10 results on '"Lang EJM"'
Search Results
2. Multiscale Workflow for Modeling Ligand Complexes of Zinc Metalloproteins.
- Author
-
Yang Z, Twidale RM, Gervasoni S, Suardíaz R, Colenso CK, Lang EJM, Spencer J, and Mulholland AJ
- Subjects
- Ligands, Quantum Theory, Workflow, Zinc, Metalloproteins
- Abstract
Zinc metalloproteins are ubiquitous, with protein zinc centers of structural and functional importance, involved in interactions with ligands and substrates and often of pharmacological interest. Biomolecular simulations are increasingly prominent in investigations of protein structure, dynamics, ligand interactions, and catalysis, but zinc poses a particular challenge, in part because of its versatile, flexible coordination. A computational workflow generating reliable models of ligand complexes of biological zinc centers would find broad application. Here, we evaluate the ability of alternative treatments, using (nonbonded) molecular mechanics (MM) and quantum mechanics/molecular mechanics (QM/MM) at semiempirical (DFTB3) and density functional theory (DFT) levels of theory, to describe the zinc centers of ligand complexes of six metalloenzyme systems differing in coordination geometries, zinc stoichiometries (mono- and dinuclear), and the nature of interacting groups (specifically the presence of zinc-sulfur interactions). MM molecular dynamics (MD) simulations can overfavor octahedral geometries, introducing additional water molecules to the zinc coordination shell, but this can be rectified by subsequent semiempirical (DFTB3) QM/MM MD simulations. B3LYP/MM geometry optimization further improved the accuracy of the description of coordination distances, with the overall effectiveness of the approach depending upon factors, including the presence of zinc-sulfur interactions that are less well described by semiempirical methods. We describe a workflow comprising QM/MM MD using DFTB3 followed by QM/MM geometry optimization using DFT (e.g., B3LYP) that well describes our set of zinc metalloenzyme complexes and is likely to be suitable for creating accurate models of zinc protein complexes when structural information is more limited.
- Published
- 2021
- Full Text
- View/download PDF
3. Constructing ion channels from water-soluble α-helical barrels.
- Author
-
Scott AJ, Niitsu A, Kratochvil HT, Lang EJM, Sengel JT, Dawson WM, Mahendran KR, Mravic M, Thomson AR, Brady RL, Liu L, Mulholland AJ, Bayley H, DeGrado WF, Wallace MI, and Woolfson DN
- Subjects
- Amino Acid Sequence, Molecular Dynamics Simulation, Protein Conformation, alpha-Helical, Protein Engineering, Solubility, Water chemistry, Ion Channels chemistry, Lipid Bilayers chemistry, Peptides chemistry
- Abstract
The design of peptides that assemble in membranes to form functional ion channels is challenging. Specifically, hydrophobic interactions must be designed between the peptides and at the peptide-lipid interfaces simultaneously. Here, we take a multi-step approach towards this problem. First, we use rational de novo design to generate water-soluble α-helical barrels with polar interiors, and confirm their structures using high-resolution X-ray crystallography. These α-helical barrels have water-filled lumens like those of transmembrane channels. Next, we modify the sequences to facilitate their insertion into lipid bilayers. Single-channel electrical recordings and fluorescent imaging of the peptides in membranes show monodisperse, cation-selective channels of unitary conductance. Surprisingly, however, an X-ray structure solved from the lipidic cubic phase for one peptide reveals an alternative state with tightly packed helices and a constricted channel. To reconcile these observations, we perform computational analyses to compare the properties of possible different states of the peptide.
- Published
- 2021
- Full Text
- View/download PDF
4. Structural resolution of switchable states of a de novo peptide assembly.
- Author
-
Dawson WM, Lang EJM, Rhys GG, Shelley KL, Williams C, Brady RL, Crump MP, Mulholland AJ, and Woolfson DN
- Subjects
- Crystallography, X-Ray, Hydrophobic and Hydrophilic Interactions, Molecular Dynamics Simulation, Protein Conformation, Protein Conformation, alpha-Helical, Protein Engineering, Proteins chemistry, Solvents, Peptides chemistry
- Abstract
De novo protein design is advancing rapidly. However, most designs are for single states. Here we report a de novo designed peptide that forms multiple α-helical-bundle states that are accessible and interconvertible under the same conditions. Usually in such designs amphipathic α helices associate to form compact structures with consolidated hydrophobic cores. However, recent rational and computational designs have delivered open α-helical barrels with functionalisable cavities. By placing glycine judiciously in the helical interfaces of an α-helical barrel, we obtain both open and compact states in a single protein crystal. Molecular dynamics simulations indicate a free-energy landscape with multiple and interconverting states. Together, these findings suggest a frustrated system in which steric interactions that maintain the open barrel and the hydrophobic effect that drives complete collapse are traded-off. Indeed, addition of a hydrophobic co-solvent that can bind within the barrel affects the switch between the states both in silico and experimentally.
- Published
- 2021
- Full Text
- View/download PDF
5. Resistance to the "last resort" antibiotic colistin: a single-zinc mechanism for phosphointermediate formation in MCR enzymes.
- Author
-
Lythell E, Suardíaz R, Hinchliffe P, Hanpaibool C, Visitsatthawong S, Oliveira ASF, Lang EJM, Surawatanawong P, Lee VS, Rungrotmongkol T, Fey N, Spencer J, and Mulholland AJ
- Subjects
- Ethanolamines chemistry, Lipid A chemistry, Molecular Dynamics Simulation, Alkaline Phosphatase chemistry, Anti-Bacterial Agents chemistry, Bacterial Proteins chemistry, Colistin chemistry, Drug Resistance, Bacterial, Zinc chemistry
- Abstract
MCR (mobile colistin resistance) enzymes catalyse phosphoethanolamine (PEA) addition to bacterial lipid A, threatening the "last-resort" antibiotic colistin. Molecular dynamics and density functional theory simulations indicate that monozinc MCR supports PEA transfer to the Thr285 acceptor, positioning MCR as a mono- rather than multinuclear member of the alkaline phosphatase superfamily.
- Published
- 2020
- Full Text
- View/download PDF
6. Domain cross-talk within a bifunctional enzyme provides catalytic and allosteric functionality in the biosynthesis of aromatic amino acids.
- Author
-
Bai Y, Lang EJM, Nazmi AR, and Parker EJ
- Subjects
- Alkyl and Aryl Transferases genetics, Alkyl and Aryl Transferases metabolism, Allosteric Regulation, Amino Acids, Aromatic chemistry, Amino Acids, Aromatic genetics, Bacterial Proteins genetics, Bacterial Proteins metabolism, Catalysis, Crystallography, X-Ray, Prevotella nigrescens genetics, Protein Domains, Scattering, Small Angle, X-Ray Diffraction, Alkyl and Aryl Transferases chemistry, Amino Acids, Aromatic biosynthesis, Bacterial Proteins chemistry, Prevotella nigrescens enzymology
- Abstract
Because of their special organization, multifunctional enzymes play crucial roles in improving the performance of metabolic pathways. For example, the bacterium Prevotella nigrescens contains a distinctive bifunctional protein comprising a 3-deoxy-d- arabino heptulosonate-7-phosphate synthase (DAH7PS), catalyzing the first reaction of the biosynthetic pathway of aromatic amino acids, and a chorismate mutase (CM), functioning at a branch of this pathway leading to the synthesis of tyrosine and phenylalanine. In this study, we characterized this P. nigrescens enzyme and found that its two catalytic activities exhibit substantial hetero-interdependence and that the separation of its two distinct catalytic domains results in a dramatic loss of both DAH7PS and CM activities. The protein displayed a unique dimeric assembly, with dimerization solely via the CM domain. Small angle X-ray scattering (SAXS)-based structural analysis of this protein indicated a DAH7PS-CM hetero-interaction between the DAH7PS and CM domains, unlike the homo-association between DAH7PS domains normally observed for other DAH7PS proteins. This hetero-interaction provides a structural basis for the functional interdependence between the two domains observed here. Moreover, we observed that DAH7PS is allosterically inhibited by prephenate, the product of the CM-catalyzed reaction. This allostery was accompanied by a striking conformational change as observed by SAXS, implying that altering the hetero-domain interaction underpins the allosteric inhibition. We conclude that for this C-terminal CM-linked DAH7PS, catalytic function and allosteric regulation appear to be delivered by a common mechanism, revealing a distinct and efficient evolutionary strategy to utilize the functional advantages of a bifunctional enzyme., (© 2019 Bai et al.)
- Published
- 2019
- Full Text
- View/download PDF
7. Structural and functional characterisation of the entry point to pyocyanin biosynthesis in Pseudomonas aeruginosa defines a new 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase subclass.
- Author
-
Sterritt OW, Lang EJM, Kessans SA, Ryan TM, Demeler B, Jameson GB, and Parker EJ
- Subjects
- 3-Deoxy-7-Phosphoheptulonate Synthase genetics, 3-Deoxy-7-Phosphoheptulonate Synthase metabolism, Amino Acid Sequence genetics, Binding Sites, Crystallography, X-Ray, Phosphates metabolism, Protein Binding, Pseudomonas aeruginosa chemistry, Pseudomonas aeruginosa genetics, Pyocyanine chemistry, Pyocyanine genetics, Shikimic Acid chemistry, Shikimic Acid metabolism, 3-Deoxy-7-Phosphoheptulonate Synthase chemistry, Allosteric Regulation genetics, Pseudomonas aeruginosa enzymology, Pyocyanine biosynthesis
- Abstract
In Pseudomonas aeruginosa ( Pae ), the shikimate pathway end product, chorismate, serves as the last common precursor for the biosynthesis of both primary aromatic metabolites, including phenylalanine, tyrosine and tryptophan, and secondary aromatic metabolites, including phenazine-1-carboxylic acid (PCA) and pyocyanin (PYO). The enzyme 3-deoxy-d- arabino -heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, en route to chorismate. P. aeruginosa expresses multiple, distinct DAH7PSs that are associated with either primary or secondary aromatic compound biosynthesis. Here we report the structure of a type II DAH7PS, encoded by phzC as part of the duplicated phenazine biosynthetic cluster, from P. aeruginosa (PAO1) revealing for the first time the structure of a type II DAH7PS involved in secondary metabolism. The omission of the structural elements α
2a and α2b , relative to other characterised type II DAH7PSs, leads to the formation of an alternative, dimeric, solution-state structure for this type II DAH7PS with an oligomeric interface that has not previously been characterised and that does not facilitate the formation of aromatic amino acid allosteric binding sites. The sequence similarity and, in particular, the common N-terminal extension suggest a common origin for the type II DAH7PSs from P. aeruginosa. The results described in the present study support an expanded classification of the type II DAH7PSs as type IIA and type IIB based on sequence characteristics, structure and function of the resultant proteins, and on defined physiological roles within primary or secondary metabolism., (© 2018 The Author(s).)- Published
- 2018
- Full Text
- View/download PDF
8. Maintaining and breaking symmetry in homomeric coiled-coil assemblies.
- Author
-
Rhys GG, Wood CW, Lang EJM, Mulholland AJ, Brady RL, Thomson AR, and Woolfson DN
- Subjects
- Amino Acid Sequence, Chromatography, High Pressure Liquid, Crystallography, X-Ray, Peptides chemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Water chemistry, Models, Molecular, Protein Folding, Protein Multimerization, Protein Structure, Secondary
- Abstract
In coiled-coil (CC) protein structures α-helices wrap around one another to form rope-like assemblies. Most natural and designed CCs have two-four helices and cyclic (C
n ) or dihedral (Dn ) symmetry. Increasingly, CCs with five or more helices are being reported. A subset of these higher-order CCs is of interest as they have accessible central channels that can be functionalised; they are α-helical barrels. These extended cavities are surprising given the drive to maximise buried hydrophobic surfaces during protein folding and assembly in water. Here, we show that α-helical barrels can be maintained by the strategic placement of β-branched aliphatic residues lining the lumen. Otherwise, the structures collapse or adjust to give more-complex multi-helix assemblies without Cn or Dn symmetry. Nonetheless, the structural hallmark of CCs-namely, knobs-into-holes packing of side chains between helices-is maintained leading to classes of CCs hitherto unobserved in nature or accessed by design.- Published
- 2018
- Full Text
- View/download PDF
9. De Novo-Designed α-Helical Barrels as Receptors for Small Molecules.
- Author
-
Thomas F, Dawson WM, Lang EJM, Burton AJ, Bartlett GJ, Rhys GG, Mulholland AJ, and Woolfson DN
- Subjects
- Amino Acid Sequence, Diphenylhexatriene chemistry, Hydrophobic and Hydrophilic Interactions, Molecular Dynamics Simulation, Protein Conformation, alpha-Helical, Protein Structure, Secondary, Peptides chemistry
- Abstract
We describe de novo-designed α-helical barrels (αHBs) that bind and discriminate between lipophilic biologically active molecules. αHBs have five or more α-helices arranged around central hydrophobic channels the diameters of which scale with oligomer state. We show that pentameric, hexameric, and heptameric αHBs bind the environmentally sensitive dye 1,6-diphenylhexatriene (DPH) in the micromolar range and fluoresce. Displacement of the dye is used to report the binding of nonfluorescent molecules: palmitic acid and retinol bind to all three αHBs with submicromolar inhibitor constants; farnesol binds the hexamer and heptamer; but β-carotene binds only the heptamer. A co-crystal structure of the hexamer with farnesol reveals oriented binding in the center of the hydrophobic channel. Charged side chains engineered into the lumen of the heptamer facilitate binding of polar ligands: a glutamate variant binds a cationic variant of DPH, and introducing lysine allows binding of the biosynthetically important farnesol diphosphate.
- Published
- 2018
- Full Text
- View/download PDF
10. Interdomain Conformational Changes Provide Allosteric Regulation en Route to Chorismate.
- Author
-
Nazmi AR, Lang EJM, Bai Y, Allison TM, Othman MH, Panjikar S, Arcus VL, and Parker EJ
- Subjects
- 3-Deoxy-7-Phosphoheptulonate Synthase genetics, Allosteric Regulation, Amino Acids, Aromatic metabolism, Chorismate Mutase genetics, Crystallography, X-Ray, Geobacillus enzymology, Shikimic Acid metabolism, 3-Deoxy-7-Phosphoheptulonate Synthase metabolism, Chorismate Mutase metabolism
- Abstract
Multifunctional proteins play a variety of roles in metabolism. Here, we examine the catalytic function of the combined 3-deoxy-d- arabino heptulosonate-7-phosphate synthase (DAH7PS) and chorismate mutase (CM) from Geobacillus sp. DAH7PS operates at the start of the biosynthetic pathway for aromatic metabolites, whereas CM operates in a dedicated branch of the pathway for the biosynthesis of amino acids tyrosine and phenylalanine. In line with sequence predictions, the two catalytic functions are located in distinct domains, and these two activities can be separated and retain functionality. For the full-length protein, prephenate, the product of the CM reaction, acts as an allosteric inhibitor for the DAH7PS. The crystal structure of the full-length protein with prephenate bound and the accompanying small angle x-ray scattering data reveal the molecular mechanism of the allostery. Prephenate binding results in the tighter association between the dimeric CM domains and the tetrameric DAH7PS, occluding the active site and therefore disrupting DAH7PS function. Acquisition of a physical gating mechanism to control catalytic function through gene fusion appears to be a general mechanism for providing allostery for this enzyme., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2016
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.