Browsing by Subject "CRYSTAL-STRUCTURE"

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  • Domanska, Ausra; Guryanov, Sergey; Butcher, Sarah J. (2021)
    Parechoviruses belong to the genus Parechovirus within the family Picornaviridae and are non-enveloped icosahedral viruses with a single-stranded RNA genome. Parechoviruses include human and animal pathogens classified into six species. Those that infect humans belong to the Parechovirus A species and can cause infections ranging from mild gastrointestinal or respiratory illness to severe neonatal sepsis. There are no approved antivirals available to treat parechovirus (nor any other picornavirus) infections. In this parechovirus review, we focus on the cleaved protein products resulting from the polyprotein processing after translation comparing and contrasting their known or predicted structures and functions to those of other picornaviruses. The review also includes our original analysis from sequence and structure prediction. This review highlights significant structural differences between parechoviral and other picornaviral proteins, suggesting that parechovirus drug development should specifically be directed to parechoviral targets.
  • Das, Biswanath; Al-Hunaiti, Afnan; Carey, Akina; Lidin, Sven; Demeshko, Serhiy; Repo, Timo; Nordlander, Ebbe (2022)
    The oxido-bridged diiron(III) complex [Fe-2(mu-O)(mu-OAc)(DPEAMP)(2)](OCH3) (1), based on a new unsymmetrical ligand with an N4O donor set, viz. [2-((bis(pyridin-2-ylmethyl)amino)methyl)-6-((ethylamino)methyl)-4-meth-ylphenol (HDPEAMP)], has been prepared and characterized by spectroscopic methods and X-ray crystallog-raphy. The crystal structure of the complex reveals that each Fe(III) ion is coordinated by three nitrogen and three oxygen donors, two of which are the bridging oxido and acetate ligands. Employing H2O2 as a terminal oxidant, 1 is capable of oxidizing a number of alkanes and alkenes with high activity. The catalytic oxidation of 1,2-dimethylcyclohexane results in excellent retention of configuration. Monitoring of the reaction of 1 with H2O2 and acetic acid in the absence of substrate, using low-temperature UV-Vis spectroscopy, suggests the in situ formation of a transient Fe(III)(2)-peroxido species. While the selectivity and nature of oxidation products implicate a high-valent iron-oxido complex as a key intermediate, the low alcohol/ketone ratios suggest a simultaneous radical-based process.
  • Vidilaseris, Keni; Kellosalo, Juho; Goldman, Adrian (2018)
    Membrane-bound pyrophosphatases (mPPases) are homodimeric integral membrane proteins that hydrolyse pyrophosphate into orthophosphates coupled to the active transport of protons or sodium ions across membranes. They occur in bacteria, archaea, plants, and protist parasites. As they are essential in protist parasites and there are no homologous proteins in animals and humans, these enzymes represent an excellent drug target for treating protistal diseases. Experimental screening to find drug candidates is an important step to discover new hit compounds. For that, a cheap, simple, and robust assay is needed. Here we report the application of the molybdenum blue reaction method for a medium throughput microplate activity assay of the hyperthermophilic bacterium Thermotoga maritima mPPase and the possible application of the assay to screen inhibitors of membrane-bound pyrophosphatases.
  • Pryce, Rhys; Azarm, Kristopher; Rissanen, Ilona; Harlos, Karl; Bowden, Thomas A.; Lee, Benhur (2020)
    The emergent zoonotic henipaviruses, Hendra, and Nipah are responsible for frequent and fatal disease outbreaks in domestic animals and humans. Specificity of henipavirus attachment glycoproteins (G) for highly species-conserved ephrin ligands underpins their broad host range and is associated with systemic and neurological disease pathologies. Here, we demonstrate that Cedar virus (CedV)—a related henipavirus that is ostensibly nonpathogenic—possesses an idiosyncratic entry receptor repertoire that includes the common henipaviral receptor, ephrin-B2, but, distinct from pathogenic henipaviruses, does not include ephrin-B3. Uniquely among known henipaviruses, CedV can use ephrin-B1 for cellular entry. Structural analyses of CedV-G reveal a key region of molecular specificity that directs ephrin-B1 utilization, while preserving a universal mode of ephrin-B2 recognition. The structural and functional insights presented uncover diversity within the known henipavirus receptor repertoire and suggest that only modest structural changes may be required to modulate receptor specificities within this group of lethal human pathogens.
  • Cecchetti, Cristina; Strauss, Jannik; Stohrer, Claudia; Naylor, Claire; Pryor, Edward; Hobbs, Jeanette; Tanley, Simon; Goldman, Adrian; Byrne, Bernadette (2021)
    Membrane proteins have a range of crucial biological functions and are the target of about 60% of all prescribed drugs. For most studies, they need to be extracted out of the lipid-bilayer, e.g. by detergent solubilisation, leading to the loss of native lipids, which may disturb important protein-lipid/bilayer interactions and thus functional and structural integrity. Relipidation of membrane proteins has proven extremely successful for studying challenging targets, but the identification of suitable lipids can be expensive and laborious. Therefore, we developed a screen to aid the high-throughput identification of beneficial lipids. The screen covers a large lipid space and was designed to be suitable for a range of stability assessment methods. Here, we demonstrate its use as a tool for identifying stabilising lipids for three membrane proteins: a bacterial pyrophosphatase (Tm-PPase), a fungal purine transporter (UapA) and a human GPCR (A(2A)R). A(2A)R is stabilised by cholesteryl hemisuccinate, a lipid well known to stabilise GPCRs, validating the approach. Additionally, our screen also identified a range of new lipids which stabilised our test proteins, providing a starting point for further investigation and demonstrating its value as a novel tool for membrane protein research. The pre-dispensed screen will be made commercially available to the scientific community in future and has a number of potential applications in the field.
  • Fewer, David P.; Metsä-Ketelä, Mikko (2020)
    Abstract Microbes are talented chemists with the ability to generate tremendously complex and diverse natural products which harbor potent biological activities. Natural products are produced using sets of specialized biosynthetic enzymes encoded by secondary metabolism pathways. Here, we present a two-step evolutionary model to explain the diversification of biosynthetic pathways that account for the proliferation of these molecules. We argue that the appearance of natural product families has been a slow and infrequent process. The first step led to the original emergence of bioactive molecules and different classes of natural products. However, much of the chemical diversity observed today has resulted from the endless modification of the ancestral biosynthetic pathways. The second step rapidly modulates the pre-existing biological activities to increase their potency and to adapt to changing environmental conditions. We highlight the importance of enzyme promiscuity in this process, as it facilitates both the incorporation of horizontally transferred genes into secondary metabolic pathways and the functional differentiation of proteins to catalyze novel chemistry. We provide examples where single point mutations or recombination events have been sufficient for new enzymatic activities to emerge. A unique feature in the evolution of microbial secondary metabolism is that gene duplication is not essential but offers opportunities to synthesize more complex metabolites. Microbial natural products are highly important for the pharmaceutical industry due to their unique bioactivities. Therefore, understanding the natural mechanisms leading to the formation of diverse metabolic pathways is vital for future attempts to utilize synthetic biology for the generation of novel molecules.
  • Gonzalez de Cozar, Jose M.; Carretero-Junquera, Maria; Ciesielski, Grzegorz L.; Miettinen, Sini M.; Varjosalo, Markku; Kaguni, Laurie S.; Dufour, Eric; Jacobs, Howard T. (2020)
    In eukaryotes, ribonuclease H1 (RNase H1) is involved in the processing and removal of RNA/DNA hybrids in both nuclear and mitochondrial DNA. The enzyme comprises a C-terminal catalytic domain and an N-terminal hybrid-binding domain (HBD), separated by a linker of variable length, 115 amino acids in Drosophila melanogaster (Dm). Molecular modelling predicted this extended linker to fold into a structure similar to the conserved HBD. Based on a deletion series, both the catalytic domain and the conserved HBD were required for high-affinity binding to heteroduplex substrates, while loss of the novel HBD led to an similar to 90% drop in K-cat with a decreased K-M, and a large increase in the stability of the RNA/DNA hybrid-enzyme complex, supporting a bipartite-binding model in which the second HBD facilitates processivity. Shotgun proteomics following in vivo cross-linking identified single-stranded DNA-binding proteins from both nuclear and mitochondrial compartments, respectively RpA-70 and mtSSB, as prominent interaction partners of Dm RNase H1. However, we were not able to document direct and stable interactions with mtSSB when the proteins were cooverexpressed in S2 cells, and functional interactions between them in vitro were minor.
  • Purhonen, Janne; Grigorjev, Vladislav; Ekiert, Robert; Aho, Noora; Rajendran, Jayasimman; Pietras, Rafal; Truve, Katarina; Wikström, Mårten; Sharma, Vivek; Osyczka, Artur; Fellman, Vineta; Kallijärvi, Jukka (2020)
    We previously observed an unexpected fivefold (35 vs. 200 days) difference in the survival of respiratory chain complex III (CIII) deficient Bcs1/(p.S78G) mice between two congenic backgrounds. Here, we identify a spontaneous homoplasmic mtDNA variant (m.G14904A, mt-Cyb(p.D254N)), affecting the CIII subunit cytochrome b (MT-CYB), in the background with short survival. We utilize maternal inheritance of mtDNA to confirm this as the causative variant and show that it further decreases the low CIII activity in Bcs1/(p.S78G) tissues to below survival threshold by 35 days of age. Molecular dynamics simulations predict D254N to restrict the flexibility of MT-CYB ef loop, potentially affecting RISP dynamics. In Rhodobacter cytochrome bc(1) complex the equivalent substitution causes a kinetics defect with longer occupancy of RISP head domain towards the quinol oxidation site. These findings represent a unique case of spontaneous mitonuclear epistasis and highlight the role of mtDNA variation as modifier of mitochondrial disease phenotypes.
  • Matovic, Jelena; Järvinen, Juulia; Bland, Helena C.; Sokka, Iris K.; Imlimthan, Surachet; Ferrando, Ruth Mateu; Huttunen, Kristiina M.; Timonen, Juri; Peräniemi, Sirpa; Aitio, Olli; Airaksinen, Anu J.; Sarparanta, Mirkka; Johansson, Mikael P.; Rautio, Jarkko; Ekholm, Filip S. (2020)
    Boron neutron capture therapy (BNCT) for cancer is on the rise worldwide due to recent developments of in-hospital neutron accelerators which are expected to revolutionize patient treatments. There is an urgent need for improved boron delivery agents, and herein we have focused on studying the biochemical foundations upon which a successful GLUT1-targeting strategy to BNCT could be based. By combining synthesis and molecular modeling with affinity and cytotoxicity studies, we unravel the mechanisms behind the considerable potential of appropriately designed glucoconjugates as boron delivery agents for BNCT. In addition to addressing the biochemical premises of the approach in detail, we report on a hit glucoconjugate which displays good cytocompatibility, aqueous solubility, high transporter affinity, and, crucially, an exceptional boron delivery capacity in the in vitro assessment thereby pointing toward the significant potential embedded in this approach.
  • Happonen, Lotta J.; Erdmann, Susanne; Garrett, Roger A.; Butcher, Sarah J. (2014)
  • Stadelmann, B.; Bender, J.; Förster, D.; Frey, W.; Nieger, M.; Gudat, D. (2015)
    A unique anionic phosphenium complex was prepared from reaction of an N-heterocyclic chlorophosphine with Collman's reagent or K[HFe(CO)(4)]/NaH and characterized by spectral and XRD data. The complex behaves as an ambident nucleophile. Reactions with acetic acid, ClSnPh3, and a further equivalent of an N-heterocyclic chlorophosphine proceed via electrophilic functionalization at the metal site to yield appropriate mono- or bis-phosphenium complexes. Reaction with MeI at -70 degrees C produces a P-alkylation product as the first spectroscopically detectable intermediate, which decays at a higher temperature to give a mixture of free P-methylated N-heterocyclic phosphine and its Fe(CO)(4) complex. The different reaction products were characterized by spectral and XRD data. Computational studies indicate that the NHP units in all complexes display p-acceptor behaviour but show no disposition to adopt phosphide-like character or formally oxidize the metal centre.
  • Kaddouri, Yassine; Benabbes, Redouane; Ouahhoud, Sabir; Abdellattif, Magda; Hammouti, Belkheir; Touzani, Rachid (2022)
    Bayoud disease affects date palms in North Africa and the Middle East, and many researchers have used various methods to fight it. One of those methods is the chemical use of synthetic compounds, which raises questions centred around the compounds and common features used to prepare targeted molecules. In this review, 100 compounds of tested small molecules, collected from 2002 to 2022 in Web of Sciences, were divided into ten different classes against the main cause of Bayoud disease pathogen Fusarium oxysporum f. sp. albedinis (F.o.a.) with structure-activity relationship (SAR) interpretations for pharmacophore site predictions as (delta(-)center dot center dot center dot delta(-)), where 12 compounds are the most efficient (one compound from each group). The compounds, i.e., (Z)-1-(1.5-Dimethyl-1H-pyrazole-3-yl)-3-hydroxy but-2-en-1-one 7, (Z)-3-(phenyl)-1-(1,5-dimethyl-1H-pyrazole-3-yl)-3-hydroxyprop-2-en-1-one 23, (Z)-1-(1,5-Dimethyl-1H-pyrazole-3-yl)-3-hydroxy-3-(pyridine-2-yl)prop-2-en-1-one 29, and 2,3-bis-[(2-hydroxy-2-phenyl)ethenyl]-6-nitro-quinoxaline 61, have antifungal pharmacophore sites (delta(-)center dot center dot center dot delta(-)) in common in N1---O4, whereas other compounds have only one delta(-) pharmacophore site pushed by the donor effect of the substituents on the phenyl rings. This specificity interferes in the biological activity against F.o.a. Further understanding of mechanistic drug-target interactions on this subject is currently underway.
  • Byggmästar, Jesper Johan André; Nagel, Morten Jesper; Albe, Karsten; Henriksson, Krister Olof Edvin; Nordlund, Kai Henrik (2019)
    We present an analytical bond-order potential for the Fe-O system, capable of reproducing the basic properties of wustite as well as the energetics of oxygen impurities in alpha-iron. The potential predicts binding energies of various small oxygen-vacancy clusters in alpha-iron in good agreement with density functional theory results, and is therefore suitable for simulations of oxygen-based defects in iron. We apply the potential in simulations of the stability and structure of Fe/FeO interfaces and FeO precipitates in iron, and observe that the shape of FeO precipitates can change due to formation of well-defined Fe/FeO interfaces. The interface with crystalline Fe also ensures that the precipitates never become fully amorphous, no matter how small they are.
  • Molina, V.; Rauhalahti, M.; Hurtado, J.; Fliegl, H.; Sundholm, D.; Munoz-Castro, A. (2017)
    [trans-Cu(mu-OH)(mu-dmpz)](6) (1) exhibits six Cu(II) centers effectively coupled through a ligand mediated mechanism leading to a diamagnetic ground state over a wide temperature range. Here we investigate further magneto-structural correlations based on the possible free electron precession along such a copper-based ring-like nanocoil mediated by bridging ligands. We find that in 1, mediated antiferro-magnetic coupling leads to characteristics that induce aromatic ring behavior through evaluation of both induced currents and shielding of cones from a relativistic density functional theory level. According to our gauge calculations including magnetically induced current densities and an induced magnetic field, a sizable ring current strength susceptibility is obtained for the cyclic Cu-N-N-Cu and Cu-O-Cu pathways, allowing a magnetic exchange between the copper centers. Our study suggests that [Cu-6(dmPz)(6)(OH)(6)] consisting of an aromatic ring structure displays aromaticity and superexchange along the Cu-O-Cu and Cu-N-N-Cu backbones, which accounts for 80% and 20% of the overall ring current strength susceptibility, respectively. This reveals the presence of particular aromatic ring characteristics in coordination compounds without a direct metal-metal bond, where several formally paramagnetic centers are antiferromagnetically-coupled through supporting ligands. We envisage that our findings can be extended to other examples depicting ligand-mediated interaction between metal centers.
  • Pantsar, Tatu; Rissanen, Sami; Dauch, Daniel; Laitinen, Tuomo; Vattulainen, Ilpo; Poso, Antti (2018)
    A mutated KRAS protein is frequently observed in human cancers. Traditionally, the oncogenic properties of KRAS missense mutants at position 12 (G12X) have been considered as equal. Here, by assessing the probabilities of occurrence of all KRAS G12X mutations and KRAS dynamics we show that this assumption does not hold true. Instead, our findings revealed an outstanding mutational bias. We conducted a thorough mutational analysis of KRAS G12X mutations and assessed to what extent the observed mutation frequencies follow a random distribution. Unique tissue-specific frequencies are displayed with specific mutations, especially with G12R, which cannot be explained by random probabilities. To clarify the underlying causes for the nonrandom probabilities, we conducted extensive atomistic molecular dynamics simulations (170 its) to study the differences of G12X mutations on a molecular level. The simulations revealed an allosteric hydrophobic signaling network in KRAS, and that protein dynamics is altered among the G12X mutants and as such differs from the wild-type and is mutation-specific. The shift in long-timescale conformational dynamics was confirmed with Markov state modeling. A G12X mutation was found to modify KRAS dynamics in an allosteric way, which is especially manifested in the switch regions that are responsible for the effector protein binding. The findings provide a basis to understand better the oncogenic properties of KRAS G12X mutants and the consequences of the observed nonrandom frequencies of specific G12X mutations.
  • Oliinyk, Olena S.; Chernov, Konstantin G.; Verkhusha, Vladislav V. (2017)
    Bacterial photoreceptors absorb light energy and transform it into intracellular signals that regulate metabolism. Bacterial phytochrome photoreceptors (BphPs), some cyanobacteriochromes (CBCRs) and allophycocyanins (APCs) possess the near-infrared (NIR) absorbance spectra that make them promising molecular templates to design NIR fluorescent proteins (FPs) and biosensors for studies in mammalian cells and whole animals. Here, we review structures, photochemical properties and molecular functions of several families of bacterial photoreceptors. We next analyze molecular evolution approaches to develop NIR FPs and biosensors. We then discuss phenotypes of current BphP-based NIR FPs and compare them with FPs derived from CBCRs and APCs. Lastly, we overview imaging applications of NIR FPs in live cells and in vivo. Our review provides guidelines for selection of existing NIR FPs, as well as engineering approaches to develop NIR FPs from the novel natural templates such as CBCRs.
  • Duyvesteyn, Helen M. E.; Santos-Pérez, Isaac; Peccati , Francesca; Martinez-Castillo, Ane; Walter, Thomas S.; Reguera , David; Goni, Felix M; Jiménez-Osés , Gonzalo; Oksanen, Hanna M; Stuart, David I.; Abrescia, Nicola GA (2021)
    Viruses are very attractive biomaterials owing to their capability as nanocarriers of genetic material. Efforts have been made to functionalize self-assembling viral protein capsids on their exterior or interior to selectively take up different payloads. PRD1 is a double-stranded DNA bacteriophage comprising an icosahedral protein outer capsid and an inner lipidic vesicle. Here, we report the three-dimensional structure of PRD1 in complex with the antipsychotic drug chlorpromazine (CPZ) by cryo-electron microscopy. We show that the jellyrolls of the viral major capsid protein P3, protruding outwards from the capsid shell, serve as scaffolds for loading heterocyclic CPZ molecules. Additional X-ray studies and molecular dynamics simulations show the binding modes and organization of CPZ molecules when complexed with P3 only and onto the virion surface. Collectively, we provide a proof of concept for the possible use of the lattice-like organisation and the quasi-symmetric morphology of virus capsomers for loading heterocyclic drugs with defined properties.
  • Isenberg, Stefan; Weller, Stefan; Kargin, Denis; Valic, Srecko; Schwederski, Brigitte; Kelemen, Zsolt; Bruhn, Clemens; Krekic, Kristijan; Maurer, Martin; Feil, Christoph M.; Nieger, Martin; Gudat, Dietrich; Nyulaszi, Laszlo; Pietschnig, Rudolf (2019)
    Invited for this month's cover picture are the groups of Professors Rudolf Pietschnig at the University of Kassel, Professor Dietrich Gudat at the University of Stuttgart and Professor Laszlo Nyulaszi at the Budapest University of Technology and Economics. The cover picture shows the thermally induced homolytic cleavage of the central P-P bond in a phosphorus-rich bis-ferrocenophane furnishing P-centered radicals (as evidenced by the computed spin-density highlighted in blue). The central P-6 unit in the title compound is a structural analog of the connecting unit in Hittorf's violet phosphorus, which links the orthogonally arranged tubular entities. A portrait of the German physicist Johann Wilhelm Hittorf is included. Read the full text of their Full Paper at 10.1002/open.201900182.
  • Hontani, Yusaku; Shcherbakova, Daria M.; Baloban, Mikhail; Zhu, Jingyi; Verkhusha, Vladislav; Kennis, John T. M. (2016)
    Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes (BphPs) are of great interest for in vivo imaging. They utilize biliverdin (BV) as a chromophore, which is a heme degradation product, and therefore they are straightforward to use in mammalian tissues. Here, we report on fluorescence properties of NIR FPs with key alterations in their BV binding sites. BphP1-FP, iRFP670 and iRFP682 have Cys residues in both PAS and GAF domains, rather than in the PAS domain alone as in wild-type BphPs. We found that NIR FP variants with Cys in the GAF or with Cys in both PAS and GAF show blue-shifted emission with long fluorescence lifetimes. In contrast, mutants with Cys in the PAS only or no Cys residues at all exhibit red-shifted emission with shorter lifetimes. Combining these results with previous biochemical and BphP1-FP structural data, we conclude that BV adducts bound to Cys in the GAF are the origin of bright blue-shifted fluorescence. We propose that the long fluorescence lifetime follows from (i) a sterically more constrained thioether linkage, leaving less mobility for ring A than in canonical BphPs, and (ii) that pi-electron conjugation does not extend on ring A, making excited-state deactivation less sensitive to ring A mobility.
  • Seifert, Tina; Malo, Marcus; Kokkola, Tarja; Engen, Karin; Friden-Saxin, Maria; Wallen, Erik A. A.; Lahtela-Kakkonen, Maija; Jarho, Elina M.; Luthman, Kristina (2014)