oligopeptide transporters, PepTSo and PepTSt. Our results identify several salt bridges that stabilize outward-facing conformations and we show that, for all the current structures of MFS transporters, the first two helices of each of the four inverted-topology repeat units form half of either the periplasmic or cytoplasmic gate and that these function cooperatively in a scissor-like motion to control access to the peptide binding site during transport.
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Feb 2015
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[7796]
Open Access
Abstract: Despite the marked increase in the number of membrane-protein structures solved using crystals grown by the lipid cubic phase or in meso method, only ten have been determined by SAD/MAD. This is likely to be a consequence of the technical difficulties associated with handling proteins and crystals in the sticky and viscous hosting mesophase that is usually incubated in glass sandwich plates for the purposes of crystallization. Here, a four-year campaign aimed at phasing the in meso structure of the integral membrane diacylglycerol kinase (DgkA) from Escherichia coli is reported. Heavy-atom labelling of this small hydrophobic enzyme was attempted by pre-labelling, co-crystallization, soaking, site-specific mercury binding to genetically engineered single-cysteine mutants and selenomethionine incorporation. Strategies and techniques for special handling are reported, as well as the typical results and the lessons learned for each of these approaches. In addition, an assay to assess the accessibility of cysteine residues in membrane proteins for mercury labelling is introduced. The various techniques and strategies described will provide a valuable reference for future experimental phasing of membrane proteins where crystals are grown by the lipid cubic phase method.
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Jan 2015
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I03-Macromolecular Crystallography
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Open Access
Abstract: Magnesium is the most abundant divalent cation in living cells and is crucial to several biological processes. MgtE is a Mg2+ channel distributed in all domains of life that contributes to the maintenance of cellular Mg2+ homeostasis. Here we report the high-resolution crystal structures of the transmembrane domain of MgtE, bound to Mg2+, Mn2+ and Ca2+. The high-resolution Mg2+-bound crystal structure clearly visualized the hydrated Mg2+ ion within its selectivity filter. Based on those structures and biochemical analyses, we propose a cation selectivity mechanism for MgtE in which the geometry of the hydration shell of the fully hydrated Mg2+ ion is recognized by the side-chain carboxylate groups in the selectivity filter. This is in contrast to the K+-selective filter of KcsA, which recognizes a dehydrated K+ ion. Our results further revealed a cation-binding site on the periplasmic side, which regulate channel opening and prevents conduction of near-cognate cations.
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Nov 2014
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[7796]
Abstract: The prokaryotic lysine-specific permease (LysP) belongs to the amino acid-polyamine-organocation (APC) transporter superfamily. In the cell, members of this family are responsible for the uptake and recycling of nutrients, for the maintenance of a constant internal ion concentration and for cell volume regulation. The detailed mechanism of substrate selectivity and transport of L-lysine by LysP is not understood. A high-resolution crystal structure would enormously facilitate such an understanding. To this end, LysP from Pseudomonas aeruginosa was recombinantly expressed in Escherichia coli and purified to near homogeneity by immobilized metal ion-affinity chromatography (IMAC) and size-exclusion chromatography (SEC). Hexagonal- and rod-shaped crystals were obtained in the presence of L-lysine and the L-lysine analogue L-4-thialysine by vapour diffusion and diffracted to 7.5 Å resolution. The diffraction data were indexed in space group P21, with unit-cell parameters a = 169.53, b = 169.53, c = 290.13 Å, γ = 120°.
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Oct 2014
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