- About this book
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- PDB Learn: Structural Biology Highlights: SARS Coronavirus Nonstructural Protein 1
- Biochemistry and biology of coronaviruses.
Eventually most of the protein sequences, except the disordered C-terminal tail in nsp10 and artificial tags generated from vectors, were involved in the final structure, and ordered water molecules were added. All the RNA substrates were extracted with phenol-chloroform and precipitated with ethanol. Purified recombinant or mutant proteins 0. The extent of 32 P-labeled cap was determined by scanning the chromatogram with a PhosphorImager .
The reaction mixtures were incubated on ice and irradiated with nm UV light in a Hoefer UVC cross-linking oven for 30 min. The distance of samples from the UV tubes was 4 cm. The gels were soaked in Enlightning Solution PerkinElmer and used for fluorography . The gel shift assay provides a simple and rapid method for detecting RNA-binding proteins. This method has been widely used in the study of sequence-specific RNA-binding proteins. The reactions were incubated at room temperature for 25 min, and separated by nondenaturing polyacrylamide gel N-PAGE. Dilution heats of SAM were measured by injecting SAM solution into buffer alone and were subtracted from the experimental curves prior to data analysis.
Dilution heats of purified proteins were measured by injecting purified proteins solution into buffer alone and were subtracted from the experimental curves prior to data analysis.
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The resulting data were fitted to a single set of identical sites model using MicroCal ORIGIN software supplied with the instrument, and the binding stoichiometry, N , the standard molar enthalpy change for the binding, , and the dissociation constant, K d , were thus obtained. A The SAM binding cleft of nsp16 built by three loop regions. Nsp10 is show as ribbon and colored in green. Nsp16 is shown as surface and colored in cyan. Loop 71—79, loop — and loop — regions are colored in yellow. SAM is shown as sticks and colored in magenta.
Structure-based sequence alignments of nsp16 and nsp The secondary structure of VP39 is shown above and that of nsp16 below the alignment. K-D-K-E surface site in the central groove of nsp Lys, Asp, Lys and Glu are shown as sticks and colored in magenta.
We thank Dr. China for the technical advices in structural analysis and molecular modeling. YC and DG thankfully acknowledge the Institute of Biotechnology, University of Helsinki and Academy of Finland for providing research facilities and support during their visits. Author Summary The distinctive feature of eukaryotic mRNAs is the presence of methylated cap structure that is required for mRNA stability and protein translation. Download: PPT. Figure 1. Figure 2. Figure 3. Figure 4. Figure 6. Structural mechanisms of nsp10 in stimulating the SAM binding of nsp Figure 7.
Structural mechanisms of nsp10 in stimulating the binding of capped RNA to nsp Crystallization and data collection Crystals were grown by the hanging-drop vapor diffusion method. Structure determination and refinement The structure was solved by the multi-wavelength anomalous diffraction MAD method based on two sets of derivative data and one set of native data. Biochemical assays for MTase activity Purified recombinant or mutant proteins 0. Gel shift assay The gel shift assay provides a simple and rapid method for detecting RNA-binding proteins. Supporting Information. Figure S1. Figure S2.
Figure S3. Table S1.
Table S2. Acknowledgments We thank Dr. References 1. J Mol Biol — View Article Google Scholar 2. J Virol — View Article Google Scholar 3. PLoS Biol 6: e View Article Google Scholar 4. PLoS Pathog 4: e View Article Google Scholar 5. View Article Google Scholar 6. J Biochem Mol Biol — View Article Google Scholar 7. View Article Google Scholar 8. View Article Google Scholar 9. View Article Google Scholar Bhardwaj K, Guarino L, Kao CC The severe acute respiratory syndrome coronavirus Nsp15 protein is an endoribonuclease that prefers manganese as a cofactor.
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PDB Learn: Structural Biology Highlights: SARS Coronavirus Nonstructural Protein 1
The viral spike glycoprotein S utilizes angiotensin-converting enzyme 2 ACE2 as a host protein receptor and mediates fusion of the viral and host membranes, making S essential to viral entry into host cells and host species tropism. As SARS-CoV enters host cells, the viral S is believed to undergo a number of conformational transitions as it is cleaved by host proteases and binds to host receptors.
We recently developed stabilizing mutations for coronavirus spikes that prevent the transition from the pre-fusion to post-fusion states. Severe acute respiratory syndrome coronavirus SARS-CoV emerged in humans from an animal reservoir in and rapidly spread globally causing 8, cases and associated deaths in 26 countries through July 1.
Biochemistry and biology of coronaviruses.
SARS-CoV reappeared in a second smaller outbreak in , but has since disappeared from human circulation. However, closely related coronaviruses, such as WIV1, currently circulate in bat reservoirs and are capable of utilizing human receptors to enter cells 2 and there are no vaccines or virus-specific treatments available for human use. The more recent emergence of Middle East respiratory syndrome coronavirus MERS-CoV 1 and the likelihood of future zoonotic transmission of novel coronaviruses to humans from animal reservoirs makes robust reagent development for the display of neutralizing epitopes of great importance to human health.
Understanding how coronavirus S glycoproteins are processed and bind to host receptors is key to the development of coronavirus vaccines and therapeutics. Coronaviruses are enveloped viruses possessing large, trimeric spike glycoproteins S required for the recognition of host receptors for many coronaviruses as well as the fusion of viral and host cell membranes for viral entry into cells 3. During viral egress from infected host cells, some coronavirus S proteins are cleaved into S1 and S2 subunits. The S1 subunit is responsible for host-receptor binding while the S2 subunit contains the membrane-fusion machinery.
Subsequent conformational changes in the second heptad repeat region HR2 of S2 form a six-helix bundle with HR1, fusing the viral and host membranes and allowing for release of the viral genome into host cells Coronavirus S is also the target of neutralizing antibodies 11 , making an understanding of S structure and conformational states pertinent for investigating S antigenic surfaces and designing vaccines.
Each of these domains have been implicated in binding to host receptors, depending on the coronavirus in question. Recent examination using cryo-electron microscopy cryo-EM has illuminated the prefusion structures of coronavirus spikes 15 , 16 , 17 , 18 , 19 , 20 , 21 , The instability of the prefusion state presents a significant challenge for the production of protein antigens for antigenic presentation of the prefusion antibody epitopes that are most likely to lead to neutralizing responses.
Recently, we presented the design of two proline mutations 2P for the prefusion stabilization of coronavirus S proteins The stabilized MERS-CoV S 2P ectodomain was shown to maintain the prefusion spike conformation, have similar antibody recognition as wild-type S and possess higher immunogenicity.
This included a 3. The S1 domains surround the helical S2 subunits and interdigitate at the membrane-distal apex of the trimeric spike. The structure of the S2 subunit appears to be highly conserved across coronavirus genera 18 , 20 , Coordinate models derived from C3 symmetry cryo-EM reconstructions are shown.
S1 regions are shown in blue and S2 regions are shown in green. Comparison of putative bi-partite fusion peptide regions. Subunits are colored as in Fig. Sequence alignment was prepared with Clustal Omega Gui et al. Some possibilities for these different observations may be the differences in spike expression system yielding alternate glycosylation patterns or alternate protein constructs, but the true source remains unclear.
The labeled description of each class begins at the lower RBD when viewed at the membrane distal apex and proceeds clockwise. Side views and membrane-distal top views are shown for each reconstruction. S1 regions are shown in blue, S2 regions are shown in green and soluble ACE2 is shown in orange. The significance of this difference remains unclear. This poor saturation is illustrated by the small proportion of triple-bound ACE2 and the majority of spikes that are unbound by receptor.
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This difference is likely due to poor density in the hinge regions between the S1 RBD and subdomain 1 SD-1 in these previous reconstructions 15 , 22 rather than the presentation of a unique receptor-bound conformation or the influence of the stabilizing mutations. This yielded a 7. ACE2-receptor binding and induced conformational changes. Colors are as in Fig. The observation that host-receptor engagement of the S1 RBD disrupts interactions within spike in a manner independent of direct receptor-spike contacts suggests a flexible mechanism for how different coronavirus spikes may bind to diverse protein receptors with their S1 RBD and facilitate fusion with host cells.
However, fine examination of the ACE2-bound S structure revealed a modest conformational change in the S2 central helix where the S2 had been uncapped by a receptor-bound RBD. This subtle transition may indicate that conformational changes towards fusion are initiated in the central helix.
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N-terminal to the central helix are the 2P mutations that stabilize the spike in its prefusion conformation. These stabilizing mutations may block the propagation of conformational changes beyond the subtle change to a short 3 10 -helix, providing a possible mechanism for their prefusion stabilizing effect. However, SARS-CoV S can be cleaved in vitro by exogenous trypsin at an equivalent site, and it has been proposed that a similar cleavage event may occur in vivo by trypsin-like proteases during viral entry 27 , This site is thought to be cleaved by host proteases after receptor recognition 4 , 5 , 6 and liberates the viral fusion peptide to insert into host membranes in a S2 pre-hairpin intermediate To examine the trypsin protease sensitivity of our prefusion-stabilized coronavirus spikes, we carried out limited proteolysis experiments of both wild-type and 2P SARS-CoV S ectodomains in the presence and absence of soluble ACE2 receptor.
A time course of the proteolysis revealed that all four samples are cleaved to S1 and S2 products at equivalent rates at similar sites Supplementary Fig. Nearing the end of the time course additional lower molecular weight bands are observed which we interpret to be degradation of the S1 subunit. Using all-particles and C3 symmetry yielded a reconstruction at 3. The last residue in S1 T and the first residue in S2 K visualized in the coordinates are labeled. Exposure of this site for cleavage may require remodeling of this penultimate loop or HR1 beyond the conformation observed in the prefusion state and hence possibly not accessible in our prefusion-stabilized S 2P ectodomains.
Our structures of the SARS-CoV S 2P ectodomain bound to a soluble form of human ACE2 receptor show that any conformational changes induced in S by receptor binding are more likely to be due to the disruption of protein-protein interactions rather than the formation of additional contacts between the S1 RBD and other regions of S Supplementary Fig.
Though more extensive changes are likely to drive conformational rearrangements accompanying membrane fusion, the 2P prefusion stabilizing mutations adjacent to the short 3 10 -helix may block these changes from occurring. However, both wild-type and 2P SARS-CoV S ectodomains bound to ACE2 were cleaved equally by trypsin, indicating that at least at the level of trypsin protease susceptibility, these two receptor-bound complexes appear equivalent. Strategies similar to those we have used have been employed to stabilize HIV envelope glycoprotein Env trimers in prefusion conformation 34 , Similarly, the soluble, stabilized versions of Env undergo large conformational changes upon CD4 binding 37 , 38 , Conversely, the Ebola virus glycoprotein GP undergoes only modest conformational changes upon binding its receptor, NPC-1 40 , Thus, these class I fusion machines likely have fundamental differences in the fusion process that requires further study.
These structural and biochemical data show that the introduction of the 2P prefusion-stabilizing mutations does not interfere with receptor binding or recognition by trypsin-like proteases. Our structural examinations of both receptor-bound and trypsin-cleaved prefusion-stabilized SARS-CoV S 2P proteins indicate that neither receptor binding nor trypsin cleavage induce large conformational changes in stabilized spikes.
This work increases the body of knowledge surrounding these prefusion-stabilized spikes as presenting native surfaces for interactions with host factors while maintaining their prefusion conformation. Proline-substituted variant harboring KP and VP mutations was generated based on this construct. Cultures were harvested after 6 d, and protein was purified from the supernatant using Strep-Tactin resin IBA.