Publications by authors named "Ehmke Pohl"

65 Publications

Going to extremes - a metagenomic journey into the dark matter of life.

FEMS Microbiol Lett 2021 06;368(12)

Matis ohf, Vinlandsleid 12, Reykjavik 113, Iceland.

The Virus-X-Viral Metagenomics for Innovation Value-project was a scientific expedition to explore and exploit uncharted territory of genetic diversity in extreme natural environments such as geothermal hot springs and deep-sea ocean ecosystems. Specifically, the project was set to analyse and exploit viral metagenomes with the ultimate goal of developing new gene products with high innovation value for applications in biotechnology, pharmaceutical, medical, and the life science sectors. Viral gene pool analysis is also essential to obtain fundamental insight into ecosystem dynamics and to investigate how viruses influence the evolution of microbes and multicellular organisms. The Virus-X Consortium, established in 2016, included experts from eight European countries. The unique approach based on high throughput bioinformatics technologies combined with structural and functional studies resulted in the development of a biodiscovery pipeline of significant capacity and scale. The activities within the Virus-X consortium cover the entire range from bioprospecting and methods development in bioinformatics to protein production and characterisation, with the final goal of translating our results into new products for the bioeconomy. The significant impact the consortium made in all of these areas was possible due to the successful cooperation between expert teams that worked together to solve a complex scientific problem using state-of-the-art technologies as well as developing novel tools to explore the virosphere, widely considered as the last great frontier of life.
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http://dx.doi.org/10.1093/femsle/fnab067DOI Listing
June 2021

Structure-functional relationship of cellular retinoic acid-binding proteins I and II interacting with natural and synthetic ligands.

Acta Crystallogr D Struct Biol 2021 Feb 5;77(Pt 2):164-175. Epub 2021 Feb 5.

Department of Chemistry, Durham University, Lower Mountjoy, South Road, Durham DH1 3LE, United Kingdom.

A detailed understanding of the interactions between small-molecule ligands and their proposed binding targets is of the utmost importance for modern drug-development programs. Cellular retinoic acid-binding proteins I and II (CRABPI and CRABPII) facilitate a number of vital retinoid signalling pathways in mammalian cells and offer a gateway to manipulation of signalling that could potentially reduce phenotypes in serious diseases, including cancer and neurodegeneration. Although structurally very similar, the two proteins possess distinctly different biological functions, with their signalling influence being exerted through both genomic and nongenomic pathways. In this article, crystal structures are presented of the L29C mutant of Homo sapiens CRABPI in complex with naturally occurring fatty acids (1.64 Å resolution) and with the synthetic retinoid DC645 (2.41 Å resolution), and of CRABPII in complex with the ligands DC479 (1.80 Å resolution) and DC645 (1.71 Å resolution). DC645 and DC479 are two potential drug compounds identified in a recent synthetic retinoid development program. In particular, DC645 has recently been shown to have disease-modifying capabilities in neurodegenerative disease models by activating both genomic and nongenomic signalling pathways. These co-crystal structures demonstrate a canonical binding behaviour akin to that exhibited with all-trans-retinoic acid and help to explain how the compounds are able to exert an influence on part of the retinoid signalling cascade.
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http://dx.doi.org/10.1107/S2059798320015247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7869897PMC
February 2021

Crystal structure of the GDP-bound GTPase domain of Rab5a from Leishmania donovani.

Acta Crystallogr F Struct Biol Commun 2020 Nov 29;76(Pt 11):544-556. Epub 2020 Oct 29.

Molecular and Structural Biology Division, CSIR - Central Drug Research Institute, Lucknow 226 031, India.

Eukaryotic Rab5s are highly conserved small GTPase-family proteins that are involved in the regulation of early endocytosis. Leishmania donovani Rab5a regulates the sorting of early endosomes that are involved in the uptake of essential nutrients through fluid-phase endocytosis. Here, the 1.80 Å resolution crystal structure of the N-terminal GTPase domain of L. donovani Rab5a in complex with GDP is presented. The crystal structure determination was enabled by the design of specific single-site mutations and two deletions that were made to stabilize the protein for previous NMR studies. The structure of LdRab5a shows the canonical GTPase fold, with a six-stranded central mixed β-sheet surrounded by five α-helices. The positions of the Switch I and Switch II loops confirm an open conformation, as expected in the absence of the γ-phosphate. However, in comparison to other GTP-bound and GDP-bound homologous proteins, the Switch I region traces a unique disposition in LdRab5a. One magnesium ion is bound to the protein at the GTP-binding site. Molecular-dynamics simulations indicate that the GDP-bound structure exhibits higher stability than the apo structure. The GDP-bound LdRab5a structure presented here will aid in efforts to unravel its interactions with its regulators, including the guanine nucleotide-exchange factor, and will lay the foundation for a structure-based search for specific inhibitors.
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http://dx.doi.org/10.1107/S2053230X20013722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605105PMC
November 2020

Preface.

Authors:
Ehmke Pohl

Methods Enzymol 2020 ;637:xix-xx

Department of Chemistry; Department of Bioscience; Biophysical Sciences Institute, Durham University, Durham, United Kingdom.

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http://dx.doi.org/10.1016/S0076-6879(20)30204-4DOI Listing
June 2021

Classical pathways of gene regulation by retinoids.

Methods Enzymol 2020 9;637:151-173. Epub 2020 Apr 9.

Department of Chemistry, Durham University, Durham, United Kingdom.

Retinoic acid receptors were discovered during early studies of the actions and mechanisms of essential vitamins. Vitamin A is metabolized in the body to retinoic acid (RA) which is a key compound in the control of many developmental processes in chordates. These functions are mediated by a subfamily of nuclear receptors, divided into two classes, the retinoic acid receptors (RAR) and the retinoid X receptors (RXR). Each class is encoded by three closely related genes that are located on different chromosomes. The three proteins in each class are designated α, β and γ, respectively. A wealth of structural studies have shown that they all share the same architecture including a DNA-binding domain connected by a flexible linker to the ligand and co-activator binding domain. Retinoic acid incorporation into the ligand-binding domain leads to a conformational change enabling the formation of RAR homodimers or RAR/RXR heterodimers that in turn bind specifically to target DNA sequences. The consensus sequences located on the promotors of regulated genes are known as retinoic acid response elements (RARE). The activated RAR/RXR homodimers recruit co-activators with histone acetylase activity leading to an opening of the chromatin structure and enabling downstream transcription of regulated genes. These canonical pathways describe the control mechanism for the majority of developmental processes mediated by retinoic acid and its derivatives.
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http://dx.doi.org/10.1016/bs.mie.2020.03.008DOI Listing
June 2021

GSP4PDB: a web tool to visualize, search and explore protein-ligand structural patterns.

BMC Bioinformatics 2020 Mar 11;21(Suppl 2):85. Epub 2020 Mar 11.

Department of Chemistry, Durham University, Durham, DH1 3LE, United Kingdom.

Background: In the field of protein engineering and biotechnology, the discovery and characterization of structural patterns is highly relevant as these patterns can give fundamental insights into protein-ligand interaction and protein function. This paper presents GSP4PDB, a bioinformatics web tool that enables the user to visualize, search and explore protein-ligand structural patterns within the entire Protein Data Bank.

Results: We introduce the notion of graph-based structural pattern (GSP) as an abstract model for representing protein-ligand interactions. A GSP is a graph where the nodes represent entities of the protein-ligand complex (amino acids and ligands) and the edges represent structural relationships (e.g. distances ligand - amino acid). The novel feature of GSP4PDB is a simple and intuitive graphical interface where the user can "draw" a GSP and execute its search in a relational database containing the structural data of each PDB entry. The results of the search are displayed using the same graph-based representation of the pattern. The user can further explore and analyse the results using a wide range of filters, or download their related information for external post-processing and analysis.

Conclusions: GSP4PDB is a user-friendly and efficient application to search and discover new patterns of protein-ligand interaction.
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http://dx.doi.org/10.1186/s12859-020-3352-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7068854PMC
March 2020

Obtaining Tertiary Protein Structures by the ab Initio Interpretation of Small Angle X-ray Scattering Data.

J Chem Theory Comput 2020 Mar 18;16(3):1985-2001. Epub 2020 Feb 18.

Department of Biosciences Durham University, Durham DH1 3LE, United Kingdom.

Small angle X-ray scattering (SAXS) is an important tool for investigating the structure of proteins in solution. We present a novel ab initio method representing polypeptide chains as discrete curves used to derive a meaningful three-dimensional model from the primary sequence and SAXS data. High resolution structures were used to generate probability density functions for each common secondary structural element found in proteins, which are used to place realistic restraints on the model curve's geometry. This is coupled with a novel explicit hydration shell model in order to derive physically meaningful three-dimensional models by optimizing against experimental SAXS data. The efficacy of this model is verified on an established benchmark protein set, and then it is used to predict the lysozyme structure using only its primary sequence and SAXS data. The method is used to generate a biologically plausible model of the coiled-coil component of the human synaptonemal complex central element protein.
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http://dx.doi.org/10.1021/acs.jctc.9b01010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145352PMC
March 2020

Crystal structures of the Bacillus subtilis prophage lytic cassette proteins XepA and YomS.

Acta Crystallogr D Struct Biol 2019 Nov 1;75(Pt 11):1028-1039. Epub 2019 Nov 1.

Department of Chemistry, Durham University, South Road, Durham DH1 3LE, England.

As part of the Virus-X Consortium that aims to identify and characterize novel proteins and enzymes from bacteriophages and archaeal viruses, the genes of the putative lytic proteins XepA from Bacillus subtilis prophage PBSX and YomS from prophage SPβ were cloned and the proteins were subsequently produced and functionally characterized. In order to elucidate the role and the molecular mechanism of XepA and YomS, the crystal structures of these proteins were solved at resolutions of 1.9 and 1.3 Å, respectively. XepA consists of two antiparallel β-sandwich domains connected by a 30-amino-acid linker region. A pentamer of this protein adopts a unique dumbbell-shaped architecture consisting of two discs and a central tunnel. YomS (12.9 kDa per monomer), which is less than half the size of XepA (30.3 kDa), shows homology to the C-terminal part of XepA and exhibits a similar pentameric disc arrangement. Each β-sandwich entity resembles the fold of typical cytoplasmic membrane-binding C2 domains. Only XepA exhibits distinct cytotoxic activity in vivo, suggesting that the N-terminal pentameric domain is essential for this biological activity. The biological and structural data presented here suggest that XepA disrupts the proton motive force of the cytoplasmatic membrane, thus supporting cell lysis.
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http://dx.doi.org/10.1107/S2059798319013330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834076PMC
November 2019

A C-terminal CXCL8 peptide based on chemokine-glycosaminoglycan interactions reduces neutrophil adhesion and migration during inflammation.

Immunology 2019 06;157(2):173-184

Applied Immunobiology and Transplantation Research Group, Institute of Cellular Medicine, Medical School, Newcastle University, Newcastle upon Tyne, UK.

Leucocyte recruitment is critical during many acute and chronic inflammatory diseases. Chemokines are key mediators of leucocyte recruitment during the inflammatory response, by signalling through specific chemokine G-protein-coupled receptors (GPCRs). In addition, chemokines interact with cell-surface glycosaminoglycans (GAGs) to generate a chemotactic gradient. The chemokine interleukin-8/CXCL8, a prototypical neutrophil chemoattractant, is characterized by a long, highly positively charged GAG-binding C-terminal region, absent in most other chemokines. To examine whether the CXCL8 C-terminal peptide has a modulatory role in GAG binding during neutrophil recruitment, we synthesized the wild-type CXCL8 C-terminal [CXCL8 (54-72)] (Peptide 1), a peptide with a substitution of glutamic acid (E) 70 with lysine (K) (Peptide 2) to increase positive charge; and also, a scrambled sequence peptide (Peptide 3). Surface plasmon resonance showed that Peptide 1, corresponding to the core CXCL8 GAG-binding region, binds to GAG but Peptide 2 binding was detected at lower concentrations. In the absence of cellular GAG, the peptides did not affect CXCL8-induced calcium signalling or neutrophil chemotaxis along a diffusion gradient, suggesting no effect on GPCR binding. All peptides equally inhibited neutrophil adhesion to endothelial cells under physiological flow conditions. Peptide 2, with its greater positive charge and binding to polyanionic GAG, inhibited CXCL8-induced neutrophil transendothelial migration. Our studies suggest that the E70K CXCL8 peptide, may serve as a lead molecule for further development of therapeutic inhibitors of neutrophil-mediated inflammation based on modulation of chemokine-GAG binding.
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http://dx.doi.org/10.1111/imm.13063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7662424PMC
June 2019

Relative Binding Energies Predict Crystallographic Binding Modes of Ethionamide Booster Lead Compounds.

J Phys Chem Lett 2019 May 23;10(9):2244-2249. Epub 2019 Apr 23.

Department of Chemistry , Durham University , South Road , Durham DH1 3LE , U.K.

Transcriptional repressor EthR from Mycobacterium tuberculosis is a valuable target for antibiotic booster drugs. We previously reported a virtual screening campaign to identify EthR inhibitors for development. Two ligand binding orientations were often proposed, though only the top scoring pose was utilized for filtering of the large data set. We obtained biophysically validated hits, some of which yielded complex crystal structures. In some cases, the crystallized binding mode and top scoring mode agree, while for others an alternate ligand binding orientation was found. In this contribution, we combine rigid docking, molecular dynamics simulations, and the linear interaction energy method to calculate binding free energies and derive relative binding energies for a number of EthR inhibitors in both modes. This strategy allowed us to correctly predict the most favorable orientation. Therefore, this widely applicable approach will be suitable to triage multiple binding modes within EthR and other potential drug targets with similar characteristics.
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http://dx.doi.org/10.1021/acs.jpclett.9b00741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503467PMC
May 2019

A unique dynamin-related protein is essential for mitochondrial fission in Toxoplasma gondii.

PLoS Pathog 2019 04 4;15(4):e1007512. Epub 2019 Apr 4.

Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity & Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, United Kingdom.

The single mitochondrion of apicomplexan protozoa is thought to be critical for all stages of the life cycle, and is a validated drug target against these important human and veterinary parasites. In contrast to other eukaryotes, replication of the mitochondrion is tightly linked to the cell cycle. A key step in mitochondrial segregation is the fission event, which in many eukaryotes occurs by the action of dynamins constricting the outer membrane of the mitochondria from the cytosolic face. To date, none of the components of the apicomplexan fission machinery have been identified and validated. We identify here a highly divergent, dynamin-related protein (TgDrpC), conserved in apicomplexans as essential for mitochondrial biogenesis and potentially for fission in Toxoplasma gondii. We show that TgDrpC is found adjacent to the mitochondrion, and is localised both at its periphery and at its basal part, where fission is expected to occur. We demonstrate that depletion or dominant negative expression of TgDrpC results in interconnected mitochondria and ultimately in drastic changes in mitochondrial morphology, as well as in parasite death. Intriguingly, we find that the canonical adaptor TgFis1 is not required for mitochondrial fission. The identification of an Apicomplexa-specific enzyme required for mitochondrial biogenesis and essential for parasite growth highlights parasite adaptation. This work paves the way for future drug development targeting TgDrpC, and for the analysis of additional partners involved in this crucial step of apicomplexan multiplication.
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http://dx.doi.org/10.1371/journal.ppat.1007512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6448817PMC
April 2019

How to Stabilize Protein: Stability Screens for Thermal Shift Assays and Nano Differential Scanning Fluorimetry in the Virus-X Project.

J Vis Exp 2019 02 11(144). Epub 2019 Feb 11.

Department of Biosciences, Durham University; Department of Chemistry, Durham University;

The Horizon2020 Virus-X project was established in 2015 to explore the virosphere of selected extreme biotopes and discover novel viral proteins. To evaluate the potential biotechnical value of these proteins, the analysis of protein structures and functions is a central challenge in this program. The stability of protein sample is essential to provide meaningful assay results and increase the crystallizability of the targets. The thermal shift assay (TSA), a fluorescence-based technique, is established as a popular method for optimizing the conditions for protein stability in high-throughput. In TSAs, the employed fluorophores are extrinsic, environmentally-sensitive dyes. An alternative, similar technique is nano differential scanning fluorimetry (nanoDSF), which relies on protein native fluorescence. We present here a novel osmolyte screen, a 96-condition screen of organic additives designed to guide crystallization trials through preliminary TSA experiments. Together with previously-developed pH and salt screens, the set of three screens provides a comprehensive analysis of protein stability in a wide range of buffer systems and additives. The utility of the screens is demonstrated in the TSA and nanoDSF analysis of lysozyme and Protein X, a target protein of the Virus-X project.
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http://dx.doi.org/10.3791/58666DOI Listing
February 2019

Fluorescent Retinoic Acid Analogues as Probes for Biochemical and Intracellular Characterization of Retinoid Signaling Pathways.

ACS Chem Biol 2019 03 13;14(3):369-377. Epub 2019 Feb 13.

Department of Biosciences , Durham University , South Road , Durham DH1 3LE , U.K.

Retinoids, such as all- trans-retinoic acid (ATRA), are endogenous signaling molecules derived from vitamin A that influence a variety of cellular processes through mediation of transcription events in the cell nucleus. Because of these wide-ranging and powerful biological activities, retinoids have emerged as therapeutic candidates of enormous potential. However, their use has been limited, to date, due to a lack of understanding of the complex and intricate signaling pathways that they control. We have designed and synthesized a family of synthetic retinoids that exhibit strong, intrinsic, solvatochromatic fluorescence as multifunctional tools to interrogate these important biological activities. We utilized the unique photophysical characteristics of these fluorescent retinoids to develop a novel in vitro fluorometric binding assay to characterize and quantify their binding to their cellular targets, including cellular retinoid binding protein II (CRABPII). The dihydroquinoline retinoid, DC360, exhibited particularly strong binding ( K = 34.0 ± 2.5 nM), and we further used X-ray crystallography to determine the structure of the DC360-CRABPII complex to 1.8 Å, which showed that DC360 occupies the known hydrophobic retinoid binding pocket. Finally, we used confocal fluorescence microscopy to image the cellular behavior of the compounds in cultured human epithelial cells, highlighting a fascinating nuclear localization, and used RNA sequencing to confirm that the compounds regulate cellular processes similar to those of ATRA. We anticipate that the unique properties of these fluorescent retinoids can now be used to cast new light on the vital and highly complex retinoid signaling pathway.
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http://dx.doi.org/10.1021/acschembio.8b00916DOI Listing
March 2019

Novel Fluorescence Competition Assay for Retinoic Acid Binding Proteins.

ACS Med Chem Lett 2018 Dec 9;9(12):1297-1300. Epub 2018 Nov 9.

Department of Chemistry, Durham University, Science Laboratories, South Road, Durham, DH1 3LE, U.K.

Vitamin A derived retinoid compounds have multiple, powerful roles in the cellular growth and development cycle and, as a result, have attracted significant attention from both academic and pharmaceutical research in developing and characterizing synthetic retinoid analogues. Simplifying the hit development workflow for retinoid signaling will improve options available for tackling related pathologies, including tumor growth and neurodegeneration. Here, we present a novel assay that employs an intrinsically fluorescent synthetic retinoid, DC271, which allows direct measurement of the binding of nonlabeled compounds to relevant proteins. The method allows for straightforward initial measurement of binding using existing compound libraries and is followed by calculation of binding constants using a dilution series of plausible hits. The ease of use, high throughput format, and measurement of both qualitative and quantitative binding offer a new direction for retinoid-related pharmacological development.
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http://dx.doi.org/10.1021/acsmedchemlett.8b00420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6295855PMC
December 2018

Functional Analyses of a Putative, Membrane-Bound, Peroxisomal Protein Import Mechanism from the Apicomplexan Protozoan .

Genes (Basel) 2018 Aug 29;9(9). Epub 2018 Aug 29.

Department of Biosciences and Centre for Global Infectious Disease, Stockton Road, Durham DH1 3LE, UK.

Peroxisomes are central to eukaryotic metabolism, including the oxidation of fatty acids-which subsequently provide an important source of metabolic energy-and in the biosynthesis of cholesterol and plasmalogens. However, the presence and nature of peroxisomes in the parasitic apicomplexan protozoa remains controversial. A survey of the available genomes revealed that genes encoding peroxisome biogenesis factors, so-called peroxins (Pex), are only present in a subset of these parasites, the coccidia. The basic principle of peroxisomal protein import is evolutionarily conserved, proteins harbouring a peroxisomal-targeting signal 1 (PTS1) interact in the cytosol with the shuttling receptor Pex5 and are then imported into the peroxisome via the membrane-bound protein complex formed by Pex13 and Pex14. Surprisingly, whilst Pex5 is clearly identifiable, Pex13 and, perhaps, Pex14 are apparently absent from the coccidian genomes. To investigate the functionality of the PTS1 import mechanism in these parasites, expression of Pex5 from the model coccidian was shown to rescue the import defect of Pex5-deleted . In support of these data, green fluorescent protein (GFP) bearing the enhanced (e)PTS1 known to efficiently localise to peroxisomes in yeast, localised to peroxisome-like bodies when expressed in Furthermore, the PTS1-binding domain of Pex5 and a PTS1 ligand from the putatively peroxisome-localised sterol carrier protein (SCP2) were shown to interact in vitro. Taken together, these data demonstrate that the Pex5⁻PTS1 interaction is functional in the coccidia and indicate that a nonconventional peroxisomal import mechanism may operate in the absence of Pex13 and Pex14.
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http://dx.doi.org/10.3390/genes9090434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6162456PMC
August 2018

Probing biological activity through structural modelling of ligand-receptor interactions of 2,4-disubstituted thiazole retinoids.

Bioorg Med Chem 2018 05 10;26(8):1560-1572. Epub 2018 Feb 10.

Department of Biosciences, Durham University, South Road, Durham DH1 3LE, UK.

Retinoids, such as all-trans-retinoic acid (ATRA), regulate cellular differentiation and signalling pathways in chordates by binding to nuclear retinoic acid receptors (RARα/β/γ). Polar interactions between receptor and ligand are important for binding and facilitating the non-polar interactions and conformational changes necessary for RAR-mediated transcriptional regulation. The constraints on activity and RAR-type specificity with respect to the structural link between the polar and non-polar functions of synthetic retinoids are poorly understood. To address this, predictions from in silico ligand-RAR docking calculations and molecular dynamics simulations for a small library of stable, synthetic retinoids (designated GZ series) containing a central thiazole linker structure and different hydrophobic region substituents, were tested using a ligand binding assay and a range of cellular biological assays. The docking analysis showed that these thiazole-containing retinoids were well suited to the binding pocket of RARα, particularly via a favorable hydrogen bonding interaction between the thiazole and Ser232 of RARα. A bulky hydrophobic region (i.e., present in compounds GZ23 and GZ25) was important for interaction with the RAR binding pockets. Ligand binding assays generally reflected the findings from in silico docking, and showed that GZ25 was a particularly strongly binding ligand for RARα/β. GZ25 also exhibited higher activity as an inducer of neuronal differentiation than ATRA and other GZ derivatives. These data demonstrate that GZ25 is a stable synthetic retinoid with improved activity which efficiently regulates neuronal differentiation and help to define the key structural requirements for retinoid activity enabling the design and development of the next generation of more active, selective synthetic retinoids as potential therapeutic regulators of neurogenesis.
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http://dx.doi.org/10.1016/j.bmc.2018.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5933457PMC
May 2018

The calcium-dependent protein kinase 1 from Toxoplasma gondii as target for structure-based drug design.

Parasitology 2018 02 1;145(2):210-218. Epub 2017 Dec 1.

Department of Biosciences,Durham University,Lower Mountjoy Durham DH1 3LE,UK.

The apicomplexan protozoan parasites include the causative agents of animal and human diseases ranging from malaria (Plasmodium spp.) to toxoplasmosis (Toxoplasma gondii). The complex life cycle of T. gondii is regulated by a unique family of calcium-dependent protein kinases (CDPKs) that have become the target of intensive efforts to develop new therapeutics. In this review, we will summarize structure-based strategies, recent successes and future directions in the pursuit of specific and selective inhibitors of T. gondii CDPK1.
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http://dx.doi.org/10.1017/S0031182017001901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964469PMC
February 2018

New active leads for tuberculosis booster drugs by structure-based drug discovery.

Org Biomol Chem 2017 Dec;15(48):10245-10255

Department of Chemistry, Durham University, South Road, Durham DH1 3LE, UK.

The transcriptional repressor EthR from Mycobacterium tuberculosis, a member of the TetR family of prokaryotic homo-dimeric transcription factors, controls the expression of the mycobacterial mono-oxygenase EthA. EthA is responsible for the bio-activation of the second-line tuberculosis pro-drug ethionamide, and consequently EthR inhibitors boost drug efficacy. Here, we present a comprehensive in silico structure-based screening protocol that led to the identification of a number of novel scaffolds of EthR inhibitors in subsequent biophysical screening by thermal shift assay. Growth inhibition assays demonstrated that five of the twenty biophysical hits were capable of boosting ethionamide activity in vitro, with the best novel scaffold displaying an EC of 34 μM. In addition, the co-crystal structures of EthR with four new ligands at resolution ranging from 2.1 to 1.4 Å confirm the binding and inactivation mode, and will enable future lead development.
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http://dx.doi.org/10.1039/c7ob00910kDOI Listing
December 2017

Air/Liquid Interfacial Nanoassembly of Molecular Building Blocks into Preferentially Oriented Porous Organic Nanosheet Crystals via Hydrogen Bonding.

ACS Nano 2017 11 24;11(11):10875-10882. Epub 2017 Oct 24.

European Synchrotron Radiation Facility (ESRF) , 38000 Grenoble, France.

Nanosheets with highly regulated nanopores are ultimately thin functional materials for diverse applications including molecular separation and detection, catalysis, and energy conversion and storage. However, their availability has hitherto been restricted to layered parent materials, covalently bonded sheets, which are layered via relatively weak electrostatic interactions. Here, we report a rational bottom-up methodology that enables nanosheet creation beyond the layered systems. We employ the air/liquid interface to assemble a triphenylbenzene derivative into perfectly oriented highly crystalline noncovalent-bonded organic nanosheets under ambient conditions. Each molecular building unit connects laterally by hydrogen bonding, endowing the nanosheets with size- and position-regulated permanent nanoporosity, as established by in situ synchrotron X-ray surface crystallography and gas sorption measurements. Notably, the nanosheets are constructed specifically by interfacial synthesis, which suppresses the intrinsic complex interpenetrated structure of the bulk crystal. Moreover, they possess exceptional long-term and thermal stability and are easily transferrable to numerous substrates without loss of structural integrity. Our work shows the power of interfacial synthesis using a suitably chosen molecular component to create two-dimensional (2D) nanoassemblies not accessible by conventional bulk crystal exfoliation techniques.
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http://dx.doi.org/10.1021/acsnano.7b04447DOI Listing
November 2017

Functional and phylogenetic evidence of a bacterial origin for the first enzyme in sphingolipid biosynthesis in a phylum of eukaryotic protozoan parasites.

J Biol Chem 2017 07 2;292(29):12208-12219. Epub 2017 Jun 2.

Department of Biosciences, Durham University, Durham DH1 3LE, United Kingdom. Electronic address:

is an obligate, intracellular eukaryotic apicomplexan protozoan parasite that can cause fetal damage and abortion in both animals and humans. Sphingolipids are essential and ubiquitous components of eukaryotic membranes that are both synthesized and scavenged by the Apicomplexa. Here we report the identification, isolation, and analyses of the serine palmitoyltransferase, an enzyme catalyzing the first and rate-limiting step in sphingolipid biosynthesis: the condensation of serine and palmitoyl-CoA. In all eukaryotes analyzed to date, serine palmitoyltransferase is a highly conserved heterodimeric enzyme complex. However, biochemical and structural analyses demonstrated the apicomplexan orthologue to be a functional, homodimeric serine palmitoyltransferase localized to the endoplasmic reticulum. Furthermore, phylogenetic studies indicated that it was evolutionarily related to the prokaryotic serine palmitoyltransferase, identified in the Sphingomonadaceae as a soluble homodimeric enzyme. Therefore this enzyme, conserved throughout the Apicomplexa, is likely to have been obtained via lateral gene transfer from a prokaryote.
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http://dx.doi.org/10.1074/jbc.M117.792374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519370PMC
July 2017

A tight tunable range for Ni(II) sensing and buffering in cells.

Nat Chem Biol 2017 04 6;13(4):409-414. Epub 2017 Feb 6.

Department of Biosciences, Durham University, DH1 3LE, UK.

The metal affinities of metal-sensing transcriptional regulators co-vary with cellular metal concentrations over more than 12 orders of magnitude. To understand the cause of this relationship, we determined the structure of the Ni(II) sensor InrS and then created cyanobacteria (Synechocystis PCC 6803) in which transcription of genes encoding a Ni(II) exporter and a Ni(II) importer were controlled by InrS variants with weaker Ni(II) affinities. Variant strains were sensitive to elevated nickel and contained more nickel, but the increase was small compared with the change in Ni(II) affinity. All of the variant sensors retained the allosteric mechanism that inhibits DNA binding following metal binding, but a response to nickel in vivo was observed only when the sensitivity was set to respond in a relatively narrow (less than two orders of magnitude) range of nickel concentrations. Thus, the Ni(II) affinity of InrS is attuned to cellular metal concentrations rather than the converse.
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http://dx.doi.org/10.1038/nchembio.2310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5365139PMC
April 2017

Mutantelec: An In Silico mutation simulation platform for comparative electrostatic potential profiling of proteins.

J Comput Chem 2017 03;38(7):467-474

Centro de Bioinformática y Simulación Molecular, Facultad de Ingeniería, Universidad de Talca, Talca, 346 5548, Chile.

The electrostatic potential plays a key role in many biological processes like determining the affinity of a ligand to a given protein target, and they are responsible for the catalytic activity of many enzymes. Understanding the effect that amino acid mutations will have on the electrostatic potential of a protein, will allow a thorough understanding of which residues are the most important in a protein. MutantElec, is a friendly web application for in silico generation of site-directed mutagenesis of proteins and the comparison of electrostatic potential between the wild type protein and the mutant(s), based on the three-dimensional structure of the protein. The effect of the mutation is evaluated using different approach to the traditional surface map. MutantElec provides a graphical display of the results that allows the visualization of changes occurring at close distance from the mutation and thus uncovers the local and global impact of a specific change. © 2017 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jcc.24712DOI Listing
March 2017

The molecular basis of the interactions between synthetic retinoic acid analogues and the retinoic acid receptors.

Medchemcomm 2017 Mar 20;8(3):578-592. Epub 2017 Jan 20.

Department of Chemistry Durham University , South Road , Durham , DH1 3LE , UK . Email:

All--retinoic acid (ATRA) and its synthetic analogues EC23 and EC19 direct cellular differentiation by interacting as ligands for the retinoic acid receptor (RARα, β and γ) family of nuclear receptor proteins. To date, a number of crystal structures of natural and synthetic ligands complexed to their target proteins have been solved, providing molecular level snap-shots of ligand binding. However, a deeper understanding of receptor and ligand flexibility and conformational freedom is required to develop stable and effective ATRA analogues for clinical use. Therefore, we have used molecular modelling techniques to define RAR interactions with ATRA and two synthetic analogues, EC19 and EC23, and compared their predicted biochemical activities to experimental measurements of relative ligand affinity and recruitment of coactivator proteins. A comprehensive molecular docking approach that explored the conformational space of the ligands indicated that ATRA is able to bind the three RAR proteins in a number of conformations with one extended structure being favoured. In contrast the biologically-distinct isomer, 9--retinoic acid (; 9CRA), showed significantly less conformational flexibility in the RAR binding pockets. These findings were used to inform docking studies of the synthetic retinoids EC23 and EC19, and their respective methyl esters. EC23 was found to be an excellent mimic for ATRA, and occupied similar binding modes to ATRA in all three target RAR proteins. In comparison, EC19 exhibited an alternative binding mode which reduces the strength of key polar interactions in RARα/γ but is well-suited to the larger RARβ binding pocket. In contrast, docking of the corresponding esters revealed the loss of key polar interactions which may explain the much reduced biological activity. Our computational results were complemented using an binding assay based on FRET measurements, which showed that EC23 was a strongly binding, pan-agonist of the RARs, while EC19 exhibited specificity for RARβ, as predicted by the docking studies. These findings can account for the distinct behaviour of EC23 and EC19 in cellular differentiation assays, and additionally, the methods described herein can be further applied to the understanding of the molecular basis for the selectivity of different retinoids to RARα, β and γ.
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http://dx.doi.org/10.1039/c6md00680aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6072416PMC
March 2017

Practical synthetic strategies towards lipophilic 6-iodotetrahydroquinolines and -dihydroquinolines.

Beilstein J Org Chem 2016 16;12:1851-1862. Epub 2016 Aug 16.

Centre for Sustainable Chemical Processes, Department of Chemistry, Durham University, South Road, Durham, DH1 3LE, UK.

The synthesis of novel tetrahydroquinolines (THQ) and dihydroquinolines (DHQ) are reported using three practical, scalable synthetic approaches to access highly lipophilic analogues bearing a 6-iodo substituent, each with a different means of cyclisation. A versatile and stable quinolin-2-one intermediate was identified, which could be reduced to the corresponding THQ with borane reagents, or to the DHQ with diisobutylaluminium hydride via a novel elimination that is more favourable at higher temperatures. Coupling these strongly electron-donating scaffolds to electron-accepting moieties caused the resulting structures to exhibit strong fluorescence.
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http://dx.doi.org/10.3762/bjoc.12.174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5082455PMC
August 2016

The Effectors and Sensory Sites of Formaldehyde-responsive Regulator FrmR and Metal-sensing Variant.

J Biol Chem 2016 09 29;291(37):19502-16. Epub 2016 Jul 29.

From the Department of Chemistry, School of Biological and Biomedical Sciences, Durham University, Durham DH1 3LE, United Kingdom.

The DUF156 family of DNA-binding transcriptional regulators includes metal sensors that respond to cobalt and/or nickel (RcnR, InrS) or copper (CsoR) plus CstR, which responds to persulfide, and formaldehyde-responsive FrmR. Unexpectedly, the allosteric mechanism of FrmR from Salmonella enterica serovar Typhimurium is triggered by metals in vitro, and variant FrmR(E64H) gains responsiveness to Zn(II) and cobalt in vivo Here we establish that the allosteric mechanism of FrmR is triggered directly by formaldehyde in vitro Sensitivity to formaldehyde requires a cysteine (Cys(35) in FrmR) conserved in all DUF156 proteins. A crystal structure of metal- and formaldehyde-sensing FrmR(E64H) reveals that an FrmR-specific amino-terminal Pro(2) is proximal to Cys(35), and these residues form the deduced formaldehyde-sensing site. Evidence is presented that implies that residues spatially close to the conserved cysteine tune the sensitivities of DUF156 proteins above or below critical thresholds for different effectors, generating the semblance of specificity within cells. Relative to FrmR, RcnR is less responsive to formaldehyde in vitro, and RcnR does not sense formaldehyde in vivo, but reciprocal mutations FrmR(P2S) and RcnR(S2P), respectively, impair and enhance formaldehyde reactivity in vitro Formaldehyde detoxification by FrmA requires S-(hydroxymethyl)glutathione, yet glutathione inhibits formaldehyde detection by FrmR in vivo and in vitro Quantifying the number of FrmR molecules per cell and modeling formaldehyde modification as a function of [formaldehyde] demonstrates that FrmR reactivity is optimized such that FrmR is modified and frmRA is derepressed at lower [formaldehyde] than required to generate S-(hydroxymethyl)glutathione. Expression of FrmA is thereby coordinated with the accumulation of its substrate.
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http://dx.doi.org/10.1074/jbc.M116.745174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5016687PMC
September 2016

Conjugate Addition of 3-Buytn-2-one to Anilines in Ethanol: Alkene Geometric Insights through In Situ FTIR Monitoring.

J Org Chem 2016 09 10;81(17):7557-65. Epub 2016 Aug 10.

Centre for Sustainable Chemical Processes, Department of Chemistry, Durham University , South Road, Durham, DH1 3LE, U.K.

A convenient, mild and effective conjugate addition of 3-butyn-2-one to a variety of anilines in ethanol is reported. The reaction was monitored and characterized through in situ FTIR, and the dynamics of the facile E/Z alkene geometry interconversion of the resultant aniline-derived enaminones was explored through NMR, FTIR and X-ray crystallography. A straightforward purification protocol that employs direct Kugelrohr distillation was identified, and the method was further extended to other amines and ynones, allowing rapid access to these interesting compounds.
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http://dx.doi.org/10.1021/acs.joc.6b01110DOI Listing
September 2016

The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein.

J Biol Chem 2015 Sep 16;290(36):22225-35. Epub 2015 Jul 16.

From the School of Biological and Biomedical Sciences, the Biophysical Sciences Institute, and

Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring.
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http://dx.doi.org/10.1074/jbc.M115.669267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4571973PMC
September 2015

Crystal Structure of a Hidden Protein, YcaC, a Putative Cysteine Hydrolase from Pseudomonas aeruginosa, with and without an Acrylamide Adduct.

Int J Mol Sci 2015 Jul 14;16(7):15971-84. Epub 2015 Jul 14.

School of Biological and Biomedical Sciences, Durham University, Durham DH1 3LE, UK.

As part of the ongoing effort to functionally and structurally characterize virulence factors in the opportunistic pathogen Pseudomonas aeruginosa, we determined the crystal structure of YcaC co-purified with the target protein at resolutions of 2.34 and 2.56 Å without a priori knowledge of the protein identity or experimental phases. The three-dimensional structure of YcaC adopts a well-known cysteine hydrolase fold with the putative active site residues conserved. The active site cysteine is covalently bound to propionamide in one crystal form, whereas the second form contains an S-mercaptocysteine. The precise biological function of YcaC is unknown; however, related prokaryotic proteins have functions in antibacterial resistance, siderophore production and NADH biosynthesis. Here, we show that YcaC is exceptionally well conserved across both bacterial and fungal species despite being non-ubiquitous. This suggests that whilst YcaC may not be part of an integral pathway, the function could confer a significant evolutionary advantage to microbial life.
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http://dx.doi.org/10.3390/ijms160715971DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4519933PMC
July 2015

Global low-frequency motions in protein allostery: CAP as a model system.

Biophys Rev 2015;7(2):175-182. Epub 2015 Feb 4.

Biophysical Sciences Institute, Durham University, Durham, UK ; School of Biological and Biomedical Sciences, Durham University, Durham, UK.

Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. There is considerable evidence that allosteric cooperativity can be communicated by the modulation of protein dynamics without conformational change. The Catabolite Activator Protein (CAP) of is an important experimental exemplar for entropically driven allostery. Here we discuss recent experimentally supported theoretical analysis that highlights the role of global low-frequency dynamics in allostery in CAP and identify how allostery arises as a natural consequence of changes in global low-frequency protein fluctuations on ligand binding.
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http://dx.doi.org/10.1007/s12551-015-0163-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4432019PMC
February 2015

Corrigendum to "High-level over-expression, purification, and crystallization of a novel phospholipase C/sphingomyelinase from Pseudomonas aeruginosa" [Protein Expr. Purif. 90 (2013) 40-46].

Protein Expr Purif 2015 Apr 15;108:115. Epub 2014 Aug 15.

Department of Chemistry & School of Biological and Biomedical Sciences, Durham University, Durham DH1 3LE, UK. Electronic address:

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http://dx.doi.org/10.1016/j.pep.2014.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5595910PMC
April 2015
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