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39 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I04-Macromolecular Crystallography	Characterisation of an unusual cysteine pair in the Rieske carnitine monooxygenase CntA catalytic site Mussa Quareshy , Muralidharan Shanmugam , Alexander D Cameron , Timothy D. H. Bugg , Yin Chen The Febs Journal DOI: 10.1111/febs.16722 Diamond Proposal Number(s): [19880] Show Abstract ▼ Abstract: Rieske monooxygenases undertake complex catalysis integral to marine, terrestrial and human gut-ecosystems. Group-I to IV Rieske monooxygenases accept aromatic substrates and have well characterised catalytic mechanisms. Nascent to our understanding are Group-V members catalysing the oxidation/breakdown of quaternary ammonium substrates. Phylogenetic analysis of Group V highlights a cysteine residue-pair adjacent to the mononuclear Fe active site with no established role. Following our elucidation of the carnitine monooxygenase CntA structure, we probed the function of the cysteine pair Cys206/Cys209. Utilising biochemical and biophysical techniques, we found the cysteine residues do not play a structural role nor influence the electron transfer pathway, but rather are used in a non-stoichiometric role to ensure the catalytic iron centre remains in an Fe(II) state.	Jan 2023
I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structures of lactaldehyde reductase, FucO, link enzyme activity to hydrogen bond networks and conformational dynamics Alberto Zavarise , Shruthi Sridhar , Tiila-Riikka Kiema , Rikkert K. Wierenga , Mikael Widersten The Febs Journal DOI: 10.1111/febs.16603 Diamond Proposal Number(s): [24732, 19951] Open Access Show Abstract ▼ Abstract: A group-III iron containing 1,2-propanediol oxidoreductase, FucO, (also known as lactaldehyde reductase) from Escherichia coli was examined regarding its structure-dynamics-function relationships in the catalysis of the NADH dependent reduction of (2S)-lactaldehyde. Crystal structures of FucO variants in the presence or absence of cofactors have been determined, illustrating large domain movements between the apo and holo enzyme structures. Different structures of FucO variants co-crystallized with NAD+ or NADH together with substrate further suggest dynamic properties of the nicotinamide moiety of the coenzyme that are important for the reaction mechanism. Modeling of the native substrate (2S)-lactaldehyde into the active site can explain the stereoselectivity exhibited by the enzyme, with a critical hydrogen bond interaction between the (2S)-hydroxyl and the side-chain of N151, as well as the previously experimentally demonstrated pro-(R) selectivity in hydride transfer from NADH to the aldehydic carbon. Furthermore, the deuterium kinetic isotope effect of hydride transfer suggests that reduction chemistry is the main rate limiting step for turnover which is not the case in FucO catalyzed alcohol oxidation. We further propose that a water molecule in the active site – hydrogen bonded to a conserved histidine (H267) and the 2’-hydroxyl of the coenzyme ribose – functions as a catalytic proton donor in the protonation of the product alcohol. A hydrogen bond network of water molecules and the side chains of amino acid residues D360 and H267 links bulk solvent to this proposed catalytic water molecule.	Aug 2022
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Structural basis for the inhibition of human angiotensin‐1 converting enzyme by fosinoprilat Gyles E. Cozier , Emma C. Newby , Sylva L. U. Schwager , R. Elwyn Isaac , Edward D. Sturrock , K. Ravi Acharya The Febs Journal DOI: 10.1111/febs.16543 Diamond Proposal Number(s): [17212] Show Abstract ▼ Abstract: Human angiotensin-I converting enzyme (ACE) has two isoforms, somatic ACE (sACE) and testis ACE (tACE). The functions of sACE are widespread, with its involvement in blood pressure regulation most extensively studied. sACE is composed of an N-domain (nACE) and a C-domain (cACE), both catalytically active but have significant structural differences, resulting in different substrate specificities. Even though ACE inhibitors are used clinically, they need much improvement because of serious side effects seen in patients (~25-30%) with long-term treatment due to non-selective inhibition of nACE and cACE. Investigation into the distinguishing structural features of each domain is therefore of vital importance for the development of domain-specific inhibitors with minimal side effects. Here we report kinetic data and high resolution crystal structures of both nACE (1.75 Å) and cACE (1.85 Å) in complex with fosinoprilat, a clinically used inhibitor. These structures allowed detailed analysis of the molecular features conferring domain selectivity by fosinoprilat. Particularly, altered hydrophobic interactions were observed to be a contributing factor. These experimental data contribute to improved understanding of the structural features that dictate ACE inhibitor domain selectivity, allowing further progress towards designing novel 2nd generation domain-specific potent ACE inhibitors suitable for clinical administration, with a variety of potential future therapeutic benefits.	Jun 2022
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Understanding the interaction of 14‐3‐3 proteins with hDMX and hDM2: a structural and biophysical study Sonja Srdanovic , Madita Wolter , Chi H. Trinh , Christian Ottmann , Stuart L. Warriner , Andrew J. Wilson The Febs Journal DOI: 10.1111/febs.16433 Diamond Proposal Number(s): [19248] Show Abstract ▼ Abstract: p53 plays a critical role in regulating diverse biological processes: DNA repair, cell cycle arrest, apoptosis, and senescence. The p53 pathway has therefore served as the focus of multiple drug-discovery efforts. p53 is negatively regulated by hDMX and hDM2; prior studies have identified 14-3-3 proteins as hDMX and hDM2 client proteins. 14-3-3 proteins are adaptor proteins that modulate localisation, degradation and interactions of their targets in response to phosphorylation. Thus, 14-3-3 proteins may indirectly modulate the interaction between hDMX or hDM2 and p53 and represent potential targets for modulation of the p53 pathway. In this manuscript, we report on the biophysical and structural characterization of peptide/protein interactions that are representative of the interaction between 14-3-3 and hDMX or hDM2. The data establish that proximal phosphosites spaced ~20-25 residues apart in both hDMX and hDM2 co-operate to facilitate high-affinity 14-3-3 binding and provide structural insight that can be utilized in future stabilizer/inhibitor discovery efforts.	Mar 2022
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	The structural basis for high‐affinity uptake of lignin‐derived aromatic compounds by proteobacterial TRAP transporters Claudine Bisson , Robert C. Salmon , Laura West , John B. Rafferty , Andrew Hitchcock , Gavin H. Thomas , David J. Kelly The Febs Journal DOI: 10.1111/febs.16156 Diamond Proposal Number(s): [8987] Show Abstract ▼ Abstract: The organic polymer lignin is a component of plant cell walls, which like (hemi)-cellulose is highly abundant in nature and relatively resistant to degradation. However, extracellular enzymes released by natural microbial consortia can cleave the β-aryl ether linkages in lignin, releasing monoaromatic phenylpropanoids that can be further catabolised by diverse species of bacteria. Biodegradation of lignin is therefore important in global carbon cycling, and its natural abundance also makes it an attractive biotechnological feedstock for the industrial production of commodity chemicals. While the pathways for degradation of lignin-derived aromatics have been extensively characterised, much less is understood about how they are recognised and taken up from the environment. The purple phototrophic bacterium Rhodopseudomonas (Rps.) palustris can grow on a range of phenylpropanoid monomers and is a model organism for studying their uptake and breakdown. Rps. palustris encodes a tripartite ATP-independent periplasmic (TRAP) transporter (TarPQM) linked to genes encoding phenylpropanoid-degrading enzymes. The periplasmic solute binding protein component of this transporter, TarP, has previously been shown to bind aromatic substrates. Here, we determine the high-resolution crystal structure of TarP from Rps. palustris as well as the structures of homologous proteins from the salt marsh bacterium Sagittula stellata and the halophile Chromohalobacter salexigens, which also grow on lignin-derived aromatics. In combination with tryptophan fluorescence ligand-binding assays, our ligand-bound co-crystal structures reveal the molecular basis for high-affinity recognition of phenylpropanoids by these TRAP transporters, which have potential for improving uptake of these compounds for biotechnological transformations of lignin.	Aug 2021
B21-High Throughput SAXS	Human myelin protein P2: From crystallography to time‐lapse membrane imaging and neuropathy‐associated variants Maiju Uusitalo , Martin Berg Klenow , Saara Laulumaa , Matthew P. Blakeley , Adam Cohen Simonsen , Salla Ruskamo , Petri Kursula The Febs Journal DOI: 10.1111/febs.16079 Diamond Proposal Number(s): [27081] Show Abstract ▼ Abstract: Peripheral myelin protein 2 (P2) is a fatty acid-binding protein expressed in vertebrate peripheral nervous system myelin, as well as in human astrocytes. Suggested functions of P2 include membrane stacking and lipid transport. Mutations in the PMP2 gene, encoding P2, are associated with Charcot-Marie-Tooth disease (CMT). Recent studies have revealed three novel PMP2 mutations in CMT patients. To shed light on the structure and function of these P2 variants, we used X-ray and neutron crystallography, small-angle X-ray scattering, circular dichroism spectroscopy, computer simulations and lipid binding assays. The crystal and solution structures of the I50del, M114T and V115A variants of P2 showed minor differences to the wild-type protein, whereas their thermal stability was reduced. Vesicle aggregation assays revealed no change in membrane stacking characteristics, while the variants showed altered fatty acid binding. Time-lapse imaging of lipid bilayers indicated formation of double membrane structures induced by P2, which could be related to its function in stacking of two myelin membrane surfaces in vivo. In order to better understand the links between structure, dynamics and function, the crystal structure of perdeuterated P2 was refined from room temperature data using neutrons and X-rays and the results were compared to simulations and cryocooled crystal structures. Our data indicate similar properties for all known human P2 CMT variants; while crystal structures are nearly identical, thermal stability and function of CMT variants are impaired. Our data provide new insights into the structure-function relationships and dynamics of P2 in health and disease.	Jun 2021
I03-Macromolecular Crystallography	Crystal structure of an inulosucrase from Halalkalicoccus jeotgali B3T‐a halophilic archaeal strain Komal Ghauri , Tjaard Pijning , Nayla Munawar , Hazrat Ali , Muhammad A. Ghauri , Munir A. Anwar , Russell Wallis The Febs Journal DOI: 10.1111/febs.15843 Show Abstract ▼ Abstract: Several archaea harbour genes that code for fructosyltransferase (FTF) enzymes. These enzymes have not been characterized yet at structure‐function level, but are of extreme interest in view of their potential role in the synthesis of novel compounds for food, nutrition and pharmaceutical applications. In this study, 3D structure of an inulin‐type fructan producing enzyme, inulosucrase (InuHj), from the archaeon Halalkalicoccus jeotgali was resolved in its apo form as well as with bound substrate (sucrose) molecule and first transglycosylation product (1‐kestose). This is the first crystal structure of an FTF from halophilic archaea. Its overall five‐bladed β‐propeller fold is conserved with previously reported FTFs, but also shows some unique features. The InuHj structure is closer to those of Gram‐negative bacteria, with exceptions such as residue E266, which is conserved in FTFs of Gram‐positive bacteria and has possible role in fructan polymer synthesis in these bacteria as compared to fructooligosaccharide (FOS) production by FTFs of Gram‐negative bacteria. Highly negative electrostatic surface potential of InuHj, due to a large amount of acidic residues, likely contributes to its halophilicity. The complex of InuHj with 1‐kestose indicates that the residues D287 in the 4B‐4C loop, Y330 in 4D‐5A and D361 in the unique α2 helix may interact with longer FOSs and facilitate the binding of longer FOS chains during synthesis. The outcome of this work will provide targets for future structure‐function studies of FTF enzymes, particularly those from archaea.	Mar 2021
Krios II-Titan Krios II at Diamond	SAP domain forms a flexible part of DNA aperture in Ku70/80 Ales Hnizda , Petr Tesina , Than-Binh Nguyen , Zdeněk Kukačka , Lukas Kater , Amanda K. Chaplin , Roland Beckmann , David B. Ascher , Petr Novák , Tom L. Blundell The Febs Journal DOI: 10.1111/febs.15732 Diamond Proposal Number(s): [17057] Open Access Show Abstract ▼ Abstract: Nonhomologous end joining (NHEJ) is a DNA repair mechanism that religates double‐strand DNA breaks to maintain genomic integrity during the entire cell cycle. The Ku70/80 complex recognizes DNA breaks and serves as an essential hub for recruitment of NHEJ components. Here, we describe intramolecular interactions of the Ku70 C‐terminal domain, known as the SAP domain. Using single‐particle cryo‐electron microscopy, mass spectrometric analysis of intermolecular cross‐linking and molecular modelling simulations, we captured variable positions of the SAP domain depending on DNA binding. The first position was localized at the DNA aperture in the Ku70/80 apo form but was not observed in the DNA‐bound state. The second position, which was observed in both apo and DNA‐bound states, was found below the DNA aperture, close to the helical arm of Ku70. The localization of the SAP domain in the DNA aperture suggests a function as a flexible entry gate for broken DNA.	Feb 2021
B21-High Throughput SAXS	Crystal and solution structure of NDRG1, a membrane‐binding protein linked to myelination and tumour suppression Venla Mustonen , Gopinath Muruganandam , Remy Loris , Petri Kursula , Salla Ruskamo The Febs Journal 21 DOI: 10.1111/febs.15660 Open Access Show Abstract ▼ Abstract: N‐myc downstream‐regulated gene 1 (NDRG1) is a tumour suppressor involved in vesicular trafficking and stress response. NDRG1 participates in peripheral nerve myelination, and mutations in the NDRG1 gene lead to Charcot‐Marie‐Tooth neuropathy. The 43‐kDa NDRG1 is considered as an inactive member of the α/β hydrolase superfamily. In addition to a central α/β hydrolase fold domain, NDRG1 consists of a short N terminus and a C‐terminal region with three 10‐residue repeats. We determined the crystal structure of the α/β hydrolase domain of human NDRG1 and characterised the structure and dynamics of full‐length NDRG1. The structure of the α/β hydrolase domain resembles the canonical α/β hydrolase fold with a central β sheet surrounded by α helices. Small‐angle X‐ray scattering and CD spectroscopy indicated a variable conformation for the N‐ and C‐terminal regions. NDRG1 binds to various types of lipid vesicles, and the conformation of the C‐terminal region is modulated upon lipid interaction. Intriguingly, NDRG1 interacts with metal ions, such as nickel, but is prone to aggregation in their presence. Our results uncover the structural and dynamic features of NDRG1, as well as elucidate its interactions with metals and lipids, and encourage studies to identify a putative hydrolase activity of NDRG1.	Jan 2021
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Mutational and structural analysis of an ancestral fungal dye‐decolorizing peroxidase Ulises A. Zitare , Mohamed H. Habib , Henriette Rozeboom , Maria L. Mascotti , Smilja Todorovic , Marco W. Fraaije The Febs Journal 41 DOI: 10.1111/febs.15687 Diamond Proposal Number(s): [19800] Open Access Show Abstract ▼ Abstract: Dye‐decolorizing peroxidases (DyPs) constitute a superfamily of heme‐containing peroxidases that are related neither to animal nor to plant peroxidase families. These are divided into four classes (types A, B, C, and D) based on sequence features. The active site of DyPs contains two highly conserved distal ligands, an aspartate and an arginine, the roles of which are still controversial. These ligands have mainly been studied in class A‐C bacterial DyPs, largely because no effective recombinant expression systems have been developed for the fungal (D‐type) DyPs. In this work, we employ ancestral sequence reconstruction (ASR) to resurrect a D‐type DyP ancestor, AncDyPD‐b1. Expression of AncDyPD‐b1 in Escherichia coli results in large amounts of a heme‐containing soluble protein and allows for the first mutagenesis study on the two distal ligands of a fungal DyP. UV‐Vis and resonance Raman (RR) spectroscopic analyses, in combination with steady‐state kinetics and the crystal structure, reveal fine pH‐dependent details about the heme active site structure and show that both the aspartate (D222) and the arginine (R390) are crucial for hydrogen peroxide reduction. Moreover, the data indicate that these two residues play important but mechanistically different roles on the intraprotein long‐range electron transfer process.	Jan 2021

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