Architecture and self‐assembly of the SARS‐CoV‐2 nucleocapsid protein

The COVID‐2019 pandemic is the most severe acute public health threat of the twenty‐first century. To properly address this crisis with both robust testing and novel treatments, we require a deep understanding of the life cycle of the causative agent, the SARS‐CoV‐2 coronavirus. Here, we examine the architecture and self‐assembly properties of the SARS‐CoV‐2 nucleocapsid protein, which packages viral RNA into new virions. We determined a 1.4 Å resolution crystal structure of this protein's N2b domain, revealing a compact, intertwined dimer similar to that of related coronaviruses including SARS‐CoV. While the N2b domain forms a dimer in solution, addition of the C‐terminal spacer B/N3 domain mediates formation of a homotetramer. Using hydrogen‐deuterium exchange mass spectrometry, we find evidence that at least part of this putatively disordered domain is structured, potentially forming an α‐helix that self‐associates and cooperates with the N2b domain to mediate tetramer formation. Finally, we map the locations of amino acid substitutions in the N protein from over 38,000 SARS‐CoV‐2 genome sequences. We find that these substitutions are strongly clustered in the protein's N2a linker domain, and that substitutions within the N1b and N2b domains cluster away from their functional RNA binding and dimerization interfaces. Overall, this work reveals the architecture and self‐assembly properties of a key protein in the SARS‐CoV‐2 life cycle, with implications for both drug design and antibody‐based testing.     

Structural basis for regulation of poly‐SUMO chain by a SUMO‐like domain of Nip45

Post‐translational modification by small ubiquitin‐like modifier (SUMO) provides an important regulatory mechanism in diverse cellular processes. Modification of SUMO has been shown to target proteins involved in systems ranging from DNA repair pathways to the ubiquitin‐proteasome degradation system by the action of SUMO‐targeted ubiquitin ligases (STUbLs). STUbLs recognize target proteins modified with a poly‐SUMO chain through their SUMO‐interacting motifs (SIMs). STUbLs are also associated with RENi family proteins, which commonly have two SUMO‐like domains (SLD1 and SLD2) at their C terminus. We have determined the crystal structures of SLD2 of mouse RENi protein, Nip45, in a free form and in complex with a mouse E2 sumoylation enzyme, Ubc9. While Nip45 SLD2 shares a β‐grasp fold with SUMO, the SIM interaction surface conserved in SUMO paralogues does not exist in SLD2. Biochemical data indicates that neither tandem SLDs or SLD2 of Nip45 bind to either tandem SIMs from either mouse STUbL, RNF4 or to those from SUMO‐binding proteins, whose interactions with SUMO have been well characterized. On the other hand, Nip45 SLD2 binds to Ubc9 in an almost identical manner to that of SUMO and thereby inhibits elongation of poly‐SUMO chains. This finding highlights a possible role of the RENi proteins in the modulation of Ubc9‐mediated poly‐SUMO formation. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Structural and biochemical characterization of a metagenome‐derived esterase with a long N‐terminal extension

The genes encoding six novel esterolytic/lipolytic enzymes, termed LC‐Est1～6, were isolated from a fosmid library of a leaf‐branch compost metagenome by functional screening using tributyrin agar plates. These enzymes greatly vary in size and amino acid sequence. The highest identity between the amino acid sequence of each enzyme and that available from the database varies from 44 to 73%. Of these metagenome‐derived enzymes, LC‐Est1 is characterized by the presence of a long N‐terminal extension (LNTE, residues 26–283) between a putative signal peptide (residues 1–25) and a C‐terminal esterase domain (residues 284–510). A putative esterase from Candidatus Solibacter usitatus (CSu‐Est) is the only protein, which shows the significant amino acid sequence identity (46%) to the entire region of LC‐Est1. To examine whether LC‐Est1 exhibits activity and its LNTE is important for activity and stability of the esterase domain, LC‐Est1 (residues 26–510), LC‐Est1C (residues 284–510), and LC‐Est1C* (residues 304–510) were overproduced in E. coli, purified, and characterized. LC‐Est1C* was only used for structural analysis. The crystal structure of LC‐Est1C* highly resembles that of the catalytic domain of Thermotoga maritima esterase, suggesting that LNTE is not required for folding of the esterase domain. The enzymatic activity of LC‐Est1C was lower than that of LC‐Est1 by 60%, although its substrate specificity was similar to that of LC‐Est1. LC‐Est1C was less stable than LC‐Est1 by 3.3°C. These results suggest that LNTE of LC‐Est1 rather exists as an independent domain but is required for maximal activity and stability of the esterase domain.     

Correlation between TGF‐β1 expression and proteomic profiling induced by severe acute respiratory syndrome coronavirus papain‐like protease

Severe acute respiratory syndrome (SARS) coronavirus (SARS‐CoV) papain‐like protease (PLpro), a deubiquitinating enzyme, demonstrates inactivation of interferon (IFN) regulatory factor 3 and NF‐κB, reduction of IFN induction, and suppression of type I IFN signaling pathway. This study investigates cytokine expression and proteomic change induced by SARS‐CoV PLpro in human promonocyte cells. PLpro significantly increased TGF‐β1 mRNA expression (greater than fourfold) and protein production (greater than threefold). Proteomic analysis, Western blot, and quantitative real‐time PCR assays indicated PLpro upregulating TGF‐β1‐associated genes: HSP27, protein disulfide isomerase A3 precursor, glial fibrillary acidic protein, vimentin, retinal dehydrogenase 2, and glutathione transferase omega‐1. PLpro‐activated ubiquitin proteasome pathway via upregulation of ubiquitin‐conjugating enzyme E2–25k and proteasome subunit alpha type 5. Proteasome inhibitor MG‐132 significantly reduced expression of TGF‐β1 and vimentin. PLpro upregulated HSP27, linking with activation of p38 MAPK and ERK1/2 signaling. Treatment with SB203580 and U0126 reduced PLpro‐induced expression of TGF‐β1, vimentin, and type I collagen. Results point to SARS‐CoV PLpro triggering TGF‐β1 production via ubiquitin proteasome, p38 MAPK, and ERK1/2‐mediated signaling.     

Crystal structure of Nsp15 endoribonuclease NendoU from SARS‐CoV‐2

Severe Acute Respiratory Syndrome coronavirus 2 (SARS‐CoV‐2) is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS‐CoVs and Middle East Respiratory Syndrome coronavirus (MERS‐CoVs), the detailed information about SARS‐CoV‐2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies, and antivirals. We applied high‐throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS‐CoV‐2 proteins and structures. Here we report two high‐resolution crystal structures of endoribonuclease Nsp15/NendoU. We compare these structures with previously reported homologs from SARS and MERS coronaviruses.     

Comparing the binding properties of peptides mimicking the Envelope protein of SARS‐CoV and SARS‐CoV‐2 to the PDZ domain of the tight junction‐associated PALS1 protein

The Envelope protein (E) is one of the four structural proteins encoded by the genome of SARS‐CoV and SARS‐CoV‐2 Coronaviruses. It is an integral membrane protein, highly expressed in the host cell, which is known to have an important role in Coronaviruses maturation, assembly and virulence. The E protein presents a PDZ‐binding motif at its C‐terminus. One of the key interactors of the E protein in the intracellular environment is the PDZ containing protein PALS1. This interaction is known to play a key role in the SARS‐CoV pathology and suspected to affect the integrity of the lung epithelia. In this paper we measured and compared the affinity of peptides mimicking the E protein from SARS‐CoV and SARS‐CoV‐2 for the PDZ domain of PALS1, through equilibrium and kinetic binding experiments. Our results support the hypothesis that the increased virulence of SARS‐CoV‐2 compared to SARS‐CoV may rely on the increased affinity of its Envelope protein for PALS1.     

Structural analysis of the polo‐box domain of human Polo‐like kinase 2

Polo‐like kinases (Plks) are the key regulators of cell cycle progression, the members of which share a kinase domain and a polo‐box domain (PBD) that serves as a protein‐binding module. While Plk1 is a promising target for antitumor therapy, Plk2 is regarded as a tumor suppressor even though the two Plks commonly recognize the S‐pS/T‐P motif through their PBD. Herein, we report the crystal structure of the PBD of Plk2 at 2.7 Å. Despite the overall structural similarity with that of Plk1 reflecting their high sequence homology, the crystal structure also contains its own features including the highly ordered loop connecting two subdomains and the absence of 3₁₀‐helices in the N‐terminal region unlike the PBD of Plk1. Based on the three‐dimensional structure, we furthermore could model its interaction with two types of phosphopeptides, one of which was previously screened as the optimal peptide for the PBD of Plk2. Proteins 2015; 83:1201–1208. © 2015 Wiley Periodicals, Inc.     

Systematic profiling of ACE2 expression in diverse physiological and pathological conditions for COVID‐19/SARS‐CoV‐2

Recent retrospective studies of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) disease (COVID‐19) revealed that the patients with common comorbidities of cancers and chronic diseases face significantly poorer clinical outcomes than those without. Since the expression profile of ACE2, a crucial cell entry receptor for SARS‐CoV‐2, could indicate the susceptibility to SARS‐CoV‐2 infection, here we systematically dissected ACE2 expression using large‐scale multi‐omics data from 30 organs/tissues, 33 cancer types and some common chronic diseases involving >28 000 samples. It was found that sex and age could be correlated with the susceptibility of SARS‐CoV‐2 infection for certain tissues. Strikingly, ACE2 was up‐regulated in cervical squamous cell carcinoma and endocervical adenocarcinoma, colon adenocarcinoma, oesophageal carcinoma, kidney renal papillary cell carcinoma, lung adenocarcinoma and uterine corpus endometrial carcinoma compared to controls. Furthermore, the patients with common chronic diseases regarding angiocardiopathy, type 2 diabetes, liver, pneumonia and hypertension were also with higher ACE2 expression compared to related controls, which were validated using independent data sets. Collectively, our study may reveal a novel important mechanism that the patients with certain cancers and chronic diseases may express higher ACE2 expression compared to the individuals without diseases, which could lead to their higher susceptibility to multi‐organ injury of SARS‐CoV‐2 infection.     

Molecular characterization of novel pyridoxal‐5′‐phosphate‐dependent enzymes from the human microbiome

Pyridoxal‐5′‐phosphate or PLP, the active form of vitamin B6, is a highly versatile cofactor that participates in a large number of mechanistically diverse enzymatic reactions in basic metabolism. PLP‐dependent enzymes account for ～1.5% of most prokaryotic genomes and are estimated to be involved in ～4% of all catalytic reactions, making this an important class of enzymes. Here, we structurally and functionally characterize three novel PLP‐dependent enzymes from bacteria in the human microbiome: two are from Eubacterium rectale, a dominant, nonpathogenic, fecal, Gram‐positive bacteria, and the third is from Porphyromonas gingivalis, which plays a major role in human periodontal disease. All adopt the Type I PLP‐dependent enzyme fold and structure‐guided biochemical analysis enabled functional assignments as tryptophan, aromatic, and probable phosphoserine aminotransferases.     

Solution structure of the N‐terminal domain of DC‐UbP/UBTD2 and its interaction with ubiquitin

DC‐UbP/UBTD2 is a ubiquitin (Ub) domain‐containing protein first identified from dendritic cells, and is implicated in ubiquitination pathway. The solution structure and backbone dynamics of the C‐terminal Ub‐like (UbL) domain were elucidated in our previous work. To further understand the biological function of DC‐UbP, we then solved the solution structure of the N‐terminal domain of DC‐UbP (DC‐UbP_N) and studied its Ub binding properties by NMR techniques. The results show that DC‐UbP_N holds a novel structural fold and acts as a Ub‐binding domain (UBD) but with low affinity. This implies that the DC‐UbP protein, composing of a combination of both UbL and UBD domains, might play an important role in regulating protein ubiquitination and delivery of ubiquitinated substrates in eukaryotic cells.     

Structural basis for receptor recognition and pore formation of a zebrafish aerolysin‐like protein

Various aerolysin‐like pore‐forming proteins have been identified from bacteria to vertebrates. However, the mechanism of receptor recognition and/or pore formation of the eukaryotic members remains unknown. Here, we present the first crystal and electron microscopy structures of a vertebrate aerolysin‐like protein from Danio rerio, termed Dln1, before and after pore formation. Each subunit of Dln1 dimer comprises a β‐prism lectin module followed by an aerolysin module. Specific binding of the lectin module toward high‐mannose glycans triggers drastic conformational changes of the aerolysin module in a pH‐dependent manner, ultimately resulting in the formation of a membrane‐bound octameric pore. Structural analyses combined with computational simulations and biochemical assays suggest a pore‐forming process with an activation mechanism distinct from the previously characterized bacterial members. Moreover, Dln1 and its homologs are ubiquitously distributed in bony fishes and lamprey, suggesting a novel fish‐specific defense molecule.     

Covid‐19.bioreproducibility.org: A web resource for SARS‐CoV‐2‐related structural models

The COVID‐19 pandemic has triggered numerous scientific activities aimed at understanding the SARS‐CoV‐2 virus and ultimately developing treatments. Structural biologists have already determined hundreds of experimental X‐ray, cryo‐EM, and NMR structures of proteins and nucleic acids related to this coronavirus, and this number is still growing. To help biomedical researchers, who may not necessarily be experts in structural biology, navigate through the flood of structural models, we have created an online resource, covid19.bioreproducibility.org, that aggregates expert‐verified information about SARS‐CoV‐2‐related macromolecular models. In this article, we describe this web resource along with the suite of tools and methodologies used for assessing the structures presented therein.     

Channel‐like crystal structure of cinchoninium L‐O‐phosphoserine salt dihydrate

Studies on the interactions between L ‐O‐ phosphoserine, as one of the simplest fragments of membrane components, and the Cinchona alkaloid cinchonine, in the crystalline state were performed. Cinchoninium L ‐O‐phosposerine salt dihydrate (PhSerCin) crystallizes in a monoclinic crystal system, space group P2₁, with unit cell parameters: a = 8.45400(10) Å, b = 7.17100(10) Å, c = 20.7760(4) Å, α = 90°, β = 98.7830(10)°, γ = 90°, Z = 2. The asymmetric unit consists of the cinchoninium cation linked by hydrogen bonds to a phosphoserine anion and two water molecules. Intermolecular hydrogen bonds connecting phosphoserine anions via water molecules form chains extended along the b axis. Two such chains symmetrically related by twofold screw axis create a “channel.” On both sides of this channel cinchonine cations are attached by hydrogen bonds in which the atoms N1, O12, and water molecules participate. This arrangement mimics the system of bilayer biological membrane. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Assembling an ion channel: ORF 3a from SARS‐CoV

Protein 3a is a 274 amino acid polytopic channel protein with three putative transmembrane domains (TMDs) encoded by severe acute respiratory syndrome corona virus (SARS‐CoV). Synthetic peptides corresponding to each of its three individual transmembrane domains (TMDs) are reconstituted into artificial lipid bilayers. Only TMD2 and TMD3 induce channel activity. Reconstitution of the peptides as TMD1 + TMD3 as well as TMD2 + TMD3 in a 1 : 1 mixture induces membrane activity for both mixtures. In a 1 : 1 : 1 mixture, channel like behavior is almost restored. Expression of full length 3a and reconstitution into artificial lipid bilayers reveal a weak cation selective (P_K ≈ 2 P_Cl) rectifying channel. In the presence of nonphysiological concentration of Ca‐ions the channel develops channel activity. © 2013 Wiley Periodicals, Inc. Biopolymers 99:628–635, 2013.     

Predicting susceptibility for SARS‐CoV‐2 infection in domestic and wildlife animals using ACE2 protein sequence homology

The article is presenting a bioinformatics based method predicting susceptibility for SARS‐CoV‐2 infection in domestic and wildlife animals. Recently, there were reports of cats and ferrets, dogs, minks, golden hamster, rhesus monkeys, tigers, and lions testing for SARS‐CoV‐2 RNA which indicated for the possible interspecies viral transmission. Our method successfully predicted the susceptibility of these animals for contracting SARS‐CoV‐2 infection. This method can be used as a screening tool for guiding viral RNA testing for domestic and wildlife animals at risk of getting COVID‐19. We provide a list of the animals at risk of developing COVID‐19 based on the susceptibility score.     

Mimicking a p53‐MDM2 interaction based on a stable immunoglobulin‐like domain scaffold

Antibodies recognize protein targets with great affinity and specificity. However, posttranslational modifications and the presence of intrinsic disulfide‐bonds pose difficulties for their industrial use. The immunoglobulin fold is one of the most ubiquitous folds in nature and it is found in many proteins besides antibodies. An example of a protein family with an immunoglobulin‐like fold is the Cysteine Protease Inhibitors (ICP) family I42 of the MEROPs database for protease and protease inhibitors. Members of this protein family are thermostable and do not present internal disulfide bonds. Crystal structures of several ICPs indicate that they resemble the Ig‐like domain of the human T cell co‐receptor CD8α As ICPs present 2 flexible recognition loops that vary accordingly to their targeted protease, we hypothesize that members of this protein family would be ideal to design peptide aptamers that mimic protein‐protein interactions. Herein, we use an ICP variant from Entamoeba histolytica (EhICP1) to mimic the interaction between p53 and MDM2. We found that a 13 amino‐acid peptide derived from p53 can be introduced in 2 variable loops (DE, FG) but not the third (BC). Chimeric EhICP1‐p53 form a stable complex with MDM2 at a micromolar range. Crystal structure of the EhICP1‐p53(FG)‐loop variant in complex with MDM2 reveals a swapping subdomain between 2 chimeric molecules, however, the p53 peptide interacts with MDM2 as in previous crystal structures. The structural details of the EhICP1‐p53(FG) interaction with MDM2 resemble the interaction between an antibody and MDM2.     

Potential anti‐bacterial drug target: Structural characterization of 3,4‐dihydroxy‐2‐butanone‐4‐phosphate synthase from Salmonella typhimurium LT2

3,4‐Dihydroxy‐2‐butanone‐4‐phosphate synthase (DHBPS) encoded by ribB gene is one of the first enzymes in riboflavin biosynthesis pathway and catalyzes the conversion of ribulose‐5‐phosphate (Ru5P) to 3,4‐dihydroxy‐2‐butanone‐4‐phosphate and formate. DHBPS is an attractive target for developing anti‐bacterial drugs as this enzyme is essential for pathogens, but absent in humans. The recombinant DHBPS enzyme of Salmonella requires magnesium ion for its activity and catalyzes the formation of 3,4‐dihydroxy‐2‐butanone‐4‐phosphate from Ru5P at a rate of 199 nmol min^?1 mg^?1 with K_m value of 116 μM at 37°C. Further, we have determined the crystal structures of Salmonella DHBPS in complex with sulfate, Ru5P and sulfate‐zinc ion at a resolution of 2.80, 2.52, and 1.86 Å, respectively. Analysis of these crystal structures reveals that the acidic loop (residues 34–39) responsible for the acid‐base catalysis is disordered in the absence of substrate or metal ion at the active site. Upon binding either substrate or sulfate and metal ions, the acidic loop becomes stabilized, adopts a closed conformation and interacts with the substrate. Our structure for the first time reveals that binding of substrate Ru5P alone is sufficient for the stabilization of the acidic active site loop into a closed conformation. In addition, the Glu38 residue from the acidic active site loop undergoes a conformational change upon Ru5P binding, which helps in positioning the second metal ion that stabilizes the Ru5P and the reaction intermediates. This is the first structural report of DHBPS in complex with either substrate or metal ion from any eubacteria. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Direct inhibitory effect on viral entry of influenza A and SARS‐CoV‐2 viruses by azithromycin

ObjectivesUsing strategy of drug repurposing, antiviral agents against influenza A virus (IAV) and newly emerging SARS‐coronavirus 2 (SARS‐CoV‐2, also as 2019‐nCoV) could be quickly screened out.Materials and MethodsA previously reported engineered replication‐competent PR8 strain carrying luciferase reporter gene (IAV‐luc) and multiple pseudotyped IAV and SARS‐CoV‐2 virus was used. To specifically evaluate the pH change of vesicles containing IAV, we constructed an A549 cell line with endosomal and lysosomal expression of pHluorin2.ResultsHere, we identified azithromycin (AZ) as an effective inhibitor against multiple IAV and SARS‐CoV‐2 strains. We found that AZ treatment could potently inhibit IAV infection in vitro. Moreover, using pseudotyped virus model, AZ could also markedly block the entry of SARS‐CoV‐2 in HEK293T‐ACE2 and Caco2 cells. Mechanistic studies further revealed that such effect was independent of interferon signalling. AZ treatment neither impaired the binding and internalization of IAV virions, nor the viral replication, but rather inhibited the fusion between viral and vacuolar membranes. Using a NPC1‐pHluorin2 reporter cell line, we confirmed that AZ treatment could alkalize the vesicles containing IAV virions, thereby preventing pH‐dependent membrane fusion.ConclusionsOverall, our findings demonstrate that AZ can exert broad‐spectrum antiviral effects against IAV and SARS‐CoV‐2, and could be served as a potential clinical anti‐SARS‐CoV‐2 drug in emergency as well as a promising lead compound for the development of next‐generation anti‐IAV drugs.     

The hypothetical role of phosphatidic acid in subverting ER membranes during SARS‐CoV infection

Positive sense (+) RNA viruses exploit membranes from a variety of cellular organelles to support the amplification of their genomes. This association concurs with the formation of vesicles whose main morphological feature is that of being wrapped by a double membrane. In the case of the SARS‐CoV virus, the outer membrane is not discrete for each vesicle, but seems to be continuous and shared between many individual vesicles, a difference with other +RNA viruses whose nature has remained elusive. I present morphological, biochemical and pharmacological arguments defending the striking analogy of this arrangement and that of entangled, nascent Lipid Droplets whose birth has been aborted by an excess of Phosphatidic Acid. Since Phosphatidic Acid can be targeted with therapeutical purposes, considering this working hypothesis may prove important in tackling SARS‐CoV infection.