Targeting G-quadruplexes in the rhinovirus genome by Pyridostatin inhibits uncoating 3 and highlights a critical role for sodium ions.

The ~ 2.4 µm long rhinovirus ss(+)RNA genome consists of roughly 7,200 nucleotides. It is tightly folded to fit into the ~ 22 nm diameter void in the protein capsid. In addition to previously predicted secondary structural elements in the RNA, using the QGRS mapper, we revealed the presence of multiple quadruplex forming G-rich sequences (QGRS) in the RV-A, B, and C clades, with four of them being exquisitely conserved. The biophysical analyses of ribooligonucleotides corresponding to selected QGRS demonstrate G-quadruplex (GQ) formation in each instance and resulted in discovering another example of an unconventional, two-layer zero-nucleotide loop RNA GQ stable at physiological conditions. By exploiting the temperature-dependent viral breathing to allow diffusion of small compounds into the virion, we demonstrate that the GQ-binding compounds PhenDC3 and pyridostatin (PDS) uniquely interfere with viral uncoating. Remarkably, this inhibition was entirely prevented in the presence of K+ but not Na+, despite the higher GQ stabilising effect of K+. Based on virus thermostability studies combined with ultrastructural imaging of isolated viral RNA, we propose a mechanism where Na+ keeps the encapsidated genome loose, allowing its penetration by PDS to promote the transition of QGRS sequestered in alternative metastable structures into GQs. The resulting conformational change then materialises in a severely compromised RNA release from the proteinaceous shell. Targeting extracellularly circulating RVs with GQ-stabilisers might thus become a novel way of combating the common cold.

Targeting G-quadruplexes in the rhinovirus genome by Pyridostatin inhibits uncoating and highlights a critical role for sodium ions.

The ~ 2.4 µm long rhinovirus ss(+)RNA genome consists of roughly 7,200 nucleotides. It is tightly folded to fit into the ~ 22 nm diameter void in the protein capsid. In addition to previously predicted secondary structural elements in the RNA, using the QGRS mapper, we revealed the presence of multiple quadruplex forming G-rich sequences (QGRS) in the RV-A, B, and C clades, with four of them being exquisitely conserved. The biophysical analyses of ribooligonucleotides corresponding to selected QGRS demonstrate G-quadruplex (GQ) formation in each instance and resulted in discovering another example of an unconventional, two-layer zero-nucleotide loop RNA GQ stable at physiological conditions. By exploiting the temperature-dependent viral breathing to allow diffusion of small compounds into the virion, we demonstrate that the GQ-binding compounds PhenDC3 and pyridostatin (PDS) uniquely interfere with viral uncoating. Remarkably, this inhibition was entirely prevented in the presence of K+ but not Na+, despite the higher GQ stabilising effect of K+. Based on virus thermostability studies combined with ultrastructural imaging of isolated viral RNA, we propose a mechanism where Na+ keeps the encapsidated genome loose, allowing its penetration by PDS to promote the transition of QGRS sequestered in alternative metastable structures into GQs. The resulting conformational change then materialises in a severely compromised RNA release from the proteinaceous shell. Targeting extracellularly circulating RVs with GQ-stabilisers might thus become a novel way of combating the common cold.

Parametric Analysis of the Shear Lag Effect in Tube Structural Systems of Tall Buildings

Horizontal loads such as earthquake and wind are considered dominant loads for the design of tall buildings. One of the most efficient structural systems in this regard is the tube structural system. Even though such systems have a high resistance when it comes to horizontal loads, the shear lag effect that is characterized by an incomplete and uneven activation of vertical elements may cause a series of problems such as the deformation of internal panels and secondary structural elements, which cumulatively grow with the height of the building. In this paper, the shear lag effect in a typical tube structure will be observed and analyzed on a series of different numerical models. A parametric analysis will be conducted with a great number of variations in the structural elements and building layout, for the purpose of giving recommendations for an optimal design of a tube structural system.

NMR resonance assignment and dynamics of profilin from Heimdallarchaeota

The origin of the eukaryotic cell is an unsettled scientific question. The Asgard superphylum has emerged as a compelling target for studying eukaryogenesis due to the previously unseen diversity of eukaryotic signature proteins. However, our knowledge about these proteins is still relegated to metagenomic data and very little is known about their structural properties. Additionally, it is still unclear if these proteins are functionally homologous to their eukaryotic counterparts. Here, we expressed, purified and structurally characterized profilin from Heimdallarchaeota in the Asgard superphylum. The structural analysis shows that while this profilin possesses similar secondary structural elements as eukaryotic profilin, it contains additional secondary structural elements that could be critical for its function and an indication of divergent evolution.

Assembly intermediates of orthoreovirus captured in the cell

Traditionally, molecular assembly pathways for viruses are inferred from high resolution structures of purified stable intermediates, low resolution images of cell sections and genetic approaches. Here, we directly visualise an unsuspected ‘single shelled’ intermediate for a mammalian orthoreovirus in cryo-preserved infected cells, by cryo-electron tomography of cellular lamellae. Particle classification and averaging yields structures to 5.6 Å resolution, sufficient to identify secondary structural elements and produce an atomic model of the intermediate, comprising 120 copies each of protein λ1 and σ2. This λ1 shell is ‘collapsed’ compared to the mature virions, with molecules pushed inwards at the icosahedral fivefolds by ~100 Å, reminiscent of the first assembly intermediate of certain prokaryotic dsRNA viruses. This supports the supposition that these viruses share a common ancestor, and suggests mechanisms for the assembly of viruses of the Reoviridae. Such methodology holds promise for dissecting the replication cycle of many viruses.

NMR backbone assignment and dynamics of Profilin from Heimdallarchaeota

The origin of the eukaryotic cell is an unsettled scientific question. The Asgard superphylum has emerged as a compelling target for studying eukaryogenesis due to the previously unseen diversity of eukaryotic signature proteins. However, our knowledge about these proteins is still relegated to metagenomic data and very little is known about their structural properties. Additionally, it is still unclear if these proteins are functionally homologous to their eukaryotic counterparts. Here, we expressed, purified and structurally characterized profilin from Heimdallarchaeota in the Asgard superphylum. The structural analysis shows that while this profilin possess similar secondary structural elements as eukaryotic profilin, it contains additional secondary structural elements that could be critical for its function and an indication of divergent evolution.

Monitoring the Site-Specific Solid-State NMR Data in Oligopeptides

Reliable values of the solid-state NMR (SSNMR) parameters together with precise structural data specific for a given amino acid site in an oligopeptide are needed for the proper interpretation of measurements aiming at an understanding of oligopeptides’ function. The periodic density functional theory (DFT)-based computations of geometries and SSNMR chemical shielding tensors (CSTs) of solids are shown to be accurate enough to support the SSNMR investigations of suitably chosen models of oriented samples of oligopeptides. This finding is based on a thorough comparison between the DFT and experimental data for a set of tripeptides with both 13Cα and 15Namid CSTs available from the single-crystal SSNMR measurements and covering the three most common secondary structural elements of polypeptides. Thus, the ground is laid for a quantitative description of local spectral parameters of crystalline oligopeptides, as demonstrated for the backbone 15Namid nuclei of samarosporin I, which is a pentadecapeptide (composed of five classical and ten nonproteinogenic amino acids) featuring a strong antimicrobial activity.

Structure of the Cytoplasmic Ring of the Xenopus laevis Nuclear Pore Complex

Nuclear pore complex (NPC) exhibits structural plasticity and has only been characterized at local resolutions of up to 15 Å for the cytoplasmic ring (CR). Here we present a single-particle cryo-electron microscopy (cryo-EM) structure of the CR from Xenopus laevis NPC at average resolutions of 5.5-7.9 Å, with local resolutions reaching 4.5 Å. Improved resolutions allow identification and placement of secondary structural elements in the majority of the CR components. The two Y complexes in each CR subunit interact with each other and associate with those from flanking subunits, forming a circular scaffold. Within each CR subunit, the Nup358-containing region wraps around the stems of both Y complexes, likely stabilizing the scaffold. Nup205 connects the short arms of the two Y complexes and associates with the stem of a neighbouring Y complex. The Nup214-containing region uses an extended coiled-coil to link Nup85 of the two Y complexes and protrudes into the axial pore of the NPC. These previously uncharacterized structural features reveal insights into NPC assembly.

Structural and conformational stability of hemocyanin from the garden snail Cornu aspersum

Various aspects of biomedical applications of molluscan hemocyanins, associated with their immunogenic properties and antitumor activity, promoted us to perform structural studies on these glycoproteins. The stability and reassociation behavior of native Cornu aspersum hemocyanin (CaH) are studied in the presence of different concentrations of Ca2+ and Mg2+ ions and pH values using electron microscopy. Higher concentrations of those ions led to a more rapid reassociation of CaH, resulting in stable multidecamers with different lengths. The conformational changes of native CaH are investigated within a wide pH-temperature range by UV circular dichroism. The relatively small changes of initial [θ]λ indicated that many secondary structural elements are preserved, even at high temperatures above 80°C, especially at neutral pH. The mechanism of thermal unfolding of CaH has a complicated character, and the process is irreversible. The conformational stability of the native didecameric aggregates of CaH toward various denaturants indicates that hydrophilic and polar forces stabilize the quaternary structure. For the first time, the unfolding of native CaH in water solutions in the presence of four different denaturants is investigated. The free energy of stabilization in water, ∆GDH2O, was calculated in the range of 15.48–16.95 kJ mol−1. The presented results will facilitate the further investigation of the properties and potential applications of CaH.