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

Instruments / Technical Area	Publication Details	Published
I24-Microfocus Macromolecular Crystallography	Thromboxane A2 G protein-coupled receptor production and crystallization for structure studies Pawel Krawinski , Martin Caffrey DOI: 10.1007/978-1-0716-2368-8 Show Abstract ▼ Abstract: G protein-coupled receptors (GPCRs) play vital roles in human physiology and pathophysiology. This makes the elucidation of the high-resolution blueprints of these high value membrane proteins of crucial importance for the structure-based design of novel therapeutics. However, the production and crystallization of GPCRs for structure determination comes with many challenges. In this chapter, we provide a comprehensive protocol for expressing and purifying the thromboxane A2 receptor (TPR), an attractive therapeutic target, for use in structure studies. Guidelines for crystallizing the TPR are also included. Together, these procedures provide a template for generating crystal structures of the TPR and indeed other GPCRs in complex with pharmacologically interesting ligands.	Jul 2022
I24-Microfocus Macromolecular Crystallography	Structural basis of the membrane intramolecular transacylase reaction responsible for lyso-form lipoprotein synthesis Samir Olatunji , Katherine Bowen , Chia-Ying Huang , Dietmar Weichert , Warispreet Singh , Irina G. Tikhonova , Eoin M. Scanlan , Vincent Olieric , Martin Caffrey Nature Communications 12 DOI: 10.1038/s41467-021-24475-0 Diamond Proposal Number(s): [23364] Open Access Show Abstract ▼ Abstract: Lipoproteins serve diverse functions in the bacterial cell and some are essential for survival. Some lipoproteins are adjuvants eliciting responses from the innate immune system of the host. The growing list of membrane enzymes responsible for lipoprotein synthesis includes the recently discovered lipoprotein intramolecular transacylase, Lit. Lit creates a lipoprotein that is less immunogenic, possibly enabling the bacteria to gain a foothold in the host by stealth. Here, we report the crystal structure of the Lit enzyme from Bacillus cereus and describe its mechanism of action. Lit consists of four transmembrane helices with an extracellular cap. Conserved residues map to the cap-membrane interface. They include two catalytic histidines that function to effect unimolecular transacylation. The reaction involves acyl transfer from the sn-2 position of the glyceryl moiety to the amino group on the N-terminal cysteine of the substrate via an 8-membered ring intermediate. Transacylation takes place in a confined aromatic residue-rich environment that likely evolved to bring distant moieties on the substrate into proximity and proper orientation for catalysis.	Jul 2021
I24-Microfocus Macromolecular Crystallography	9.8 MAG: A new host lipid for in meso (lipid cubic phase) crystallization of integral membrane proteins Leendert Van Dalsen , Luke Smithers , Coilin Boland , Dietmar Weichert , Martin Caffrey Crystal Growth & Design DOI: 10.1021/acs.cgd.0c01301 Diamond Proposal Number(s): [17810] Show Abstract ▼ Abstract: Monoolein, also referred to as 9.9 MAG, is the most commonly used monoacylglycerol for crystallizing membrane proteins by the in meso method. However, 9.9 MAG does not work for all proteins. Therefore, having available a suite of monoacylglycerols, the members of which differ in acyl chain characteristics such as chain length and position along the chain of the cis-olefinic bond, is an important screening feature. Several monoacylglycerols of this type are available and have proven their worth in enabling the structure determination of high-profile targets, including the β2-adrenoreceptor-Gs protein and the rhodopsin-arrestin complexes, and cytochrome caa3 oxidase. Here a new monoacylglycerol, 9.8 MAG, is introduced. Since the performance in crystallogenesis depends critically on the phase properties of the host lipid, the thermotropic and lyotropic mesophase behavior and microstructure of hydrated 9.8 MAG have been quantified by small-angle X-ray diffraction. The lipid is shown to be compatible with cholesterol at levels typically used in crystallization trials. Further, 9.8 MAG supports the crystallization and structure determination of two benchmark proteins: the α-helical lipoprotein N-acyltransferase, Lnt, and the β-barrel alginate transporter, AlgE. 9.8 MAG can now be included in host lipid screens to optimize the structure determination of a broader range of membrane proteins, many of which are scientifically and medically important.	Nov 2020
I24-Microfocus Macromolecular Crystallography	Effects of 2-monoacylglycerol on in meso crystallization and the crystal structures of integral membrane proteins Luke Smithers , Leendert Van Dalsen , Coilin Boland , Gerard Reid , Dietmar Weichert , Martin Caffrey Crystal Growth & Design DOI: 10.1021/acs.cgd.0c00660 Diamond Proposal Number(s): [17810] Show Abstract ▼ Abstract: Monoacylglycerols (MAGs) are the lipids most commonly used for crystallizing membrane proteins by the in meso method. The acyl chain and glycerol components of MAGs are ester-linked at the sn-1 hydroxyl of glycerol in 1-MAGs. 1-MAGs succumb to spontaneous transacylation where the chain migrates between the sn-1 and sn-2 hydroxyls. In the mesophase formed by 1-monoolein, the most commonly used MAG, equilibrium is reached in two weeks at 6 mol% 2-monoolein. Variability in 2-MAG content appears in synthetic MAGs produced for the purpose of host lipid screening. In the interests of reproducibility, extensive purification has been implemented to minimize the 2-MAG content of newly synthesized lipid. Here we show, with two membrane proteins, that such efforts are not needed. Specifically, the initial 2-MAG content can vary over wide limits without impacting negatively on the crystallization process or on the structures obtained with those crystals. Dispensing with rounds of purification means that MAGs can be produced using protocols that are simpler, faster, less expensive and more environmentally friendly. An added feature of having 2-monoolein in the crystallization mix is that it stabilizes mesophases with bigger aqueous channels, well suited to structure determination of proteins and complexes with large extramembrane domains.	Jul 2020
I24-Microfocus Macromolecular Crystallography	Structures of lipoprotein signal peptidase II from Staphylococcus aureus complexed with antibiotics globomycin and myxovirescin Samir Olatunji , Xiaoxiao Yu , Jonathan Bailey , Chia-Ying Huang , Marta Zapotoczna , Katherine Bowen , Maja Remškar , Rolf Müller , Eoin M. Scanlan , Joan A. Geoghegan , Vincent Olieric , Martin Caffrey Nature Communications 11 DOI: 10.1038/s41467-019-13724-y Diamond Proposal Number(s): [17810] Open Access Show Abstract ▼ Abstract: Antimicrobial resistance is a major global threat that calls for new antibiotics. Globomycin and myxovirescin are two natural antibiotics that target the lipoprotein-processing enzyme, LspA, thereby compromising the integrity of the bacterial cell envelope. As part of a project aimed at understanding their mechanism of action and for drug development, we provide high-resolution crystal structures of the enzyme from the human pathogen methicillin-resistant Staphylococcus aureus (MRSA) complexed with globomycin and with myxovirescin. Our results reveal an instance of convergent evolution. The two antibiotics possess different molecular structures. Yet, they appear to inhibit identically as non-cleavable tetrahedral intermediate analogs. Remarkably, the two antibiotics superpose along nineteen contiguous atoms that interact similarly with LspA. This 19-atom motif recapitulates a part of the substrate lipoprotein in its proposed binding mode. Incorporating this motif into a scaffold with suitable pharmacokinetic properties should enable the development of effective antibiotics with built-in resistance hardiness.	Jan 2020
I24-Microfocus Macromolecular Crystallography	Crystal structure of undecaprenyl-pyrophosphate phosphatase and its role in peptidoglycan biosynthesis 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 Nature Communications 9 DOI: 10.1038/s41467-018-03477-5 Diamond Proposal Number(s): [12710] Open Access Show Abstract ▼ 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.	Mar 2018
I24-Microfocus Macromolecular Crystallography	Proton movement and coupling in the POT family of peptide transporters 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 Proceedings Of The National Academy Of Sciences 63 DOI: 10.1073/pnas.1710727114 Diamond Proposal Number(s): [9406] Open Access Show Abstract ▼ 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.	Nov 2017
I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	The cubicon method for concentrating membrane proteins in the cubic mesophase Pikyee Ma , Dietmar Weichert , Luba A. Aleksandrov , Timothy J. Jensen , John R. Riordan , Xiangyu Liu , Brian K. Kobilka , Martin Caffrey Nature Protocols 12, 1745 - 1762 DOI: 10.1038/nprot.2017.057 Diamond Proposal Number(s): [11890, 10357, 9406] Show Abstract ▼ 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.	Aug 2017
I24-Microfocus Macromolecular Crystallography	Structural insights into the mechanism of the membrane integral N-acyltransferase step in bacterial lipoprotein synthesis 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 Nature Communications 8 DOI: 10.1038/ncomms15952 Diamond Proposal Number(s): [11890, 12710] Open Access Show Abstract ▼ 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.	Jul 2017
I24-Microfocus Macromolecular Crystallography	Structural basis of lipoprotein signal peptidase II action and inhibition by the antibiotic globomycin Lutz Vogeley , Toufic El Arnaout , Jonathan Bailey , Phillip J. Stansfeld , Coiiín Boland , Martin Caffrey Science 351 (6275), 876 - 880 DOI: 10.1126/science.aad3747 Show Abstract ▼ 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.	Feb 2016

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