ORCID Profile
0000-0001-6869-4414
Current Organisation
University of Leeds
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Publisher: Elsevier BV
Date: 02-2001
DOI: 10.1016/S0020-7519(01)00111-4
Abstract: Fab I, enoyl acyl carrier protein reductase (ENR), is an enzyme used in fatty acid synthesis. It is a single chain polypeptide in plants, bacteria, and mycobacteria, but is part of a complex polypeptide in animals and fungi. Certain other enzymes in fatty acid synthesis in apicomplexan parasites appear to have multiple forms, homologous to either a plastid, plant-like single chain enzyme or more like the animal complex polypeptide chain. We identified a plant-like Fab I in Plasmodium falciparum and modelled the structure on the Brassica napus and Escherichia coli structures, alone and complexed to triclosan (5-chloro-2-[2,4 dichlorophenoxy] phenol]), which confirmed all the requisite features of an ENR and its interactions with triclosan. Like the remarkable effect of triclosan on a wide variety of bacteria, this compound markedly inhibits growth and survival of the apicomplexan parasites P. falciparum and Toxoplasma gondii at low (i.e. IC50 congruent with150-2000 and 62 ng/ml, respectively) concentrations. Discovery and characterisation of an apicomplexan Fab I and discovery of triclosan as lead compound provide means to rationally design novel inhibitory compounds.
Publisher: Springer Science and Business Media LLC
Date: 17-05-2021
DOI: 10.1038/S41467-021-23151-7
Abstract: During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process in yeast, endocytic coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagination. Here, we present a cryo-EM structure of a 16-mer complex of the ANTH and ENTH membrane-binding domains from Sla2 and Ent1 bound to PIP 2 that constitutes the anchor to the plasma membrane. Detailed in vitro and in vivo mutagenesis of the complex interfaces delineate the key interactions for complex formation and deficient cell growth phenotypes demonstrate its biological relevance. A hetero-tetrameric unit binds PIP 2 molecules at the ANTH-ENTH interfaces and can form larger assemblies to contribute to membrane remodeling. Finally, a time-resolved small-angle X-ray scattering study of the interaction of these adaptor domains in vitro suggests that ANTH and ENTH domains have evolved to achieve a fast subsecond timescale assembly in the presence of PIP 2 and do not require further proteins to form a stable complex. Together, these findings provide a molecular understanding of an essential piece in the molecular puzzle of clathrin-coated endocytic sites.
Publisher: Elsevier BV
Date: 05-2020
Publisher: American Chemical Society (ACS)
Date: 13-12-2013
DOI: 10.1021/BI400945Y
Publisher: Cold Spring Harbor Laboratory
Date: 26-09-2019
DOI: 10.1101/779975
Abstract: Several types of drug-resistant epileptic encephalopathies of infancy have been associated with mutations in the KCNT1 gene, which encodes the sodium-activated potassium channel subunit K Na 1.1. These mutations are commonly gain-of-function, increasing channel activity, therefore inhibition by drugs is proposed as a stratified approach to treat disorders. To date, quinidine therapy has been trialled with several patients, but mostly with unsuccessful outcomes, which has been linked to its low potency and lack of specificity. Here we describe the use of a cryo-electron microscopy-derived K Na 1.1 structure and mutational analysis to identify the quinidine biding site and identified novel inhibitors that target this site using computational methods. We describe six compounds that inhibit K Na 1.1 channels with low- and sub-micromolar potencies, likely through binding in the intracellular pore vestibule. In preliminary hERG inhibition and cytotoxicity assays, two compounds showed little effect. These compounds may provide starting points for the development of novel pharmacophores for K Na 1.1 inhibition, with the view to treating KCNT1 -associated epilepsy and, with their potencies higher than quinidine, could become key tool compounds to further study this channel. Furthermore, this study illustrates the potential for utilising cryo-electron microscopy in ion channel drug discovery.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 02-08-2019
Abstract: qNORs that catalyze the reduction of nitric oxide to nitrous oxide are dimeric and obtain their protons from cytoplasmic end.
Publisher: International Union of Crystallography (IUCr)
Date: 18-05-2018
DOI: 10.1107/S2059798318006496
Abstract: Cryo-electron microscopy (cryo-EM) can now be used to determine high-resolution structural information on a erse range of biological specimens. Recent advances have been driven primarily by developments in microscopes and detectors, and through advances in image-processing software. However, for many single-particle cryo-EM projects, major bottlenecks currently remain at the s le-preparation stage obtaining cryo-EM grids of sufficient quality for high-resolution single-particle analysis can require the careful optimization of many variables. Common hurdles to overcome include problems associated with the s le itself (buffer components, labile complexes), s le distribution (obtaining the correct concentration, affinity for the support film), preferred orientation, and poor reproducibility of the grid-making process within and between batches. This review outlines a number of methodologies used within the electron-microscopy community to address these challenges, providing a range of approaches which may aid in obtaining optimal grids for high-resolution data collection.
Publisher: International Union of Crystallography (IUCr)
Date: 12-04-2006
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Stephen Muench.