I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[24948]
Open Access
Abstract: Chagas disease is a neglected tropical disease (NTD) caused by Trypanosoma cruzi, whilst leishmaniasis, which is caused by over 20 species of Leishmania, represents a group of NTDs endemic to most countries in the tropical and subtropical belt of the planet. These diseases remain a significant health problem both in endemic countries and globally. These parasites and other trypanosomatids, including T. theileri, a bovine pathogen, rely on cysteine biosynthesis for the production of trypanothione, which is essential for parasite survival in hosts. The de novo pathway of cysteine biosynthesis requires the conversion of O-acetyl-L-serine into L-cysteine, which is catalysed by cysteine synthase (CS). These enzymes present potential for drug development against T. cruzi, Leishmania spp. and T. theileri. To enable these possibilities, biochemical and crystallographic studies of CS from T. cruzi (TcCS), L. infantum (LiCS) and T. theileri (TthCS) were conducted. Crystal structures of the three enzymes were determined at resolutions of 1.80 Å for TcCS, 1.75 Å for LiCS and 2.75 Å for TthCS. These three homodimeric structures show the same overall fold and demonstrate that the active-site geometry is conserved, supporting a common reaction mechanism. Detailed structural analysis revealed reaction intermediates of the de novo pathway ranging from an apo structure of LiCS and holo structures of both TcCS and TthCS to the substrate-bound structure of TcCS. These structures will allow exploration of the active site for the design of novel inhibitors. Additionally, unexpected binding sites discovered at the dimer interface represent new potential for the development of protein–protein inhibitors.
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Jun 2023
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I03-Macromolecular Crystallography
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Andrius
Jasilionis
,
Magdalena
Plotka
,
Lei
Wang
,
Sebastian
Dorawa
,
Joanna
Lange
,
Hildegard
Watzlawick
,
Tom
Van Den Bergh
,
Bas
Vroling
,
Josef
Altenbuchner
,
Anna-Karina
Kaczorowska
,
Ehmke
Pohl
,
Tadeusz
Kaczorowski
,
Eva
Nordberg Karlsson
,
Stefanie
Freitag-Pohl
Diamond Proposal Number(s):
[18598]
Abstract: Bacteriophages encode a wide variety of cell wall disrupting enzymes that aid the viral escape in the final stages of infection. These lytic enzymes have accumulated notable interest due to their potential as novel antibacterials for infection treatment caused by multiple-drug resistant bacteria. Here, the detailed functional and structural characterization of Thermus parvatiensis prophage peptidoglycan lytic amidase AmiP, a globular Amidase_3 type lytic enzyme adapted to high temperatures is presented. The sequence and structure comparison with homologous lytic amidases reveals the key adaptation traits that ensure the activity and stability of AmiP at high temperatures. The crystal structure determined at a resolution of 1.8 Å displays a compact α/β-fold with multiple secondary structure elements omitted or shortened compared to protein structures of similar proteins. The functional characterisation of AmiP demonstrates high efficiency of catalytic activity and broad substrate specificity towards thermophilic and mesophilic bacteria strains containing Orn-type or DAP-type peptidoglycan. The here presented AmiP constitutes the most thermoactive and ultrathermostable Amidase_3 type lytic enzyme biochemically characterised with a temperature optimum at 85 °C. The extraordinary high melting temperature Tm 102.6 °C confirms fold stability up to approximately 100 °C. Furthermore, AmiP is shown to be more active over the alkaline pH range with pH optimum at pH 8.5 and tolerates NaCl up to 300 mM with the activity optimum at 25 mM NaCl. This set of beneficial characteristics suggests that AmiP can be further exploited in biotechnology.
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Jan 2023
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[24948]
Open Access
Abstract: The Candidate Phyla Radiation is a recently uncovered and vast expansion of the bacterial domain of life, made up of largely uncharacterized phyla that lack isolated representatives. This unexplored territory of genetic diversity presents an abundance of novel proteins with potential applications in the life-science sectors. Here we present the structural and functional elucidation of CPR-C4, a hypothetical protein from the genome of a thermophilic Candidate Phyla Radiation organism, identified through metagenomic sequencing. Our analyses revealed that CPR-C4 is a member of a family of highly conserved proteins within the Candidate Phyla Radiation. The function of CPR-C4 as a cysteine protease was predicted through remote structural similarity to the Homo sapiens vasohibins and subsequently confirmed experimentally with fluorescence-based activity assays. Furthermore, detailed structural and sequence alignment analysis enabled identification of a non-canonical cysteine-histidine-leucine(carbonyl) catalytic triad. The unexpected structural and functional similarities between CPR-C4 and the human vasohibins suggest an evolutionary relationship undetectable at the sequence level alone.
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Apr 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Maria
Schwarz
,
Rebecca F. M.
Eno
,
Stefanie
Freitag-Pohl
,
Christopher R.
Coxon
,
Hannah E.
Straker
,
David J.
Wortley
,
David J.
Hughes
,
Glynn
Mitchell
,
Jenny
Moore
,
Ian
Cummins
,
Nawaporn
Onkokesung
,
Melissa
Brazier-Hicks
,
Robert
Edwards
,
Ehmke
Pohl
,
Patrick G.
Steel
Diamond Proposal Number(s):
[24948]
Open Access
Abstract: The evolution and growth of multiple-herbicide resistance (MHR) in grass weeds continues to threaten global cereal production. While various processes can contribute to resistance, earlier work has identified the phi class glutathione-S-transferase (AmGSTF1) as a functional biomarker of MHR in black-grass (Alopecurus myosuroides). This study provides further insights into the role of AmGSTF1 in MHR using a combination of chemical and structural biology. Crystal structures of wild-type AmGSTF1, together with two specifically designed variants that allowed the co-crystal structure determination with glutathione and a glutathione adduct of the AmGSTF1 inhibitor 4-chloro-7-nitro-benzofurazan (NBD-Cl) were obtained. These studies demonstrated that the inhibitory activity of NBD-Cl was associated with the occlusion of the active site and the impediment of substrate binding. A search for other selective inhibitors of AmGSTF1, using ligand-fishing experiments, identified a number of flavonoids as potential ligands. Subsequent experiments using black-grass extracts discovered a specific flavonoid as a natural ligand of the recombinant enzyme. A series of related synthetic flavonoids was prepared and their binding to AmGSTF1 was investigated showing a high affinity for derivatives bearing a O-5-decyl-α-carboxylate. Molecular modelling based on high-resolution crystal structures allowed a binding pose to be defined which explained flavonoid binding specificity. Crucially, high binding affinity was linked to a reversal of the herbicide resistance phenotype in MHR black-grass. Collectively, these results present a nature-inspired new lead for the development of herbicide synergists to counteract MHR in weeds.
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Nov 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[24948]
Open Access
Abstract: 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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Feb 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Stefanie
Freitag-Pohl
,
Andrius
Jasilionis
,
Maria
Håkansson
,
L. Anders
Svensson
,
Rebeka
Kovačič
,
Martin
Welin
,
Hildegard
Watzlawick
,
Lei
Wang
,
Josef
Altenbuchner
,
Magdalena
Płotka
,
Anna Karina
Kaczorowska
,
Tadeusz
Kaczorowski
,
Eva
Nordberg Karlsson
,
Salam
Al-Karadaghi
,
Björn
Walse
,
Arnthór
Aevarsson
,
Ehmke
Pohl
Abstract: 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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Nov 2019
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[18598]
Abstract: 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 utilised for filtering of the large dataset. We obtained biophysically validated hits, some which yielded complex crystal structures. In some cases, the crystallised binding mode and top scoring mode agree, while for others the alternate ligand binding orientation was found. In this contribution we combine rigid docking, MD simulations and the LIE method to calculate free energies of binding and derive relative binding energies for a number of EthR inhibitors in both modes. This strategy allowed us to correctly predict the most favourable 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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Apr 2019
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I02-Macromolecular Crystallography
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David R.
Chisholm
,
Charles W. E.
Tomlinson
,
Garr-Layy
Zhou
,
Claire
Holden
,
Valerie
Affleck
,
Rebecca
Lamb
,
Katherine
Newling
,
Peter
Ashton
,
Roy
Valentine
,
Christopher
Redfern
,
János
Erostyák
,
Geza
Makkai
,
Carrie A.
Ambler
,
Andrew
Whiting
,
Ehmke
Pohl
Open Access
Abstract: Retinoids, such as all-trans-retinoic acid (ATRA), are endogenous signalling molecules derived from Vitamin A that influ-ence a variety of cellular processes through mediation of transcription events in the cell nucleus. Due to 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 con-trol. We have designed and synthesized a family of synthetic retinoids that exhibit strong, intrinsic, solvatochromatic fluo-rescence 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 quanti-fy their binding to their cellular targets, including Cellular Retinoid Binding Protein II (CRABPII). The dihydroquinoline retinoid, DC360, exhibited particularly strong binding (Kd = 34.0 ± 2.5 nM) and we further used X-ray crystallography to solve 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 behaviour of the com-pounds in cultured human epithelial cells, highlighting a fascinating nuclear localisation, and used RNA sequencing to con-firm that the compounds regulate similar cellular processes to ATRA. We anticipate that the unique properties of these fluo-rescent retinoids can now be used to shed new light on the vital and highly complex retinoid signalling pathway.
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Feb 2019
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I03-Macromolecular Crystallography
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Open Access
Abstract: 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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Nov 2018
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Abstract: The transcriptional regulator EthR from Mycobacterium tuberculosis, a member of the TetR family of prokaryotic homo-dimeric transcriptions factors, controls the expression of the mycobacterial mono-oxygenase EthA. Due to the fact that EthA is responsible for the bio-activation of the second-line tuberculosis pro-drug ethionamide, EthR inhibitors have been shown to boost drug efficacy by increasing EthA levels. 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. We present biophysical characterization of 85 potential leads, 20 of which showed binding by thermal shift assays. 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. The crystal structures include ligands with three new chemical scaffolds that will enable future lead development. Five of the lead compounds showed the desired booster effect with the most promising displaying an EC50 value of 0.76 μM.
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Nov 2017
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