The molecular basis for the severity and rapid spread of the COVID-19 disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is largely unknown. ORF8 is a rapidly evolving ...accessory protein that has been proposed to interfere with immune responses. The crystal structure of SARS-CoV-2 ORF8 was determined at 2.04-Å resolution by X-ray crystallography. The structure reveals a ∼60-residue core similar to SARS-CoV-2 ORF7a, with the addition of two dimerization interfaces unique to SARS-CoV-2 ORF8. A covalent disulfide-linked dimer is formed through an N-terminal sequence specific to SARS-CoV-2, while a separate noncovalent interface is formed by another SARS-CoV-2-specific sequence,
YIDI
Together, the presence of these interfaces shows how SARS-CoV-2 ORF8 can form unique large-scale assemblies not possible for SARS-CoV, potentially mediating unique immune suppression and evasion activities.
X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between ...pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.
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•Acoustic methods inject crystal-containing droplets directly from microplate wells•On-demand acoustic injection uses crystals efficiently without orifices or clogging•Diffraction patterns from crystals measuring several tens of μm are of high quality•Complete datasets can be obtained from fewer than 50,000 crystals
Acoustic droplet ejection provides an automated tool for efficient use of protein crystals in SFX experiments. Roessler et al. used this method to deliver crystal-containing droplets into the XFEL beam to coincide with each X-ray pulse.
Complete folding is not a prerequisite for protein function, as disordered and partially folded states of proteins frequently perform essential biological functions. In order to understand their ...functions at the molecular level, we utilized diverse experimental measurements to calculate ensemble models of three nonhomologous, intrinsically disordered proteins: I-2, spinophilin, and DARPP-32, which bind to and regulate protein phosphatase 1 (PP1). The models demonstrate that these proteins have dissimilar propensities for secondary and tertiary structure in their unbound forms. Direct comparison of these ensemble models with recently determined PP1 complex structures suggests a significant role for transient, preformed structure in the interactions of these proteins with PP1. Finally, we generated an ensemble model of partially disordered I-2 bound to PP1 that provides insight into the relationship between flexibility and biological function in this dynamic complex.
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► First ensemble comparison of three different IDPs that bind the same target, PP1 ► Ensemble models of unbound PP1 regulators show diverse transient 2° and 3° structure ► Free and bound state similarities suggest preformed structure is important ► Model of partially disordered PP1:I-2 complex provides insight into function
BST-2/tetherin is a host antiviral molecule that functions to potently inhibit the release of enveloped viruses from infected cells. In return, viruses have evolved antagonists to this activity. ...BST-2 traps budding virions by using two separate membrane-anchoring regions that simultaneously incorporate into the host and viral membranes. Here, we detailed the structural and biophysical properties of the full-length BST-2 ectodomain, which spans the two membrane anchors. The 1.6-Å crystal structure of the complete mouse BST-2 ectodomain reveals an ∼145-Å parallel dimer in an extended α-helix conformation that predominantly forms a coiled coil bridged by three intermolecular disulfides that are required for stability. Sequence analysis in the context of the structure revealed an evolutionarily conserved design that destabilizes the coiled coil, resulting in a labile superstructure, as evidenced by solution x-ray scattering displaying bent conformations spanning 150 and 180 Å for the mouse and human BST-2 ectodomains, respectively. Additionally, crystal packing analysis revealed possible curvature-sensing tetrameric structures that may aid in proper placement of BST-2 during the genesis of viral progeny. Overall, this extended coiled-coil structure with inherent plasticity is undoubtedly necessary to accommodate the dynamics of viral budding while ensuring separation of the anchors.
To take full advantage of advanced data collection techniques and high beam flux at next‐generation macromolecular crystallography beamlines, rapid and reliable methods will be needed to mount and ...align many samples per second. One approach is to use an acoustic ejector to eject crystal‐containing droplets onto a solid X‐ray transparent surface, which can then be positioned and rotated for data collection. Proof‐of‐concept experiments were conducted at the National Synchrotron Light Source on thermolysin crystals acoustically ejected onto a polyimide `conveyor belt'. Small wedges of data were collected on each crystal, and a complete dataset was assembled from a well diffracting subset of these crystals. Future developments and implementation will focus on achieving ejection and translation of single droplets at a rate of over one hundred per second.
Sequence analysis revealed phospholipase A2 (PLA2) motifs in capsid proteins of parvoviruses. Although PLA2 activity is not known to exist in viruses, putative PLA2s from divergent parvoviruses, ...human B19, porcine parvovirus, and insect GmDNV (densovirus from Galleria mellonella), can emulate catalytic properties of secreted PLA2. Mutations of critical amino acids strongly reduce both PLA2 activity and, proportionally, viral infectivity, but cell surface attachment, entry, and endocytosis by PLA2-deficient virions are not affected. PLA2 activity is critical for efficient transfer of the viral genome from late endosomes/lysosomes to the nucleus to initiate replication. These findings offer the prospect of developing PLA2 inhibitors as a new class of antiviral drugs against parvovirus infections and associated diseases.
Highlights ► In monocytes, CCL19 causes potent phosphorylation of p38, ERK1/2, and JNK. ► MAPK phosphorylation affects monocyte migration. ► PGE2 is an important activator of RhoA in monocytes. ► ...ROCK activity is essential for PGE2 -mediated migration in response to CCL19.
Rhodopseudomonas palustris metabolizes aromatic compounds derived from lignin degradation products and has the potential for bioremediation of xenobiotic compounds. We recently identified four ...possible solute-binding proteins in R. palustris that demonstrated binding to aromatic lignin monomers. Characterization of these proteins in the absence and presence of the aromatic ligands will provide unprecedented insights into the specificity and mode of aromatic ligand binding in solute-binding proteins. Here, we report the thermodynamic and structural properties of the proteins with aromatic ligands using isothermal titration calorimetry, small/wide angle x-ray scattering, and theoretical predictions. The proteins exhibit high affinity for the aromatic substrates with dissociation constants in the low micromolar to nanomolar range. The global shapes of the proteins are characterized by flexible ellipsoid-like structures with maximum dimensions in the 80–90-Å range. The data demonstrate that the global shapes remained unaltered in the presence of the aromatic ligands. However, local structural changes were detected in the presence of some ligands, as judged by the observed features in the wide angle x-ray scattering regime at q ∼0.20–0.40 Å−1. The theoretical models confirmed the elongated nature of the proteins and showed that they consist of two domains linked by a hinge. Evaluation of the protein-binding sites showed that the ligands were found in the hinge region and that ligand stabilization was primarily driven by hydrophobic interactions. Taken together, this study shows the capability of identifying solute-binding proteins that interact with lignin degradation products using high throughput genomic and biophysical approaches, which can be extended to other organisms.
Background: Aromatic binding proteins in R. palustris transport lignin degradation products and have the potential for bioremediation.
Results: The proteins bind aromatic compounds with high affinity and have dynamic elongated structures.
Conclusion: Ligand binding induces local changes of residues that stabilize the compounds through hydrophobic interactions.
Significance: The results provide thermodynamic and structural insights into solute-binding proteins for lignin degradation products.
High throughput screening technologies such as acoustic droplet ejection (ADE) greatly increase the rate at which X-ray diffraction data can be acquired from crystals. One promising high throughput ...screening application of ADE is to rapidly combine protein crystals with fragment libraries. In this approach, each fragment soaks into a protein crystal either directly on data collection media or on a moving conveyor belt which then delivers the crystals to the X-ray beam. By simultaneously handling multiple crystals combined with fragment specimens, these techniques relax the automounter duty-cycle bottleneck that currently prevents optimal exploitation of third generation synchrotrons. Two factors limit the speed and scope of projects that are suitable for fragment screening using techniques such as ADE. Firstly, in applications where the high throughput screening apparatus is located inside the X-ray station (such as the conveyor belt system described above), the speed of data acquisition is limited by the time required for each fragment to soak into its protein crystal. Secondly, in applications where crystals are combined with fragments directly on data acquisition media (including both of the ADE methods described above), the maximum time that fragments have to soak into crystals is limited by evaporative dehydration of the protein crystals during the fragment soak. Here we demonstrate that both of these problems can be minimized by using small crystals, because the soak time required for a fragment hit to attain high occupancy depends approximately linearly on crystal size.
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