Abstract
Betacoronaviruses are a genus within the Coronaviridae family of RNA viruses. They are capable of infecting vertebrates and causing epidemics as well as global pandemics in humans. ...Mitigating the threat posed by Betacoronaviruses requires an understanding of their molecular diversity. The development of novel antivirals hinges on understanding the key regulatory elements within the viral RNA genomes, in particular the 5′-proximal region, which is pivotal for viral protein synthesis. Using a combination of cryo-electron microscopy, atomic force microscopy, chemical probing, and computational modeling, we determined the structures of 5′-proximal regions in RNA genomes of Betacoronaviruses from four subgenera: OC43-CoV, SARS-CoV-2, MERS-CoV, and Rousettus bat-CoV. We obtained cryo-electron microscopy maps and determined atomic-resolution models for the stem-loop-5 (SL5) region at the translation start site and found that despite low sequence similarity and variable length of the helical elements it exhibits a remarkable structural conservation. Atomic force microscopy imaging revealed a common domain organization and a dynamic arrangement of structural elements connected with flexible linkers across all four Betacoronavirus subgenera. Together, these results reveal common features of a critical regulatory region shared between different Betacoronavirus RNA genomes, which may allow targeting of these RNAs by broad-spectrum antiviral therapeutics.
Graphical Abstract
Graphical Abstract
Symmetrical protein cages have evolved to fulfil diverse roles in nature, including compartmentalization and cargo delivery
, and have inspired synthetic biologists to create novel protein assemblies ...via the precise manipulation of protein-protein interfaces. Despite the impressive array of protein cages produced in the laboratory, the design of inducible assemblies remains challenging
. Here we demonstrate an ultra-stable artificial protein cage, the assembly and disassembly of which can be controlled by metal coordination at the protein-protein interfaces. The addition of a gold (I)-triphenylphosphine compound to a cysteine-substituted, 11-mer protein ring triggers supramolecular self-assembly, which generates monodisperse cage structures with masses greater than 2 MDa. The geometry of these structures is based on the Archimedean snub cube and is, to our knowledge, unprecedented. Cryo-electron microscopy confirms that the assemblies are held together by 120 S-Au
-S staples between the protein oligomers, and exist in two chiral forms. The cage shows extreme chemical and thermal stability, yet it readily disassembles upon exposure to reducing agents. As well as gold, mercury(II) is also found to enable formation of the protein cage. This work establishes an approach for linking protein components into robust, higher-order structures, and expands the design space available for supramolecular assemblies to include previously unexplored geometries.
W niniejszej pracy zestawiono środowiskowy i prawny aspekt rozwoju zielonej infrastruktury na terenach miejskich, ze szczególnym uwzględnieniem dachów zielonych. Wykazano, że zarówno istniejąca ...zielona infrastruktura, obejmująca parki, obszary chronione, lasy, pola itd., jak i kreowana de novo, np. w postaci dachów zielonych, minimalizuje skutki zmian klimatu i jest kluczowa dla zrównoważonego rozwoju. Zidentyfikowane korzyści płynące z wprowadzania dachów zielonych to m.in.: oszczędność energii, poprawa jakości powietrza i wody, wspieranie renaturyzacji miast i wzrost różnorodności biologicznej, które łącznie sprzyjają polepszeniu jakości życia w miastach. Dlatego też wskazano potrzebę usankcjonowania rozwoju zielonej infrastruktury na terenach miejskich w systemie prawnym. Dostrzegając aktualne zagrożenia środowiskowe oraz korzyści z zielonej infrastruktury, ustawodawca europejski podał wytyczne dotyczące jej tworzenia. W artykule dokonano przeglądu stanu prawnego i oceniono, w jaki sposób polityka Unii Europejskiej została zaimplementowana do wspólnotowego oraz krajowego porządku prawnego. W szczególności zbadano, w jakim zakresie organy samorządu terytorialnego uwzględniają zieloną infrastrukturę w miejscowych planach zagospodarowania przestrzennego.
Artificial protein cages have great potential in a number of areas including cargo capture and delivery and as artificial vaccines. Here, we investigate an artificial protein cage whose assembly is ...triggered by gold nanoparticles. Using biochemical and biophysical methods we were able to determine both the mechanical properties and the gross compositional features of the cage which, combined with mathematical models and biophysical data, allowed the structure of the cage to be predicted. The accuracy of the overall geometrical prediction was confirmed by the cryo-EM structure determined to sub-5 Å resolution. This showed the cage to be nonregular but similar to a dodecahedron, being constructed from 12 11-membered rings. Surprisingly, the structure revealed that the cage also contained a single, small gold nanoparticle at each 3-fold axis meaning that each cage acts as a synthetic framework for regular arrangement of 20 gold nanoparticles in a three-dimensional lattice.
Virus-like particles (VLPs) have significant potential as artificial vaccines and drug delivery systems. The ability to control their size has wide ranging utility but achieving such controlled ...polymorphism using a single protein subunit is challenging as it requires altering VLP geometry. Here we achieve size control of MS2 bacteriophage VLPs via insertion of amino acid sequences in an external loop to shift morphology to significantly larger forms. The resulting VLP size and geometry is controlled by altering the length and type of the insert. Cryo electron microscopy structures of the new VLPs, in combination with a kinetic model of their assembly, show that the abundance of wild type (
= 3),
= 4, D3 and D5 symmetrical VLPs can be biased in this way. We propose a mechanism whereby the insert leads to a change in the dynamic behavior of the capsid protein dimer, affecting the interconversion between the symmetric and asymmetric conformers and thus determining VLP size and morphology.
Hypusination is a unique post-translational modification of the eukaryotic translation factor 5A (eIF5A) that is essential for overcoming ribosome stalling at polyproline sequence stretches. The ...initial step of hypusination, the formation of deoxyhypusine, is catalyzed by deoxyhypusine synthase (DHS), however, the molecular details of the DHS-mediated reaction remained elusive. Recently, patient-derived variants of DHS and eIF5A have been linked to rare neurodevelopmental disorders. Here, we present the cryo-EM structure of the human eIF5A-DHS complex at 2.8 Å resolution and a crystal structure of DHS trapped in the key reaction transition state. Furthermore, we show that disease-associated DHS variants influence the complex formation and hypusination efficiency. Hence, our work dissects the molecular details of the deoxyhypusine synthesis reaction and reveals how clinically-relevant mutations affect this crucial cellular process.
Cage forming proteins have numerous potential applications in biomedicine and biotechnology, where the iron storage ferritin is a widely used example. However, controlling ferritin cage ...assembly/disassembly remains challenging, typically requiring extreme conditions incompatible with many desirable cargoes, particularly for more fragile biopharmaceuticals. Recently, a ferritin from the hyperthermophile bacterium
Thermotoga maritima
(TmFtn) has been shown to have reversible assembly under mild conditions, offering greater potential biocompatibility in terms of cargo access and encapsulation. Like
Archeoglobus fulgidus
ferritin (AfFtn), TmFtn forms 24mer cages mediated by metal ions (Mg
2+
). We have solved the crystal structure of the wild type TmFtn and several mutants displaying different assembly/disassembly properties. These data combined with other biophysical studies allow us to suggest candidate interfacial amino acids crucial in controlling assembly. This work deepens our understanding of how these ferritin complexes assemble and is a useful step towards production of triggerable ferritins in which these properties can be finely designed and controlled.
Modifications to a protein cage whose assembly depends on the presence of metal ions can modulate the extent of its dependence and in some cases convert the assembly to be salt independent.
Engineered protein cages are promising tools that can be customized for applications in medicine and nanotechnology. A major challenge is developing a straightforward strategy for endowing cages with ...bespoke, inducible disassembly. Such cages would allow release of encapsulated cargoes at desired timing and location. Here, we achieve such programmable disassembly using protein cages, in which the subunits are held together by different molecular cross-linkers. This modular system enables cage disassembly to be controlled in a condition-dependent manner. Structural details of the resulting cages were determined using cryo–electron microscopy, which allowed observation of bridging cross-linkers at intended positions. Triggered disassembly was demonstrated by high-speed atomic force microscopy and subsequent cargo release using an encapsulated Förster resonance energy transfer pair whose signal depends on the quaternary structure of the cage.
DNA glycosylases are emerging as relevant pharmacological targets in inflammation, cancer and neurodegenerative diseases. Consequently, the search for inhibitors of these enzymes has become a very ...active research field. As a continuation of previous work that showed that 2-thioxanthine (2TX) is an irreversible inhibitor of zinc finger (ZnF)-containing Fpg/Nei DNA glycosylases, we designed and synthesized a mini-library of 2TX-derivatives (TXn) and evaluated their ability to inhibit Fpg/Nei enzymes. Among forty compounds, four TXn were better inhibitors than 2TX for Fpg. Unexpectedly, but very interestingly, two dithiolated derivatives more selectively and efficiently inhibit the zincless finger (ZnLF)-containing enzymes (human and mimivirus Neil1 DNA glycosylases hNeil1 and MvNei1, respectively). By combining chemistry, biochemistry, mass spectrometry, blind and flexible docking and X-ray structure analysis, we localized new TXn binding sites on Fpg/Nei enzymes. This endeavor allowed us to decipher at the atomic level the mode of action for the best TXn inhibitors on the ZnF-containing enzymes. We discovered an original inhibition mechanism for the ZnLF-containing Fpg/Nei DNA glycosylases by disulfide cyclic trimeric forms of dithiopurines. This work paves the way for the design and synthesis of a new structural class of inhibitors for selective pharmacological targeting of hNeil1 in cancer and neurodegenerative diseases.
An artificial protein cage made from a 12-membered ring Stupka, Izabela; Biela, Artur P; Piette, Bernard ...
Journal of materials chemistry. B, Materials for biology and medicine,
01/2024, Letnik:
12, Številka:
2
Journal Article
Recenzirano
Odprti dostop
Artificial protein cages have great potential in diverse fields including as vaccines and drug delivery vehicles. TRAP-cage is an artificial protein cage notable for the way in which the interface ...between its ring-shaped building blocks can be modified such that the conditions under which cages disassemble can be controlled. To date, TRAP-cages have been constructed from homo-11mer rings,
i.e.
, hendecamers. This is interesting as convex polyhedra with identical regular faces cannot be formed from hendecamers. TRAP-cage overcomes this limitation due to intrinsic flexibility, allowing slight deformation to absorb any error. The resulting TRAP-cage made from 24 TRAP 11mer rings is very close to regular with only very small errors necessary to allow the cage to form. The question arises as to the limits of the error that can be absorbed by a protein structure in this way before the formation of an apparently regular convex polyhedral becomes impossible. Here we use a naturally occurring TRAP variant consisting of twelve identical monomers (
i.e.
, a dodecamer) to probe these limits. We show that it is able to form an apparently regular protein cage consisting of twelve TRAP rings. Comparison of the cryo-EM structure of the new cage with theoretical models and related cages gives insight into the rules of cage formation and allows us to predict other cages that may be formed given TRAP-rings consisting of different numbers of monomers.
Changing the symmetry of the constituent ring-shaped building block of an artificial cage.
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