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Structures of the SARS‐CoV‐2 nucleocapsid and their perspectives for drug design

Structures of the SARS‐CoV‐2 nucleocapsid and their perspectives for drug design Article Structures of the SARS-CoV-2 nucleocapsid and their perspectives for drug design 1,2 3 2,4 2,4 2,4 1 Ya Peng , Ning Du , Yuqing Lei , Sonam Dorje , Jianxun Qi , Tingrong Luo , 2,3,4,* 3,** George F Gao & Hao Song Abstract tail/who-director-general-s-opening-remarks-at-the-media-briefing- on-covid-19---11-march-2020). COVID-19, caused by SARS-CoV-2, has resulted in severe and 2019-nCoV, which is the seventh coronavirus capable of infecting unprecedented economic and social disruptions in the world. humans, was later named “SARS-CoV-2” by the International Nucleocapsid (N) protein, which is the major structural component Committee on Taxonomy of Viruses (ICTV) (Gorbalenya et al, 2020), of the virion and is involved in viral replication, assembly and although with some controversies (Jiang et al, 2020). The other six immune regulation, plays key roles in the viral life cycle. Here, we coronaviruses are the low-pathogenicity members HCoV-OC43, solved the crystal structures of the N- and C-terminal domains HCoV-HKU1, HCoV-NL63 and HCoV-229E and the highly pathogenic (N-NTD and N-CTD) of SARS-CoV-2 N protein, at 1.8 and 1.5 Å reso- SARS-CoV and MERS-CoV (Lu et al, 2020). Sequence comparison has lution, respectively. Both structures show conserved features from shown that SARS-CoV-2 has the closest relationship (96.2%) with the other CoV N proteins. The binding sites targeted by small mole- bat SARS-like coronavirus RaTG13 (Lu et al, 2020; Zhou et al, 2020a, cules against HCoV-OC43 and MERS-CoV, which inhibit viral infec- b), but the origin of this virus is more than likely the bat; what tion by blocking the RNA-binding activity or normal remains to be identified is a possible intermediate host. Although oligomerization of N protein, are relatively conserved in our struc- many small-molecule drugs targeting the viral polymerase and ture, indicating N protein is a promising drug target. In addition, protease, such as remdesivir, lipinavir, ritonavir and hydroxychloro- certain areas of N-NTD and N-CTD display distinct charge distribu- quine, have been shown to be promising at the beginning (Wang tion patterns in SARS-CoV-2, which may alter the RNA-binding et al, 2020c; Zhang et al, 2020a), no therapeutics have yet been modes. The specific antigenic characteristics are critical for devel- proven effective for the treatment of severe illness in recent clinical oping specific immune-based rapid diagnostic tests. Our structural trials (Beigel et al, 2020; Cao et al, 2020), and hydroxychloroquine information can aid in the discovery and development of antiviral has been found no benefit to help patients with COVID-19 recover inhibitors against SARS-CoV-2 in the future. (Cavalcanti et al, 2020; Hoffmann et al, 2020; Maisonnasse et al, 2020), indicating more effective antiviral drugs are yet to be devel- Keywords 2019-nCoV; antivirals; dimerization; nucleocapsid; RNA binding oped, and combinational therapeutic approaches are necessary to Subject Categories Microbiology, Virology & Host Pathogen Interaction; improve patient outcomes in COVID-19. Recently, several protective Structural Biology neutralization antibodies blocking viral entry were rapidly developed DOI 10.15252/embj.2020105938 | Received 20 June 2020 | Revised 14 August (Shi et al, 2020; Wu et al, 2020b,c), and diverse types of vaccine 2020 | Accepted 21 August 2020 | Published online 11 September 2020 candidates are undergoing clinical evaluation (Wang et al, 2020b). The EMBO Journal (2020) 39:e105938 However, there is still no clinically approved specific drug or vaccine available to treat the disease. Therefore, it is urgent to develop speci- fic drugs/inhibitors against distinct targets and infection processes. Introduction Among positive-sense RNA viruses, the family Coronaviridae, of which SARS-CoV-2 is a member, has the largest genome (~ 30,000 In December 2019, a new coronavirus (2019-nCoV) was detected bases). It contains two large overlapping open reading frames due to emerging viral pneumonia cases in Wuhan, China (Li et al, (ORF1a and ORF1b) and encodes four structural proteins, i.e. spike, 2020; Tan et al, 2020; Wang et al, 2020a; Wu et al, 2020a; Zhou envelope, membrane and nucleocapsid (N) proteins, and nine acces- et al, 2020b; Zhu et al, 2020). The World Health Organization sory proteins. ORF1a and ORF1b are further processed to generate named the infectious disease “COVID-19”, and it declared a global 16 nonstructural proteins (Nsp1 to 16). Among the viral proteins, pandemic on 11 March 2020 (https://www.who.int/dg/speeches/de the N protein is the central component of virions. It binds to viral 1 Laboratory of Animal Infectious Diseases, College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning, China 2 CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China 3 Research Network of Immunity and Health (RNIH), Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China 4 University of Chinese Academy of Sciences, Beijing, China *Corresponding author. Tel: +86 10 64807688; E-mail: [email protected] **Corresponding author. Tel: +86 10 64807417; E-mail: [email protected] ª 2020 The Authors The EMBO Journal 39:e105938 | 2020 1 of 12 The EMBO Journal Ya Peng et al genomic RNA to package the RNA into a ribonucleoprotein (RNP) dimerization domain and also has RNA-binding activity. To further complex. Besides assembly, N proteins also possess other functions, study the biochemical properties of SARS-CoV-2 N, we produced including roles in viral mRNA transcription and replication (Zuniga SARS-CoV-2 N-NTD and N-CTD in Escherichia coli, and we obtained et al, 2010; Cong et al, 2020), cytoskeleton organization and the soluble protein domains by metal affinity chromatography and gel immune regulation (Surjit et al, 2004, 2006; Lu et al, 2011). Espe- filtration chromatography (Fig EV1A and B). Sedimentation velocity cially, SARS-CoV-2 N protein has been found to function as a viral analytical ultracentrifugation analyses determined that SARS-CoV-2 suppressor of RNA silencing (VSR) through its double-stranded N-NTD exists as a monomer (~ 15.4 kDa) (Fig 1D), while N-CTD RNA-binding activity to combat host RNAi-mediated antiviral exists as a dimer (~ 28.7 kDa) (Fig 1E) in solution. These results responses (Mu et al, 2020). In addition, N protein could induce both further confirm that SARS-CoV-2 N protein shares the same modular humoral and cellular immune responses after infection (Ni et al, organization with other CoV N proteins. 2020; Xiang et al, 2020), making it a key target for diagnosis and vaccine development. Structure of SARS-CoV-2 N-NTD Coronavirus N proteins have two conserved and independently folded structural domains, called the N-terminal domain (NTD) and The crystal structure of SARS-CoV-2 N-NTD was solved by molecular C-terminal domain (CTD) (Chang et al, 2014). The two domains are replacement to a resolution of 1.8 A,with R and R values of work free connected by an intrinsically disordered region (IDR) called the 19.7 and 22.7%, respectively (Table 1). Each asymmetric unit central linker region (LKR). The LKR includes a Ser/Arg (SR)-rich contains four N-NTD molecules (Fig EV2A) with an overall root- region that contains putative phosphorylation sites (Peng et al, 2008). mean-square difference (RMSD) of 0.16–0.28 A between each In addition, the N-NTD and N-CTD of coronaviruses are usually protomer (Fig EV2B). Each N-NTD molecule presents a right-handed flanked by two IDRs, which are called N-arm and C-tail. Previous fist shape and consists of a four-stranded antiparallel b-sheet core studies have revealed that N-NTD is responsible for RNA binding, N- subdomain, which is sandwiched between loops or short 3 helices CTD for both RNA binding and dimerization, and the IDR for modulat- and a protruding b-hairpin region formed by b2and b3 strands out of ing the RNA-binding activity of N-NTD and N-CTD and oligomeriza- the core (Fig 2A and B). Based on the surface charge distribution, the tion (Chang et al, 2014). The structures of both N-NTD and N-CTD protruding loops are positively charged, providing a putative site for from several human-infecting coronaviruses have been solved (Yu RNA binding (Fig 2C). In addition, according to the complex struc- et al, 2006; Chen et al, 2007; Saikatendu et al, 2007; Lin et al, 2014; tures of the previously solved HCoV-NL63 N-NTD with ribonu- Papageorgiou et al, 2016; Szelazek et al, 2017; Nguyen et al, 2019), cleotide 5 -mono-phosphates (AMP/GMP/UMP/CMP) (Lin et al, but the structure of the full-length N protein remains a mystery due to 2014), the core domain also contains an RNA-binding site, which is the disordered IDR and oligomeric characteristics (Cong et al, 2017). conserved among different human-infecting coronaviruses (Fig 2D). The importance of N protein in viral assembly, replication and host The overall structure of SARS-CoV-2 N-NTD strongly resembles immune response regulation makes it an attractive target for develop- other CoV N-NTD structures, with an RMSD of 0.33–0.76 A (Fig 3A). ing broad-spectrum antiviral inhibitors. Previous studies have shown We compared the SARS-CoV-2 N-NTD structure with all of the other that small-molecule drugs can be developed by targeting the RNA- available human-infecting CoV N-NTD structures, i.e. SARS-CoV, binding activity and the oligomerization of the N protein (Lin et al, MERS-CoV, HCoV-OC43 and HCoV-NL63. Of note, among these structures, two regions show the greatest structural differences. First, 2014; Chang et al, 2016; Zhang et al, 2020b). Here, we determined the crystal structures of SARS-CoV-2 N- the orientations of the N-terminal regions are distinct. The N-term- ˚ ˚ NTD at a resolution of 1.8 A and N-CTD at a resolution of 1.5 A, inal loops of SARS-CoV-2, SARS-CoV and MERS-CoV N-NTD stretch outward, while the loops from HCoV-OC43 and HCoV-NL63 N-NTD and we compared these with other human-infecting coronavirus N- NTD and N-CTD structures to identify suitable target sites for anti- rotate towards the core subdomain. Second, the protruding b-hairpin COVID-19 drug discovery and elucidate their structural drug-binding regions are flexible; particularly, the connecting flexible loops are invisible in HCoV-OC43 and HCoV-NL63 N-NTD structures (Fig 3A). features. These studies provide important structural information to guide the development of antiviral compounds against SARS-CoV-2 The ribonucleotide-binding site is located at the core subdomain in and targeting the N protein. the HCoV-NL63 N-NTD-AMP complex structure. We then compared the residues involved in AMP binding in different coronaviruses (Fig 3B and C). The highly conserved residues S64, Y126 and R164 Results (HCoV-OC43 N-NTD numbering), located near the base of the pocket, bind to AMP via hydrogen-bonding interactions, while the Domain organization of the SARS-CoV-2 N protein residues on the side wall of the pockets, including Y124 and G68, further stabilize the binding. However, this region is not conserved According to the sequence alignment analysis of SARS-CoV-2 and in all human-infecting coronaviruses, and it could be further divided other coronaviruses, SARS-CoV-2 has a genome organization similar into two groups. The SARS-CoV-2, SARS-CoV and MERS-CoV NTD to that of SARS-CoV (Fig 1A), and the SARS-CoV-2 N protein also N-terminal regions are similar to that of HCoV-OC43, containing shares the same modular organization with the SARS-CoV N protein. conserved residues Y124 and G68 (or Ala). The phenolic hydroxyl It is composed of three IDRs (N-arm, LKR and C-tail) and two struc- group of Y124 interacts with the AMP adenine ring via a hydrogen tural domains (NTD and CTD) (Fig 1B). SARS-CoV-2 has a close rela- bond, and the backbone of G68 forms hydrogen bonds with the tionship with SARS-CoV, and both belong to lineage B beta- monophosphate group of AMP (Fig 3B). However, for HCoV-NL63, coronaviruses (Fig 1C). Previous studies have shown that coron- residues H77 and P24 are present at equivalent positions, and both avirus N-NTD is the RNA-binding domain, while N-CTD is the residues lack the capability of hydrogen bond formation, and they 2 of 12 The EMBO Journal 39:e105938 | 2020 ª 2020 The Authors HCoV HCoV-OC43 -OC43 HCoV HCoV H -NL63 -NL63 SARS-CoV SARS-CoV- -2 2 Ya Peng et al The EMBO Journal SARS-CoV-2 Genome ~ 30,000 nt Polyproteins: Non-structural proteins Accessory proteins 1a 1b 7a 3a 6 8 9b 9c 10 Nsp7 Nsp11 Nsp1 Nsp5 Nsp9 Nsp3 7b 3b Nsp2 Nsp4 Nsp6 Nsp10 Nsp8 Nsp12 Nsp14 Nsp16 Structural proteins SEM N Nsp13 Nsp15 Dimerization domain 44 RNA-binding domain 174 255 RNA-binding domain 364 N-arm LKR C-tail SR-rich motif NTD CTD 143 175 203 254 365 419 Nucleocapsid (N) protein C 10 SARS-CoV-2 N-NTD Monomer 6 ~15.4 kDa βCoV A βCoV B sedimentation coefficent [S] SARS-CoV-2 N-CTD Dimer ~28.7 kDa 0.05 αCoV βCoV C 2 sedimentation coefficent [S] Figure 1. Domain organization of the SARS-CoV-2 nucleocapsid protein. A Genome organization of SARS-CoV-2. B Schematic representation of SARS-CoV-2 N protein domains. Three intrinsically disordered regions, i.e. N-arm, linker region (LKR) and C-tail, and the N-terminal domain (NTD) and C-terminal domain (CTD) are illustrated. The charged Ser/Arg (SR)-rich motif (coloured purple) is shown. C Phylogenetic analysis of N proteins from seven human-infecting coronaviruses. SARS-CoV-2 is highlighted by a red asterisk. D Analytical ultracentrifugation absorbance data analysis of N-NTD. The clear peak at ~ 15.4 kDa molecular weight (MW) corresponds to the N-NTD monomer. E Analytical ultracentrifugation absorbance data analysis of N-CTD. The clear peak at ~ 28.7 kDa MW corresponds to the N-CTD dimer. may further occlude AMP binding (Fig 3C). Therefore, the RNA- potential surfaces also show different charge distribution patterns in binding modes must differ between these two groups. Whether this different coronavirus N-NTD structures, especially the N-terminal is a divergent characteristic between beta-coronavirus (HCoV-OC43, loop, the top tip of the protruding region and the bottom of core SARS-CoV-2, SARS-CoV and MERS-CoV) and alpha-coronaviruses subdomain (Fig 3D–H). However, all of the structures display an (HCoV-NL63) needs further investigation (Fig 1C). The electrostatic extended positively charged groove from the AMP-binding site to the ª 2020 The Authors The EMBO Journal 39:e105938 | 2020 3 of 12 HCoV HCoV-2 -229E 29E SARS-CoV SARS-CoV V HCoV HCoV H -HKU1 -HKU1 MERS-CoV MERS-CoV V c(s)(AU/S) c(s)(AU/S) The EMBO Journal Ya Peng et al Table 1. Data collection and refinement statistics. and is comprised of five a-helices, two b-strands, and two 3 helices (Fig 4C). The b-hairpin from one protomer is inserted into SARS-CoV-2 N-NTD SARS-CoV-2 N-CTD the cavity of the other protomer, resulting in the formation of the Data collection four-stranded, antiparallel b-sheet at the dimer interface. The b- Wavelength (Å) 0.97919 0.97919 sheet forms one face of the slab dimer, while on the opposite face of Space group P 121 1 P 1 the dimer, the surface is formed by a-helices and loops. The dimer Cell dimensions interface buries a surface area of 2,590 A . Extensive hydrogen bond interactions between the two hairpins and hydrophobic interactions a, b, c (Å) 59.27, 55.43, 85.68 43.73, 50.31, 69.13 between the b-sheet and the alpha helices make the dimeric struc- a, b, c (°) 90.00, 95.38, 90.00 73.88, 89.94, 82.55 ture highly stable. This is consistent with our previous biochemical Resolution (Å) 50.00–1.80 (1.86–1.80) 50.00–1.39 (1.41–1.39) characterization that N-CTD exists as a dimer in solution (Fig 1E). R 0.023 (0.330) 0.020 (0.321) p.i.m. We then compared the SARS-CoV-2 N-CTD structure with the available N-CTD structures of SARS-CoV, MERS-CoV and HCoV- I/rI 32.724 (2.538) 30.265 (2.035) NL63, and we found that the overall structure is highly similar, CC 0.998 (0.963) 0.996 (0.808) 1/2 with an RMSD of 0.31–0.98 A (Fig 5A). They all show a Completeness (%) 98.4 (97.6) 91.7 (67.2) conserved positively charged groove in the helix face of the N- Redundancy 6.6 (6.6) 3.4 (3.0) CTD dimer, due to the distribution of several positively charged Refinement residues, including K256, K257, K261 and R262 in SARS-CoV-2 N- CTD (Figs 5B and EV3). Interestingly, the electrostatic potential Resolution (Å) 29.50–1.80 26.83–1.50 surfaces in the b-sheet faces of these proteins show distinct No. reflections 46,709 81,130 features. The MERS-CoV structure displays a positively charged R /R 0.197/0.227 0.177/0.190 work free central region, whereas SARS-CoV-2 and SARS-CoV structures No. atoms both display a negatively charged region. For NL63, the central Protein 3,956 3,534 region of its b-sheet face shows a highly negatively charged region (Fig 5B). These differences may affect the binding modes of RNA Ligand/ion 00 recognition. Our structure shows SARS-CoV-2 N-CTD functions as Water 611 740 the dimerization domain and the RNA-binding domain involved in B-factors RNP assembly. Protein 29.617.2 Drug target sites in the N protein Ligand/ion –– Water 30.528.3 As the RNA-binding activity of the N protein is critical for viral RNP R.m.s. deviations formation and genome replication, blocking RNA binding of N-NTD Bond lengths (Å) 0.005 0.003 has been proved to be a good strategy to develop antiviral drugs. The compound PJ34, which targets the ribonucleotide-binding site Bond angles (°) 0.808 0.651 in N-NTD, displays potent inhibition of the RNA-binding activity of Ramachandran plot the HCoV-OC43 N protein and can inhibit viral replication of HCoV- Favoured (%) 99.40 99.77 OC43 (Lin et al, 2014). PJ34 mimics the binding of AMP to N-NTD, Allowed (%) 0.60 0.23 and it fits in the ribonucleotide-binding pocket of N-NTD with an Outliers (%) 0.00 0.00 extended trend to the N-terminal loop. We have compared the corre- sponding binding site for PJ34 in our SARS-CoV-2 N-NTD structure Values in parentheses are for highest-resolution shell. b 1/2 R = Σ [1/(N  1)] Σ |I  <I>|/Σ Σ I , where I is the observed p.i.m. hkl i i hkl i i i with that in the HCoV-OC43 N-NTD structure (Fig 6A), and we intensity and <I> is the average intensity from multiple measurements. found that the key residues that are involved in the interactions, R = Σ ||F |  |F ||/Σ |F |, where F and F are the structure-factor work o c o o c including S51, F53, Y109, Y111 and R149 (in SARS-CoV-2 N-NTD amplitudes from the data and the model, respectively. R is the R factor for free numbering), are conserved (Fig 6B). a subset (5%) of reflections that was selected prior to refinement calculations and was not included in the refinement. Another strategy is to block normal N protein oligomerization, thereby halting RNP formation or inducing abnormal aggregation. Recently, the novel inhibitor 5-benzyloxygramine (P3) was identi- protruding region, indicating this patch is responsible for RNA bind- fied by virtual screening (Lin et al, 2020). This compound could ing in different coronaviruses. mediate MERS-CoV N-NTD non-native dimerization and induce N protein aggregation. It was shown to have potent antiviral activity Structure of SARS-CoV-2 N-CTD against MERS-CoV. The complex structure of MERS-CoV N-NTD and P3 shows that P3 targets the non-native interface of dimeric N- The crystal structure of SARS-CoV-2 N-CTD was solved by molecu- NTD and binds to two hydrophobic pockets in two N-NTD proto- lar replacement to a resolution of 1.5 A, with R and R values mers. P3 occupies the N-terminal vector-fusion residue-binding work free of 17.7 and 19.0%, respectively (Table 1). SARS-CoV-2 N-CTD cavity of NTD promoter 1 in the ligand-free MERS-CoV N-NTD forms a tight homodimer and displays an overall rectangular slab structure (Fig 6C) and further stabilizes the dimeric status by shape (Fig 4A). Each protomer has the shape of the letter C (Fig 4B) massive hydrophobic interactions. We compared the binding cavity 4 of 12 The EMBO Journal 39:e105938 | 2020 ª 2020 The Authors Ya Peng et al The EMBO Journal AB β4 β4 β3 β3 β1 β5 β2 β6 β1 β5 β2 β6 η1 C η1 CD Ribonucleotide binding site 9DULDEOH &RQVHUYHG Figure 2. Structure of SARS-CoV-2 N-NTD. A Cartoon representation of SARS-CoV-2 N-NTD. B Topology diagram for SARS-CoV-2 N-NTD; g represents the 3 helix and b represents the b-sheet. C The electrostatic surface potential of SARS-CoV-2 N-NTD. Red and blue colours indicate negative and positive potential, respectively. The RNA-binding sites are highlighted in dotted circles and labelled. D Surface representation of SARS-CoV-2 N-NTD coloured according to sequence conservation based the alignment of N protein sequences from seven human-infecting CoVs using the ConSurf server (Ashkenazy et al, 2016); dark magenta indicates the most conserved, dark cyan indicates the most divergent. of P3 with the corresponding site in our SARS-CoV-2 N-NTD struc- other coronaviruses (Yu et al, 2006; Chen et al, 2007; Saikatendu ture, and we found that almost all of the residues involved in the et al, 2007; Lin et al, 2014; Papageorgiou et al, 2016; Szelazek interactions are conserved, except F135 in MERS-CoV, which is et al, 2017; Nguyen et al,2019).N-NTD has a right-handed fist replaced by I146 in SARS-CoV-2 (Fig 6D). Although both residues shape consisting of an antiparallel b-sheet core subdomain and a are nonpolar amino acids, the difference in hydrophobic force may protruding b-hairpin region. N-CTD is present as a tight inter- affect the dimerization efficiency. twined homodimer and displays an overall rectangular slab shape. Based on the surface electronic distribution and superimposition with other coronavirus N-NTD structures, we compared the Discussion ribonucleotide-binding pocket in SARS-CoV-2 N-NTD, and we found that the binding residues are highly conserved, which could In this study, we solved the crystal structures of both SARS-CoV-2 be a verified target for antiviral drugs that block the RNA-binding N-NTD and N-CTD. Both structures show conserved features with activity of the N protein. ª 2020 The Authors The EMBO Journal 39:e105938 | 2020 5 of 12 The EMBO Journal Ya Peng et al SARS-CoV-2 AB C SARS-CoV H77/Y124 Y109 A55 HCoV-OC43 Y110 A56 P24/G68 AMP AMP Y124 G68 MERS-CoV Y99 G46 HCoV-NL63 Y111 Y126 Y112 Y101 Y79/Y126 S53 S20/S64 S52 S64 F53 S42 F54 F66 R149 Y22/F66 R116/R164 Y44 R150 R164 R138 DE F SARS-CoV-2 SARS-CoV HCoV-OC43 GH MERS-CoV HCoV-NL63 Figure 3. Comparison of the structure of SARS-CoV-2 N-NTD with other human-infecting CoV N-NTD structures. A Superposition of the SARS-CoV-2 N-NTD structure with other human-infecting CoV N-NTD structures. The ribbon representation of each structure is coloured separately (SARS-CoV, PDB: 2OFZ, cyan; HCoV-OC43, PDB: 4LI4, pink; MERS-CoV, PDB: 4UD1, orange; HCoV-NL63, PDB: 5N4K, blue) (Saikatendu et al, 2007; Lin et al, 2014; Papageorgiou et al, 2016; Szelazek et al, 2017). The loops that show significant differences are highlighted by dotted circles. The AMP ligand in the HCoV- OC43 N-NTD-AMP complex is shown as a stick structure. B Structural superimposition of SARS-CoV-2, SARS-CoV and MERS-CoV N-NTDs with that of HCoV-OC43 bound to AMP to show the detailed interactions involved in AMP binding. C Structural superimposition of HCoV-NL63 N-NTD with HCoV-OC43 N-NTD bound to AMP to show the detailed interactions involved in AMP binding. D–H Comparison of the electrostatic surfaces with other human-infecting CoV N-NTD structures. The electrostatic surface of N-NTD from (D) SARS-CoV-2, (E) SARS-CoV, (F) HCoV-OC43 bound to AMP, (G) MERS-CoV and (H) HCoV-NL63 are shown, and the AMP-binding sites are highlighted in dotted circles. Red and blue colours indicate negative and positive potential, respectively. 6 of 12 The EMBO Journal 39:e105938 | 2020 ª 2020 The Authors Ya Peng et al The EMBO Journal A C N N N VLGHYLHZ η1 η1 η2 β2 β1 C η2 β1 β2 WRSYLHZ β2 N N N β1 C C C C η1 η2 β2 β1 η2 η1 N N N Figure 4. Structure of SARS-CoV-2 N-CTD. A Cartoon representation of SARS-CoV-2 N-CTD. Ribbon representation of SARS-CoV-2 N-CTD showing the separated monomer and dimer. One monomer is coloured in pink, and the other is coloured in yellow. B Rotation of (A) along the x-axis by 90° to show the top view. C Topology diagram for SARS-CoV-2 N-CTD. The a-helices are numbered A, B and C, g represents the 3 helix and b represents the b-sheet, according to a previous SARS N-CTD structural element numbering rule (Yu et al, 2006). Although both N-NTD and N-CTD structures resemble those of and specific anti-N antibodies would be critical to enhance the rapid other coronaviruses, especially SARS-CoV, the electrostatic potential molecular diagnosis for COVID-19. During our manuscript prepara- surfaces display different charge distribution patterns in some areas, tion, other groups also solved crystal structures of N-NTD or including the N-terminal loop, the top tip of the protruding region, N-CTD, which show similar structural features (Kang et al, 2020; the bottom of the core subdomain in N-NTD and the b-sheet face of Ye et al, 2020). N-CTD. These divergent features may alter the RNA-binding modes Until now, there are no structures of the full-length N proteins or binding efficiency. Of note, we found that the orientations of the from coronaviruses reported, due to their dynamic and oligomeric N-terminal loops of N-NTD were distinct. The N-terminal loops of characteristics. Based on our dimeric structure of N-CTD and previ- SARS-CoV-2, SARS-CoV and MERS-CoV N-NTD stretch outward, ous studies, the N protein forms a dimer by N-CTD interaction, while the loops from HCoV-OC43 and HCoV-NL63 N-NTD rotate and the disordered LKRs serve as the two arms connecting the two towards the core subdomain. This result suggests that different N- N-NTDs to the N-CTD dimers (Chen et al, 2007; Nguyen et al, NTD may have group-specific characteristics and that the distinc- 2019). In the RNP complex, the N-CTDs form the helical core. The tions may alter the locations of N-arms and then lead to different RNA molecule twists around the helical groove and further inter- structural and functional properties. In addition, as the N protein acts with the NTDs (Chang et al, 2014). According to previous could interact with many host proteins during the infection process cryo-electron tomography studies of the vRNP complex isolated and cause a robust humoral immune response after infection, the from murine hepatitis virus (MHV) and SARS-CoV virions, each divergent charge distribution surfaces could be critical to identify dimeric N unit packs in a long loose helical or a supercoiled orga- specific interacting cellular proteins and develop specific immune- nization (Neuman et al, 2006; Ba´rcena et al, 2009; Gui et al, based rapid diagnostic test (Burbelo et al, 2020; Ni et al, 2020). 2017). Recently, preprint: Klein et al (2020) observed that SARS- Besides the serological assays for detection of SARS-CoV-2 antibod- CoV-2 RNPs organizing like string beads, indicating the high steri- ies (Horber et al, 2020), the rapid detection of viral antigens based cal flexibility of RNP assembly. However, a lack of high-resolution on specific antibodies can provide essential information for disease structures of RNP limits our understanding of the precise RNP monitoring (Che et al, 2004). Therefore, to develop high-affinity assembly mechanism. ª 2020 The Authors The EMBO Journal 39:e105938 | 2020 7 of 12 The EMBO Journal Ya Peng et al SARS-CoV-2 SARS-CoV MERS-CoV HCoV-NL63 Figure 5. Comparison of the structure of SARS-CoV-2 N-CTD with other coronavirus N-CTD structures. A Superimposed structures in ribbon representation of N-CTD from SARS-CoV-2 (pink), SARS-CoV (PDB: 2CJR, cyan) (Chen et al, 2007), MERS-CoV (PDB: 6G13, orange) (Nguyen et al, 2019) and HCoV-NL63 (PDB: 5EPW, blue) (Szelazek et al, 2017). B Electrostatic surface views of the four N-CTD structures. Red and blue colours indicate negative and positive potential, respectively. 8 of 12 The EMBO Journal 39:e105938 | 2020 ª 2020 The Authors O Ya Peng et al The EMBO Journal AB PJ34 Y111 / Y124 F53 / F66 Y109 / Y126 S51/S64 R149 /R164 SARS-CoV-2 N-NTD HCoV-OC43 N-NTD PJ34 complex CD protomer1 T115 / T105 / T105 G116 / G106/G106 T148/ T137/ T137 A119 / A109 /A109 I146 / F135/ F135 P3 protomer2 N75 / N66/ N66 Y112 / Y102 Y102 / W52// W43 W43 SARS-CoV-2 N-NTD MERS-CoV N-NTD MERS-CoV N-NTD P3 complex Figure 6. Conserved drug target sites in SARS-CoV-2 N protein. A, B Superimposition of the structure of SARS-CoV-2 N-NTD (light blue) with HCoV-OC43 N-NTD (light pink) bound to PJ34 (magenta) (PDB: 4KXJ) (Lin et al, 2014). The detailed interactions are shown in (B). The water molecule mediating hydrogen-bonding interactions is shown as a red sphere. C, D Superimposition of the structure of SARS-CoV-2 N-NTD (light blue) with MERS-CoV N-NTD dimer (grey) or MERS-CoV N-NTD bound to P3 (cyan) (PDB: 6KL6) (Lin et al, 2020). The detailed interactions are shown in (D). Coronavirus N proteins have been attractive targets for antiviral and found that the drug targeting sites are relatively conserved, indicat- drugs as they are critical for viral replication and assembly. Few inhibi- ing further structure-based drug optimization for broad-spectrum agent tors have been identified through various approaches based on HCoV- development is very promising. Our structural information of SARS- OC43 or MERS-CoV (Lin et al, 2014; Zhang et al, 2020b). We analysed CoV-2 N-NTD and C-CTD can aid in the discovery and development of the binding sites of the representative inhibitors in our crystal structures antiviral inhibitors against SARS-CoV-2 in the future. ª 2020 The Authors The EMBO Journal 39:e105938 | 2020 9 of 12 H The EMBO Journal Ya Peng et al Materials and Methods were performed using REFMAC5 (Murshudov et al, 1997) and COOT (Emsley & Cowtan, 2004), respectively. Further refinement Gene cloning protein production and purification was performed using Phenix (Adams et al, 2010). Final statistics for data collection and structure refinement are presented in Table 1. SARS-CoV-2 N-NTD (amino acid residues 44–174, GISAID accession ID: EPI_ISL_402119) fused at its N-terminus with a hexa-histidine tag Biochemical characterization of N proteins was cloned into the pET-21a vector (Novagen) with NdeIand XhoI restriction sites (Li et al, 2019). Transformed E. coli strain BL21 (DE3) The purified protein was analysed with an analytical gel filtration clones were grown in LB medium containing 100 lg/ml ampicillin to assay with a calibrated Superdex 75 10/300 GL column (GE an OD of 0.6–0.8 at 37°C. Expression of the recombinant proteins Healthcare). The sample was further analysed with SDS–PAGE. was induced by the addition of 0.5 mM isopropyl-b-D-1-thiogalacto- The analytical ultracentrifugation assay was performed according pyranoside (IPTG), and incubation was continued for a further 16 h to a previously reported method (Wang et al, 2017). The proteins at 16°C. Cells were harvested by centrifugation at 7,000 g for 15 min were prepared in 20 mM Tris (pH 8.0) and 150 mM NaCl at a at 4°C and then resuspended in lysis buffer [20 mM Tris–HCl (pH 8.0) concentration of 0.8 mg/ml. The assay was performed on an opti- and 150 mM NaCl] and further homogenized with a low-temperature mal ProteomeLab XL-I analytical ultracentrifuge (Beckman Coulter) ultra-high pressure cell disrupter (JNBIO, China). The lysate was clari- at a speed of 228,000 g. The molecular mass analysis was performed fied by centrifugation at 20,000 g for 60 min at 4°C. The supernatant with XL-I data analysis software. was purified by metal affinity chromatography using a HisTrap HP 5 ml column (GE Healthcare). Proteins were eluted using lysis buffer supplemented with 300 mM imidazole. The proteins were further Data availability purified by gel filtration chromatography using a HiLoad 16/600 Superdex 75 PG (GE Healthcare) with a running buffer of 20 mM Atomic coordinates and structure factors have been deposited in the Tris–HCl (pH 8.0) and 50 mM NaCl, and the collected protein frac- Protein Data Bank with accession codes 7CDZ (https://www.rcsb.org/ tions were concentrated to 15 mg/ml using a membrane concentrator structure/7CDZ) and 7CE0 (https://www.rcsb.org/structure/7CE0). with a molecular weight cut-off of 10 kDa (Millipore). SARS-CoV-2 N-CTD (amino acid residues 255–364) was Expanded View for this article is available online. constructed and expressed in the same way as N-NTD. During metal affinity chromatography, N-CTDs were eluted using lysis buffer Acknowledgements supplemented with 500 mM imidazole. The proteins were further We thank the staff of BL19U1 beamlines at Shanghai Synchrotron Radiation purified by gel filtration chromatography using a HiLoad 16/600 Facility. We thank Qian Wang (Institute of Microbiology CAS) for help with Superdex 75 PG (GE Healthcare) with a running buffer of 20 mM analytical ultracentrifugation experiment. This work was supported by the Tris–HCl (pH 8.0) and 150 mM NaCl, and the collected protein frac- Ministry of Science and Technology of the People’s Republic of China tions were concentrated to 10 mg/ml using a membrane concentra- (2020YFC0845900), the National Science and Technology Major Project tor with a molecular weight cut-off of 10 kDa (Millipore). (2018ZX10733403), and the National Natural Science Foundation of China (NSFC) (81702015). H.S. is supported by the Youth Innovation Promotion Asso- Crystallization, data collection and structure determination ciation CAS (2017117). G.F.G. is supported partly by the COVID-19 Emergency Project of CAS and the External Cooperation Program of CAS Crystallization trials were set up with commercial crystallization kits (153211KYSB20160001). (Hampton Research) using the sitting drop vapour diffusion method. The resultant drop was then sealed, equilibrating against 100 ll reser- Author contributions voir solution at 18°C. Diffractable crystals of SARS-CoV-2 N-NTD GFG and HS designed and supervised the study. YP, ND, YL and SD conducted were obtained in 0.1 M SPG (succinic acid, sodium phosphate the experiments. JQ collected the data sets and solved the structures. HS, TL monobasic monohydrate and glycine mix buffer pH 6.0) (Molecular and GFG analysed the data and wrote the manuscript. Dimensions, MD2-59) with 25% w/v polyethylene glycol 1,500 at 18°C. SARS-CoV-2 N-CTD crystals were obtained in 4 M potassium Conflict of interest formate, 0.1 M Bis-Tris propane (pH 9.0) and 2% w/v polyethylene The authors declare that they have no conflict of interest. glycol monomethyl ether 2,000 at 18°C. Crystals were flash-cooled in liquid nitrogen after a brief soaking in reservoir solution with the addi- tion of 17% (v/v) glycerol. The X-ray diffraction data were collected References under cryogenic conditions (173°C) at Shanghai Synchrotron Radia- tion Facility (SSRF) beam line BL19U1 and indexed, integrated and Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, scaled with HKL3000 (Minor et al, 2006). Hung LW, Kapral GJ, Grosse-Kunstleve RW et al (2010) PHENIX: a comprehensive Python-based system for macromolecular structure SARS-CoV-2 N-NTD and N-CTD structures were solved by the molecular replacement method using Phaser (Read, 2001) from the solution. Acta Crystallogr D Biol Crystallogr 66: 213 – 221 CCP4 programme suite (Winn et al, 2011), with the structures of Ashkenazy H, Abadi S, Martz E, Chay O, Mayrose I, Pupko T, Ben-Tal N (2016) ConSurf 2016: an improved methodology to estimate and visualize SARS-CoV N-NTD (PDB: 2OFZ) (Saikatendu et al, 2007) and N-CTD (PDB: 2CJR) (Chen et al, 2007) as the search models, respectively. evolutionary conservation in macromolecules. Nucleic Acids Res 44: Initial restrained rigid-body refinement and manual model building W344 – W350 10 of 12 The EMBO Journal 39:e105938 | 2020 ª 2020 The Authors Ya Peng et al The EMBO Journal Bárcena M, Oostergetel GT, Bartelink W, Faas FG, Verkleij A, Rottier PJ, Koster Hörber S, Soldo J, Relker L, Jürgens S, Guther J, Peter S, Lehmann R, Peter A AJ, Bosch BJ (2009) Cryo-electron tomography of mouse hepatitis virus: (2020) Evaluation of three fully-automated SARS-CoV-2 antibody assays. insights into the structure of the coronavirion. Proc Natl Acad Sci USA 106: Clin Chem Lab Med https://doi.org/10.1515/cclm-2020-0975 582 – 587 Jiang S, Shi Z, Shu Y, Song J, Gao GF, Tan W, Guo D (2020) A distinct name is Beigel JH, Tomashek KM, Dodd LE, Mehta AK, Zingman BS, Kalil AC, Hohmann needed for the new coronavirus. Lancet 395: 949 E, Chu HY, Luetkemeyer A, Kline S et al (2020) Remdesivir for the Kang S, Yang M, Hong Z, Zhang L, Huang Z, Chen X, He S, Zhou Z, Zhou Z, treatment of Covid-19 – preliminary report. N Engl J Med https://doi.org/ Chen Q et al (2020) Crystal structure of SARS-CoV-2 nucleocapsid protein 10.1056/NEJMoa2007764 RNA binding domain reveals potential unique drug targeting sites. Acta Burbelo PD, Riedo FX, Morishima C, Rawlings S, Smith D, Das S, Strich JR, Pharm Sin B 10: 1228 – 1238 Chertow DS, Davey RT, Cohen JI (2020) Sensitivity in detection of Klein S, Cortese M, Winter SL, Wachsmuth-Melm M, Neufeldt CJ, Cerikan B, antibodies to nucleocapsid and spike proteins of severe acute respiratory Stanifer ML, Boulant S, Bartenschlager R, Chlanda P (2020) SARS-CoV-2 syndrome Coronavirus 2 in patients with coronavirus disease 2019. J Infect structure and replication characterized by in situ cryo-electron Dis 222: 206 – 213 tomography. bioRxiv https://doi.org/10.1101/2020.06.23.167064 [PREPRINT] Cao B, Wang Y, Wen D, Liu W, Wang J, Fan G, Ruan L, Song B, Cai Y, Wei M Li C, Chai Y, Song H, Weng C, Qi J, Sun Y, Gao GF (2019) Crystal structure of et al (2020) A trial of lopinavir-ritonavir in adults hospitalized with severe african swine fever virus dUTPase reveals a potential drug target. MBio 10: Covid-19. N Engl J Med 382: 1787 – 1799 e02483-19 Cavalcanti AB, Zampieri FG, Rosa RG, Azevedo LCP, Veiga VC, Avezum A, Li Q, Guan X, Wu P, Wang X, Zhou L, Tong Y, Ren R, Leung KSM, Lau EHY, Damiani LP, Marcadenti A, Kawano-Dourado L, Lisboa T et al (2020) Wong JY et al (2020) Early transmission dynamics in Wuhan, China, of Hydroxychloroquine with or without Azithromycin in mild-to-moderate novel coronavirus-infected pneumonia. N Engl J Med 382: 1199 – 1207 Covid-19. N Engl J Med https://doi.org/10.1056/NEJMoa2019014 Lin SY, Liu CL, Chang YM, Zhao J, Perlman S, Hou MH (2014) Structural basis Chang CK, Hou MH, Chang CF, Hsiao CD, Huang TH (2014) The SARS coronavirus for the identification of the N-terminal domain of coronavirus nucleocapsid protein–forms and functions. Antiviral Res 103: 39 – 50 nucleocapsid protein as an antiviral target. J Med Chem 57: 2247 – 2257 Chang CK, Jeyachandran S, Hu NJ, Liu CL, Lin SY, Wang YS, Chang YM, Hou Lin SM, Lin SC, Hsu JN, Chang CK, Chien CM, Wang YS, Wu HY, Jeng US, MH (2016) Structure-based virtual screening and experimental validation Kehn-Hall K, Hou MH (2020) Structure-based stabilization of non-native of the discovery of inhibitors targeted towards the human coronavirus protein-protein interactions of coronavirus nucleocapsid proteins in nucleocapsid protein. Mol BioSyst 12: 59 – 66 antiviral drug design. J Med Chem 63: 3131 – 3141 Che XY, Qiu LW, Pan YX, Wen K, Hao W, Zhang LY, Wang YD, Liao ZY, Hua X, Lu X, Pan J, Tao J, Guo D (2011) SARS-CoV nucleocapsid protein antagonizes Cheng VC et al (2004) Sensitive and specific monoclonal antibody-based IFN-b response by targeting initial step of IFN-b induction pathway, capture enzyme immunoassay for detection of nucleocapsid antigen in and its C-terminal region is critical for the antagonism. Virus Genes 42: sera from patients with severe acute respiratory syndrome. J Clin Microbiol 37 – 45 42: 2629 – 2635 Lu R, Zhao X, Li J, Niu P, Yang B, Wu H, Wang W, Song H, Huang B, Zhu N Chen CY, Chang CK, Chang YW, Sue SC, Bai HI, Riang L, Hsiao CD, Huang TH et al (2020) Genomic characterisation and epidemiology of 2019 novel (2007) Structure of the SARS coronavirus nucleocapsid protein RNA- coronavirus: implications for virus origins and receptor binding. Lancet binding dimerization domain suggests a mechanism for helical packaging 395: 565 – 574 of viral RNA. J Mol Biol 368: 1075 – 1086 Maisonnasse P, Guedj J, Contreras V, Behillil S, Solas C, Marlin R, Naninck T, Cong Y, Kriegenburg F, de Haan CAM, Reggiori F (2017) Coronavirus Pizzorno A, Lemaitre J, Goncalves A et al (2020) Hydroxychloroquine use nucleocapsid proteins assemble constitutively in high molecular against SARS-CoV-2 infection in non-human primates. Nature https://doi. oligomers. Sci Rep 7: 5740 org/10.1038/s41586-020-2558-4 Cong Y, Ulasli M, Schepers H, Mauthe M, V’Kovski P, Kriegenburg F, Thiel V, Minor W, Cymborowski M, Otwinowski Z, Chruszcz M (2006) HKL-3000: the de Haan CAM, Reggiori F (2020) Nucleocapsid protein recruitment to integration of data reduction and structure solution–from diffraction replication-transcription complexes plays a crucial role in coronaviral life images to an initial model in minutes. Acta Crystallogr D Biol Crystallogr cycle. J Virol 94:e01925-19 62: 859 – 866 Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Mu J, Xu J, Zhang L, Shu T, Wu D, Huang M, Ren Y, Li X, Geng Q, Xu Y et al Acta Crystallogr D Biol Crystallogr 60: 2126 – 2132 (2020) SARS-CoV-2-encoded nucleocapsid protein acts as a viral Gorbalenya AE, Baker SC, Baric RS, de Groot RJ, Drosten C, Gulyaeva AA, suppressor of RNA interference in cells. Sci China Life Sci 63: 1 – 4 Haagmans BL, Lauber C, Leontovich AM, Neuman BW et al.(2020)The Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular species severe acute respiratory syndrome-related coronavirus: structures by the maximum-likelihood method. Acta Crystallogr D Biol classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol 5: Crystallogr 53: 240 – 255 536 – 544. Neuman BW, Adair BD, Yoshioka C, Quispe JD, Orca G, Kuhn P, Milligan RA, Gui M, Liu X, Guo D, Zhang Z, Yin CC, Chen Y, Xiang Y (2017) Electron Yeager M, Buchmeier MJ (2006) Supramolecular architecture of severe microscopy studies of the coronavirus ribonucleoprotein complex. Protein acute respiratory syndrome coronavirus revealed by electron Cell 8: 219 – 224 cryomicroscopy. J Virol 80: 7918 – 7928 Hoffmann M, Mosbauer K, Hofmann-Winkler H, Kaul A, Kleine-Weber H, Kruger Nguyen THV, Lichiere J, Canard B, Papageorgiou N, Attoumani S, Ferron F, N, Gassen NC, Muller MA, Drosten C, Pohlmann S (2020) Chloroquine does Coutard B (2019) Structure and oligomerization state of the C-terminal not inhibit infection of human lung cells with SARS-CoV-2. Nature https:// region of the Middle East respiratory syndrome coronavirus nucleoprotein. doi.org/10.1038/s41586-020-2575-3 Acta Crystallogr D Struct Biol 75: 8 – 15 ª 2020 The Authors The EMBO Journal 39:e105938 | 2020 11 of 12 The EMBO Journal Ya Peng et al Ni L, Ye F, Cheng ML, Feng Y, Deng YQ, Zhao H, Wei P, Ge J, Gou M, Li X et al Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan (2020) Detection of SARS-CoV-2-specific humoral and cellular immunity in RM, Krissinel EB, Leslie AG, McCoy A et al (2011) Overview of the CCP4 COVID-19 convalescent individuals. Immunity 52: 971 – 977 suite and current developments. Acta Crystallogr D Biol Crystallogr 67: Papageorgiou N, Lichiere J, Baklouti A, Ferron F, Sevajol M, Canard B, Coutard 235 – 242 B(2016) Structural characterization of the N-terminal part of the MERS- Wu F, Zhao S, Yu B, Chen YM, Wang W, Song ZG, Hu Y, Tao ZW, Tian JH, Pei CoV nucleocapsid by X-ray diffraction and small-angle X-ray scattering. YY et al (2020a) A new coronavirus associated with human respiratory Acta Crystallogr D Struct Biol 72: 192 – 202 disease in China. Nature 579: 265 – 269 Peng TY, Lee KR, Tarn WY (2008) Phosphorylation of the arginine/serine Wu Y, Li C, Xia S, Tian X, Kong Y, Wang Z, Gu C, Zhang R, Tu C, Xie Y et al dipeptide-rich motif of the severe acute respiratory syndrome coronavirus (2020b) Identification of human single-domain antibodies against SARS- nucleocapsid protein modulates its multimerization, translation inhibitory CoV-2. Cell Host Microbe 27: 891 – 898 activity and cellular localization. FEBS J 275: 4152 – 4163 Wu Y, Wang F, Shen C, Peng W, Li D, Zhao C, Li Z, Li S, Bi Y, Yang Y et al Read RJ (2001) Pushing the boundaries of molecular replacement with (2020c) A noncompeting pair of human neutralizing antibodies maximum likelihood. Acta Crystallogr D Biol Crystallogr 57: 1373 – 1382 block COVID-19 virus binding to its receptor ACE2. Science 368: Saikatendu KS, Joseph JS, Subramanian V, Neuman BW, Buchmeier MJ, 1274 – 1278 Stevens RC, Kuhn P (2007) Ribonucleocapsid formation of severe acute Xiang F, Wang X, He X, Peng Z, Yang B, Zhang J, Zhou Q, Ye H, Ma Y, Li H respiratory syndrome coronavirus through molecular action of the N- et al (2020) Antibody detection and dynamic characteristics in patients terminal domain of N protein. J Virol 81: 3913 – 3921 with COVID-19. Clin Infect Dis https://doi.org/10.1093/cid/ciaa461 Shi R, Shan C, Duan X, Chen Z, Liu P, Song J, Song T, Bi X, Han C, Wu L et al Ye Q, West AMV, Silletti S, Corbett KD (2020) Architecture and self-assembly (2020) A human neutralizing antibody targets the receptor-binding site of of the SARS-CoV-2 nucleocapsid protein. Protein Sci https://doi.org/10. SARS-CoV-2. Nature 584: 120 – 124 1002/pro.3909 Surjit M, Liu B, Jameel S, Chow VT, Lal SK (2004) The SARS coronavirus Yu IM, Oldham ML, Zhang J, Chen J (2006) Crystal structure of the severe nucleocapsid protein induces actin reorganization and apoptosis in COS-1 acute respiratory syndrome (SARS) coronavirus nucleocapsid protein cells in the absence of growth factors. Biochem J 383: 13 – 18 dimerization domain reveals evolutionary linkage between corona- and Surjit M, Liu B, Chow VT, Lal SK (2006) The nucleocapsid protein of severe arteriviridae. J Biol Chem 281: 17134 – 17139 acute respiratory syndrome-coronavirus inhibits the activity of cyclin- Zhang L, Lin D, Sun X, Curth U, Drosten C, Sauerhering L, Becker S, Rox K, cyclin-dependent kinase complex and blocks S phase progression in Hilgenfeld R (2020a) Crystal structure of SARS-CoV-2 main protease mammalian cells. J Biol Chem 281: 10669 – 10681 provides a basis for design of improved alpha-ketoamide inhibitors. Szelazek B, Kabala W, Kus K, Zdzalik M, Twarda-Clapa A, Golik P, Burmistrz Science 368: 409 – 412 M, Florek D, Wladyka B, Pyrc K et al (2017) Structural characterization of Zhang X, Tan Y, Ling Y, Lu G, Liu F, Yi Z, Jia X, Wu M, Shi B, Xu S et al human coronavirus NL63 N protein. J Virol 91:e02503-16 (2020b) Viral and host factors related to the clinical outcome of COVID-19. Tan W, Zhao X, Ma X, Wang W, Niu P, Xu W, Gao GF, Wu G (2020) A novel Nature 583: 437 – 440 coronavirus genome identified in a cluster of pneumonia cases - Wuhan, Zhou H, Chen X, Hu T, Li J, Song H, Liu Y, Wang P, Liu D, Yang J, Holmes EC China 2019–2020. China CDC Wkly 2: 61 – 62 et al (2020a) A novel bat coronavirus closely related to SARS-CoV-2 Wang H, Han M, Qi J, Hilgenfeld R, Luo T, Shi Y, Gao GF, Song H (2017) contains natural insertions at the S1/S2 cleavage site of the spike protein. Crystal structure of the C-terminal fragment of NS1 protein from yellow Curr Biol 30: 2196 – 2203 fever virus. Sci China Life Sci 60: 1403 – 1406 Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, Si HR, Zhu Y, Li B, Wang C, Horby PW, Hayden FG, Gao GF (2020a) A novel coronavirus outbreak Huang CL et al (2020b) A pneumonia outbreak associated with a new of global health concern. Lancet 395: 470 – 473 coronavirus of probable bat origin. Nature 579: 270 – 273 Wang J, Peng Y, Xu H, Cui Z, Williams RO (2020b) The COVID-19 vaccine race: Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, Zhao X, Huang B, Shi W, Lu R challenges and opportunities in vaccine formulation. AAPS PharmSciTech et al (2020) A novel coronavirus from patients with pneumonia in China, 21: 225 2019. N Engl J Med 382: 727 – 733 Wang M, Cao R, Zhang L, Yang X, Liu J, Xu M, Shi Z, Hu Z, Zhong W, Xiao G Zuniga S, Cruz JL, Sola I, Mateos-Gomez PA, Palacio L, Enjuanes L (2010) (2020c) Remdesivir and chloroquine effectively inhibit the recently Coronavirus nucleocapsid protein facilitates template switching and is emerged novel coronavirus (2019-nCoV) in vitro. Cell Res 30: 269 – 271 required for efficient transcription. J Virol 84: 2169 – 2175 12 of 12 The EMBO Journal 39:e105938 | 2020 ª 2020 The Authors http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Structures of the SARS‐CoV‐2 nucleocapsid and their perspectives for drug design

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Article Structures of the SARS-CoV-2 nucleocapsid and their perspectives for drug design 1,2 3 2,4 2,4 2,4 1 Ya Peng , Ning Du , Yuqing Lei , Sonam Dorje , Jianxun Qi , Tingrong Luo , 2,3,4,* 3,** George F Gao & Hao Song Abstract tail/who-director-general-s-opening-remarks-at-the-media-briefing- on-covid-19---11-march-2020). COVID-19, caused by SARS-CoV-2, has resulted in severe and 2019-nCoV, which is the seventh coronavirus capable of infecting unprecedented economic and social disruptions in the world. humans, was later named “SARS-CoV-2” by the International Nucleocapsid (N) protein, which is the major structural component Committee on Taxonomy of Viruses (ICTV) (Gorbalenya et al, 2020), of the virion and is involved in viral replication, assembly and although with some controversies (Jiang et al, 2020). The other six immune regulation, plays key roles in the viral life cycle. Here, we coronaviruses are the low-pathogenicity members HCoV-OC43, solved the crystal structures of the N- and C-terminal domains HCoV-HKU1, HCoV-NL63 and HCoV-229E and the highly pathogenic (N-NTD and N-CTD) of SARS-CoV-2 N protein, at 1.8 and 1.5 Å reso- SARS-CoV and MERS-CoV (Lu et al, 2020). Sequence comparison has lution, respectively. Both structures show conserved features from shown that SARS-CoV-2 has the closest relationship (96.2%) with the other CoV N proteins. The binding sites targeted by small mole- bat SARS-like coronavirus RaTG13 (Lu et al, 2020; Zhou et al, 2020a, cules against HCoV-OC43 and MERS-CoV, which inhibit viral infec- b), but the origin of this virus is more than likely the bat; what tion by blocking the RNA-binding activity or normal remains to be identified is a possible intermediate host. Although oligomerization of N protein, are relatively conserved in our struc- many small-molecule drugs targeting the viral polymerase and ture, indicating N protein is a promising drug target. In addition, protease, such as remdesivir, lipinavir, ritonavir and hydroxychloro- certain areas of N-NTD and N-CTD display distinct charge distribu- quine, have been shown to be promising at the beginning (Wang tion patterns in SARS-CoV-2, which may alter the RNA-binding et al, 2020c; Zhang et al, 2020a), no therapeutics have yet been modes. The specific antigenic characteristics are critical for devel- proven effective for the treatment of severe illness in recent clinical oping specific immune-based rapid diagnostic tests. Our structural trials (Beigel et al, 2020; Cao et al, 2020), and hydroxychloroquine information can aid in the discovery and development of antiviral has been found no benefit to help patients with COVID-19 recover inhibitors against SARS-CoV-2 in the future. (Cavalcanti et al, 2020; Hoffmann et al, 2020; Maisonnasse et al, 2020), indicating more effective antiviral drugs are yet to be devel- Keywords 2019-nCoV; antivirals; dimerization; nucleocapsid; RNA binding oped, and combinational therapeutic approaches are necessary to Subject Categories Microbiology, Virology & Host Pathogen Interaction; improve patient outcomes in COVID-19. Recently, several protective Structural Biology neutralization antibodies blocking viral entry were rapidly developed DOI 10.15252/embj.2020105938 | Received 20 June 2020 | Revised 14 August (Shi et al, 2020; Wu et al, 2020b,c), and diverse types of vaccine 2020 | Accepted 21 August 2020 | Published online 11 September 2020 candidates are undergoing clinical evaluation (Wang et al, 2020b). The EMBO Journal (2020) 39:e105938 However, there is still no clinically approved specific drug or vaccine available to treat the disease. Therefore, it is urgent to develop speci- fic drugs/inhibitors against distinct targets and infection processes. Introduction Among positive-sense RNA viruses, the family Coronaviridae, of which SARS-CoV-2 is a member, has the largest genome (~ 30,000 In December 2019, a new coronavirus (2019-nCoV) was detected bases). It contains two large overlapping open reading frames due to emerging viral pneumonia cases in Wuhan, China (Li et al, (ORF1a and ORF1b) and encodes four structural proteins, i.e. spike, 2020; Tan et al, 2020; Wang et al, 2020a; Wu et al, 2020a; Zhou envelope, membrane and nucleocapsid (N) proteins, and nine acces- et al, 2020b; Zhu et al, 2020). The World Health Organization sory proteins. ORF1a and ORF1b are further processed to generate named the infectious disease “COVID-19”, and it declared a global 16 nonstructural proteins (Nsp1 to 16). Among the viral proteins, pandemic on 11 March 2020 (https://www.who.int/dg/speeches/de the N protein is the central component of virions. It binds to viral 1 Laboratory of Animal Infectious Diseases, College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning, China 2 CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China 3 Research Network of Immunity and Health (RNIH), Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China 4 University of Chinese Academy of Sciences, Beijing, China *Corresponding author. Tel: +86 10 64807688; E-mail: [email protected] **Corresponding author. Tel: +86 10 64807417; E-mail: [email protected] ª 2020 The Authors The EMBO Journal 39:e105938 | 2020 1 of 12 The EMBO Journal Ya Peng et al genomic RNA to package the RNA into a ribonucleoprotein (RNP) dimerization domain and also has RNA-binding activity. To further complex. Besides assembly, N proteins also possess other functions, study the biochemical properties of SARS-CoV-2 N, we produced including roles in viral mRNA transcription and replication (Zuniga SARS-CoV-2 N-NTD and N-CTD in Escherichia coli, and we obtained et al, 2010; Cong et al, 2020), cytoskeleton organization and the soluble protein domains by metal affinity chromatography and gel immune regulation (Surjit et al, 2004, 2006; Lu et al, 2011). Espe- filtration chromatography (Fig EV1A and B). Sedimentation velocity cially, SARS-CoV-2 N protein has been found to function as a viral analytical ultracentrifugation analyses determined that SARS-CoV-2 suppressor of RNA silencing (VSR) through its double-stranded N-NTD exists as a monomer (~ 15.4 kDa) (Fig 1D), while N-CTD RNA-binding activity to combat host RNAi-mediated antiviral exists as a dimer (~ 28.7 kDa) (Fig 1E) in solution. These results responses (Mu et al, 2020). In addition, N protein could induce both further confirm that SARS-CoV-2 N protein shares the same modular humoral and cellular immune responses after infection (Ni et al, organization with other CoV N proteins. 2020; Xiang et al, 2020), making it a key target for diagnosis and vaccine development. Structure of SARS-CoV-2 N-NTD Coronavirus N proteins have two conserved and independently folded structural domains, called the N-terminal domain (NTD) and The crystal structure of SARS-CoV-2 N-NTD was solved by molecular C-terminal domain (CTD) (Chang et al, 2014). The two domains are replacement to a resolution of 1.8 A,with R and R values of work free connected by an intrinsically disordered region (IDR) called the 19.7 and 22.7%, respectively (Table 1). Each asymmetric unit central linker region (LKR). The LKR includes a Ser/Arg (SR)-rich contains four N-NTD molecules (Fig EV2A) with an overall root- region that contains putative phosphorylation sites (Peng et al, 2008). mean-square difference (RMSD) of 0.16–0.28 A between each In addition, the N-NTD and N-CTD of coronaviruses are usually protomer (Fig EV2B). Each N-NTD molecule presents a right-handed flanked by two IDRs, which are called N-arm and C-tail. Previous fist shape and consists of a four-stranded antiparallel b-sheet core studies have revealed that N-NTD is responsible for RNA binding, N- subdomain, which is sandwiched between loops or short 3 helices CTD for both RNA binding and dimerization, and the IDR for modulat- and a protruding b-hairpin region formed by b2and b3 strands out of ing the RNA-binding activity of N-NTD and N-CTD and oligomeriza- the core (Fig 2A and B). Based on the surface charge distribution, the tion (Chang et al, 2014). The structures of both N-NTD and N-CTD protruding loops are positively charged, providing a putative site for from several human-infecting coronaviruses have been solved (Yu RNA binding (Fig 2C). In addition, according to the complex struc- et al, 2006; Chen et al, 2007; Saikatendu et al, 2007; Lin et al, 2014; tures of the previously solved HCoV-NL63 N-NTD with ribonu- Papageorgiou et al, 2016; Szelazek et al, 2017; Nguyen et al, 2019), cleotide 5 -mono-phosphates (AMP/GMP/UMP/CMP) (Lin et al, but the structure of the full-length N protein remains a mystery due to 2014), the core domain also contains an RNA-binding site, which is the disordered IDR and oligomeric characteristics (Cong et al, 2017). conserved among different human-infecting coronaviruses (Fig 2D). The importance of N protein in viral assembly, replication and host The overall structure of SARS-CoV-2 N-NTD strongly resembles immune response regulation makes it an attractive target for develop- other CoV N-NTD structures, with an RMSD of 0.33–0.76 A (Fig 3A). ing broad-spectrum antiviral inhibitors. Previous studies have shown We compared the SARS-CoV-2 N-NTD structure with all of the other that small-molecule drugs can be developed by targeting the RNA- available human-infecting CoV N-NTD structures, i.e. SARS-CoV, binding activity and the oligomerization of the N protein (Lin et al, MERS-CoV, HCoV-OC43 and HCoV-NL63. Of note, among these structures, two regions show the greatest structural differences. First, 2014; Chang et al, 2016; Zhang et al, 2020b). Here, we determined the crystal structures of SARS-CoV-2 N- the orientations of the N-terminal regions are distinct. The N-term- ˚ ˚ NTD at a resolution of 1.8 A and N-CTD at a resolution of 1.5 A, inal loops of SARS-CoV-2, SARS-CoV and MERS-CoV N-NTD stretch outward, while the loops from HCoV-OC43 and HCoV-NL63 N-NTD and we compared these with other human-infecting coronavirus N- NTD and N-CTD structures to identify suitable target sites for anti- rotate towards the core subdomain. Second, the protruding b-hairpin COVID-19 drug discovery and elucidate their structural drug-binding regions are flexible; particularly, the connecting flexible loops are invisible in HCoV-OC43 and HCoV-NL63 N-NTD structures (Fig 3A). features. These studies provide important structural information to guide the development of antiviral compounds against SARS-CoV-2 The ribonucleotide-binding site is located at the core subdomain in and targeting the N protein. the HCoV-NL63 N-NTD-AMP complex structure. We then compared the residues involved in AMP binding in different coronaviruses (Fig 3B and C). The highly conserved residues S64, Y126 and R164 Results (HCoV-OC43 N-NTD numbering), located near the base of the pocket, bind to AMP via hydrogen-bonding interactions, while the Domain organization of the SARS-CoV-2 N protein residues on the side wall of the pockets, including Y124 and G68, further stabilize the binding. However, this region is not conserved According to the sequence alignment analysis of SARS-CoV-2 and in all human-infecting coronaviruses, and it could be further divided other coronaviruses, SARS-CoV-2 has a genome organization similar into two groups. The SARS-CoV-2, SARS-CoV and MERS-CoV NTD to that of SARS-CoV (Fig 1A), and the SARS-CoV-2 N protein also N-terminal regions are similar to that of HCoV-OC43, containing shares the same modular organization with the SARS-CoV N protein. conserved residues Y124 and G68 (or Ala). The phenolic hydroxyl It is composed of three IDRs (N-arm, LKR and C-tail) and two struc- group of Y124 interacts with the AMP adenine ring via a hydrogen tural domains (NTD and CTD) (Fig 1B). SARS-CoV-2 has a close rela- bond, and the backbone of G68 forms hydrogen bonds with the tionship with SARS-CoV, and both belong to lineage B beta- monophosphate group of AMP (Fig 3B). However, for HCoV-NL63, coronaviruses (Fig 1C). Previous studies have shown that coron- residues H77 and P24 are present at equivalent positions, and both avirus N-NTD is the RNA-binding domain, while N-CTD is the residues lack the capability of hydrogen bond formation, and they 2 of 12 The EMBO Journal 39:e105938 | 2020 ª 2020 The Authors HCoV HCoV-OC43 -OC43 HCoV HCoV H -NL63 -NL63 SARS-CoV SARS-CoV- -2 2 Ya Peng et al The EMBO Journal SARS-CoV-2 Genome ~ 30,000 nt Polyproteins: Non-structural proteins Accessory proteins 1a 1b 7a 3a 6 8 9b 9c 10 Nsp7 Nsp11 Nsp1 Nsp5 Nsp9 Nsp3 7b 3b Nsp2 Nsp4 Nsp6 Nsp10 Nsp8 Nsp12 Nsp14 Nsp16 Structural proteins SEM N Nsp13 Nsp15 Dimerization domain 44 RNA-binding domain 174 255 RNA-binding domain 364 N-arm LKR C-tail SR-rich motif NTD CTD 143 175 203 254 365 419 Nucleocapsid (N) protein C 10 SARS-CoV-2 N-NTD Monomer 6 ~15.4 kDa βCoV A βCoV B sedimentation coefficent [S] SARS-CoV-2 N-CTD Dimer ~28.7 kDa 0.05 αCoV βCoV C 2 sedimentation coefficent [S] Figure 1. Domain organization of the SARS-CoV-2 nucleocapsid protein. A Genome organization of SARS-CoV-2. B Schematic representation of SARS-CoV-2 N protein domains. Three intrinsically disordered regions, i.e. N-arm, linker region (LKR) and C-tail, and the N-terminal domain (NTD) and C-terminal domain (CTD) are illustrated. The charged Ser/Arg (SR)-rich motif (coloured purple) is shown. C Phylogenetic analysis of N proteins from seven human-infecting coronaviruses. SARS-CoV-2 is highlighted by a red asterisk. D Analytical ultracentrifugation absorbance data analysis of N-NTD. The clear peak at ~ 15.4 kDa molecular weight (MW) corresponds to the N-NTD monomer. E Analytical ultracentrifugation absorbance data analysis of N-CTD. The clear peak at ~ 28.7 kDa MW corresponds to the N-CTD dimer. may further occlude AMP binding (Fig 3C). Therefore, the RNA- potential surfaces also show different charge distribution patterns in binding modes must differ between these two groups. Whether this different coronavirus N-NTD structures, especially the N-terminal is a divergent characteristic between beta-coronavirus (HCoV-OC43, loop, the top tip of the protruding region and the bottom of core SARS-CoV-2, SARS-CoV and MERS-CoV) and alpha-coronaviruses subdomain (Fig 3D–H). However, all of the structures display an (HCoV-NL63) needs further investigation (Fig 1C). The electrostatic extended positively charged groove from the AMP-binding site to the ª 2020 The Authors The EMBO Journal 39:e105938 | 2020 3 of 12 HCoV HCoV-2 -229E 29E SARS-CoV SARS-CoV V HCoV HCoV H -HKU1 -HKU1 MERS-CoV MERS-CoV V c(s)(AU/S) c(s)(AU/S) The EMBO Journal Ya Peng et al Table 1. Data collection and refinement statistics. and is comprised of five a-helices, two b-strands, and two 3 helices (Fig 4C). The b-hairpin from one protomer is inserted into SARS-CoV-2 N-NTD SARS-CoV-2 N-CTD the cavity of the other protomer, resulting in the formation of the Data collection four-stranded, antiparallel b-sheet at the dimer interface. The b- Wavelength (Å) 0.97919 0.97919 sheet forms one face of the slab dimer, while on the opposite face of Space group P 121 1 P 1 the dimer, the surface is formed by a-helices and loops. The dimer Cell dimensions interface buries a surface area of 2,590 A . Extensive hydrogen bond interactions between the two hairpins and hydrophobic interactions a, b, c (Å) 59.27, 55.43, 85.68 43.73, 50.31, 69.13 between the b-sheet and the alpha helices make the dimeric struc- a, b, c (°) 90.00, 95.38, 90.00 73.88, 89.94, 82.55 ture highly stable. This is consistent with our previous biochemical Resolution (Å) 50.00–1.80 (1.86–1.80) 50.00–1.39 (1.41–1.39) characterization that N-CTD exists as a dimer in solution (Fig 1E). R 0.023 (0.330) 0.020 (0.321) p.i.m. We then compared the SARS-CoV-2 N-CTD structure with the available N-CTD structures of SARS-CoV, MERS-CoV and HCoV- I/rI 32.724 (2.538) 30.265 (2.035) NL63, and we found that the overall structure is highly similar, CC 0.998 (0.963) 0.996 (0.808) 1/2 with an RMSD of 0.31–0.98 A (Fig 5A). They all show a Completeness (%) 98.4 (97.6) 91.7 (67.2) conserved positively charged groove in the helix face of the N- Redundancy 6.6 (6.6) 3.4 (3.0) CTD dimer, due to the distribution of several positively charged Refinement residues, including K256, K257, K261 and R262 in SARS-CoV-2 N- CTD (Figs 5B and EV3). Interestingly, the electrostatic potential Resolution (Å) 29.50–1.80 26.83–1.50 surfaces in the b-sheet faces of these proteins show distinct No. reflections 46,709 81,130 features. The MERS-CoV structure displays a positively charged R /R 0.197/0.227 0.177/0.190 work free central region, whereas SARS-CoV-2 and SARS-CoV structures No. atoms both display a negatively charged region. For NL63, the central Protein 3,956 3,534 region of its b-sheet face shows a highly negatively charged region (Fig 5B). These differences may affect the binding modes of RNA Ligand/ion 00 recognition. Our structure shows SARS-CoV-2 N-CTD functions as Water 611 740 the dimerization domain and the RNA-binding domain involved in B-factors RNP assembly. Protein 29.617.2 Drug target sites in the N protein Ligand/ion –– Water 30.528.3 As the RNA-binding activity of the N protein is critical for viral RNP R.m.s. deviations formation and genome replication, blocking RNA binding of N-NTD Bond lengths (Å) 0.005 0.003 has been proved to be a good strategy to develop antiviral drugs. The compound PJ34, which targets the ribonucleotide-binding site Bond angles (°) 0.808 0.651 in N-NTD, displays potent inhibition of the RNA-binding activity of Ramachandran plot the HCoV-OC43 N protein and can inhibit viral replication of HCoV- Favoured (%) 99.40 99.77 OC43 (Lin et al, 2014). PJ34 mimics the binding of AMP to N-NTD, Allowed (%) 0.60 0.23 and it fits in the ribonucleotide-binding pocket of N-NTD with an Outliers (%) 0.00 0.00 extended trend to the N-terminal loop. We have compared the corre- sponding binding site for PJ34 in our SARS-CoV-2 N-NTD structure Values in parentheses are for highest-resolution shell. b 1/2 R = Σ [1/(N  1)] Σ |I  <I>|/Σ Σ I , where I is the observed p.i.m. hkl i i hkl i i i with that in the HCoV-OC43 N-NTD structure (Fig 6A), and we intensity and <I> is the average intensity from multiple measurements. found that the key residues that are involved in the interactions, R = Σ ||F |  |F ||/Σ |F |, where F and F are the structure-factor work o c o o c including S51, F53, Y109, Y111 and R149 (in SARS-CoV-2 N-NTD amplitudes from the data and the model, respectively. R is the R factor for free numbering), are conserved (Fig 6B). a subset (5%) of reflections that was selected prior to refinement calculations and was not included in the refinement. Another strategy is to block normal N protein oligomerization, thereby halting RNP formation or inducing abnormal aggregation. Recently, the novel inhibitor 5-benzyloxygramine (P3) was identi- protruding region, indicating this patch is responsible for RNA bind- fied by virtual screening (Lin et al, 2020). This compound could ing in different coronaviruses. mediate MERS-CoV N-NTD non-native dimerization and induce N protein aggregation. It was shown to have potent antiviral activity Structure of SARS-CoV-2 N-CTD against MERS-CoV. The complex structure of MERS-CoV N-NTD and P3 shows that P3 targets the non-native interface of dimeric N- The crystal structure of SARS-CoV-2 N-CTD was solved by molecu- NTD and binds to two hydrophobic pockets in two N-NTD proto- lar replacement to a resolution of 1.5 A, with R and R values mers. P3 occupies the N-terminal vector-fusion residue-binding work free of 17.7 and 19.0%, respectively (Table 1). SARS-CoV-2 N-CTD cavity of NTD promoter 1 in the ligand-free MERS-CoV N-NTD forms a tight homodimer and displays an overall rectangular slab structure (Fig 6C) and further stabilizes the dimeric status by shape (Fig 4A). Each protomer has the shape of the letter C (Fig 4B) massive hydrophobic interactions. We compared the binding cavity 4 of 12 The EMBO Journal 39:e105938 | 2020 ª 2020 The Authors Ya Peng et al The EMBO Journal AB β4 β4 β3 β3 β1 β5 β2 β6 β1 β5 β2 β6 η1 C η1 CD Ribonucleotide binding site 9DULDEOH &RQVHUYHG Figure 2. Structure of SARS-CoV-2 N-NTD. A Cartoon representation of SARS-CoV-2 N-NTD. B Topology diagram for SARS-CoV-2 N-NTD; g represents the 3 helix and b represents the b-sheet. C The electrostatic surface potential of SARS-CoV-2 N-NTD. Red and blue colours indicate negative and positive potential, respectively. The RNA-binding sites are highlighted in dotted circles and labelled. D Surface representation of SARS-CoV-2 N-NTD coloured according to sequence conservation based the alignment of N protein sequences from seven human-infecting CoVs using the ConSurf server (Ashkenazy et al, 2016); dark magenta indicates the most conserved, dark cyan indicates the most divergent. of P3 with the corresponding site in our SARS-CoV-2 N-NTD struc- other coronaviruses (Yu et al, 2006; Chen et al, 2007; Saikatendu ture, and we found that almost all of the residues involved in the et al, 2007; Lin et al, 2014; Papageorgiou et al, 2016; Szelazek interactions are conserved, except F135 in MERS-CoV, which is et al, 2017; Nguyen et al,2019).N-NTD has a right-handed fist replaced by I146 in SARS-CoV-2 (Fig 6D). Although both residues shape consisting of an antiparallel b-sheet core subdomain and a are nonpolar amino acids, the difference in hydrophobic force may protruding b-hairpin region. N-CTD is present as a tight inter- affect the dimerization efficiency. twined homodimer and displays an overall rectangular slab shape. Based on the surface electronic distribution and superimposition with other coronavirus N-NTD structures, we compared the Discussion ribonucleotide-binding pocket in SARS-CoV-2 N-NTD, and we found that the binding residues are highly conserved, which could In this study, we solved the crystal structures of both SARS-CoV-2 be a verified target for antiviral drugs that block the RNA-binding N-NTD and N-CTD. Both structures show conserved features with activity of the N protein. ª 2020 The Authors The EMBO Journal 39:e105938 | 2020 5 of 12 The EMBO Journal Ya Peng et al SARS-CoV-2 AB C SARS-CoV H77/Y124 Y109 A55 HCoV-OC43 Y110 A56 P24/G68 AMP AMP Y124 G68 MERS-CoV Y99 G46 HCoV-NL63 Y111 Y126 Y112 Y101 Y79/Y126 S53 S20/S64 S52 S64 F53 S42 F54 F66 R149 Y22/F66 R116/R164 Y44 R150 R164 R138 DE F SARS-CoV-2 SARS-CoV HCoV-OC43 GH MERS-CoV HCoV-NL63 Figure 3. Comparison of the structure of SARS-CoV-2 N-NTD with other human-infecting CoV N-NTD structures. A Superposition of the SARS-CoV-2 N-NTD structure with other human-infecting CoV N-NTD structures. The ribbon representation of each structure is coloured separately (SARS-CoV, PDB: 2OFZ, cyan; HCoV-OC43, PDB: 4LI4, pink; MERS-CoV, PDB: 4UD1, orange; HCoV-NL63, PDB: 5N4K, blue) (Saikatendu et al, 2007; Lin et al, 2014; Papageorgiou et al, 2016; Szelazek et al, 2017). The loops that show significant differences are highlighted by dotted circles. The AMP ligand in the HCoV- OC43 N-NTD-AMP complex is shown as a stick structure. B Structural superimposition of SARS-CoV-2, SARS-CoV and MERS-CoV N-NTDs with that of HCoV-OC43 bound to AMP to show the detailed interactions involved in AMP binding. C Structural superimposition of HCoV-NL63 N-NTD with HCoV-OC43 N-NTD bound to AMP to show the detailed interactions involved in AMP binding. D–H Comparison of the electrostatic surfaces with other human-infecting CoV N-NTD structures. The electrostatic surface of N-NTD from (D) SARS-CoV-2, (E) SARS-CoV, (F) HCoV-OC43 bound to AMP, (G) MERS-CoV and (H) HCoV-NL63 are shown, and the AMP-binding sites are highlighted in dotted circles. Red and blue colours indicate negative and positive potential, respectively. 6 of 12 The EMBO Journal 39:e105938 | 2020 ª 2020 The Authors Ya Peng et al The EMBO Journal A C N N N VLGHYLHZ η1 η1 η2 β2 β1 C η2 β1 β2 WRSYLHZ β2 N N N β1 C C C C η1 η2 β2 β1 η2 η1 N N N Figure 4. Structure of SARS-CoV-2 N-CTD. A Cartoon representation of SARS-CoV-2 N-CTD. Ribbon representation of SARS-CoV-2 N-CTD showing the separated monomer and dimer. One monomer is coloured in pink, and the other is coloured in yellow. B Rotation of (A) along the x-axis by 90° to show the top view. C Topology diagram for SARS-CoV-2 N-CTD. The a-helices are numbered A, B and C, g represents the 3 helix and b represents the b-sheet, according to a previous SARS N-CTD structural element numbering rule (Yu et al, 2006). Although both N-NTD and N-CTD structures resemble those of and specific anti-N antibodies would be critical to enhance the rapid other coronaviruses, especially SARS-CoV, the electrostatic potential molecular diagnosis for COVID-19. During our manuscript prepara- surfaces display different charge distribution patterns in some areas, tion, other groups also solved crystal structures of N-NTD or including the N-terminal loop, the top tip of the protruding region, N-CTD, which show similar structural features (Kang et al, 2020; the bottom of the core subdomain in N-NTD and the b-sheet face of Ye et al, 2020). N-CTD. These divergent features may alter the RNA-binding modes Until now, there are no structures of the full-length N proteins or binding efficiency. Of note, we found that the orientations of the from coronaviruses reported, due to their dynamic and oligomeric N-terminal loops of N-NTD were distinct. The N-terminal loops of characteristics. Based on our dimeric structure of N-CTD and previ- SARS-CoV-2, SARS-CoV and MERS-CoV N-NTD stretch outward, ous studies, the N protein forms a dimer by N-CTD interaction, while the loops from HCoV-OC43 and HCoV-NL63 N-NTD rotate and the disordered LKRs serve as the two arms connecting the two towards the core subdomain. This result suggests that different N- N-NTDs to the N-CTD dimers (Chen et al, 2007; Nguyen et al, NTD may have group-specific characteristics and that the distinc- 2019). In the RNP complex, the N-CTDs form the helical core. The tions may alter the locations of N-arms and then lead to different RNA molecule twists around the helical groove and further inter- structural and functional properties. In addition, as the N protein acts with the NTDs (Chang et al, 2014). According to previous could interact with many host proteins during the infection process cryo-electron tomography studies of the vRNP complex isolated and cause a robust humoral immune response after infection, the from murine hepatitis virus (MHV) and SARS-CoV virions, each divergent charge distribution surfaces could be critical to identify dimeric N unit packs in a long loose helical or a supercoiled orga- specific interacting cellular proteins and develop specific immune- nization (Neuman et al, 2006; Ba´rcena et al, 2009; Gui et al, based rapid diagnostic test (Burbelo et al, 2020; Ni et al, 2020). 2017). Recently, preprint: Klein et al (2020) observed that SARS- Besides the serological assays for detection of SARS-CoV-2 antibod- CoV-2 RNPs organizing like string beads, indicating the high steri- ies (Horber et al, 2020), the rapid detection of viral antigens based cal flexibility of RNP assembly. However, a lack of high-resolution on specific antibodies can provide essential information for disease structures of RNP limits our understanding of the precise RNP monitoring (Che et al, 2004). Therefore, to develop high-affinity assembly mechanism. ª 2020 The Authors The EMBO Journal 39:e105938 | 2020 7 of 12 The EMBO Journal Ya Peng et al SARS-CoV-2 SARS-CoV MERS-CoV HCoV-NL63 Figure 5. Comparison of the structure of SARS-CoV-2 N-CTD with other coronavirus N-CTD structures. A Superimposed structures in ribbon representation of N-CTD from SARS-CoV-2 (pink), SARS-CoV (PDB: 2CJR, cyan) (Chen et al, 2007), MERS-CoV (PDB: 6G13, orange) (Nguyen et al, 2019) and HCoV-NL63 (PDB: 5EPW, blue) (Szelazek et al, 2017). B Electrostatic surface views of the four N-CTD structures. Red and blue colours indicate negative and positive potential, respectively. 8 of 12 The EMBO Journal 39:e105938 | 2020 ª 2020 The Authors O Ya Peng et al The EMBO Journal AB PJ34 Y111 / Y124 F53 / F66 Y109 / Y126 S51/S64 R149 /R164 SARS-CoV-2 N-NTD HCoV-OC43 N-NTD PJ34 complex CD protomer1 T115 / T105 / T105 G116 / G106/G106 T148/ T137/ T137 A119 / A109 /A109 I146 / F135/ F135 P3 protomer2 N75 / N66/ N66 Y112 / Y102 Y102 / W52// W43 W43 SARS-CoV-2 N-NTD MERS-CoV N-NTD MERS-CoV N-NTD P3 complex Figure 6. Conserved drug target sites in SARS-CoV-2 N protein. A, B Superimposition of the structure of SARS-CoV-2 N-NTD (light blue) with HCoV-OC43 N-NTD (light pink) bound to PJ34 (magenta) (PDB: 4KXJ) (Lin et al, 2014). The detailed interactions are shown in (B). The water molecule mediating hydrogen-bonding interactions is shown as a red sphere. C, D Superimposition of the structure of SARS-CoV-2 N-NTD (light blue) with MERS-CoV N-NTD dimer (grey) or MERS-CoV N-NTD bound to P3 (cyan) (PDB: 6KL6) (Lin et al, 2020). The detailed interactions are shown in (D). Coronavirus N proteins have been attractive targets for antiviral and found that the drug targeting sites are relatively conserved, indicat- drugs as they are critical for viral replication and assembly. Few inhibi- ing further structure-based drug optimization for broad-spectrum agent tors have been identified through various approaches based on HCoV- development is very promising. Our structural information of SARS- OC43 or MERS-CoV (Lin et al, 2014; Zhang et al, 2020b). We analysed CoV-2 N-NTD and C-CTD can aid in the discovery and development of the binding sites of the representative inhibitors in our crystal structures antiviral inhibitors against SARS-CoV-2 in the future. ª 2020 The Authors The EMBO Journal 39:e105938 | 2020 9 of 12 H The EMBO Journal Ya Peng et al Materials and Methods were performed using REFMAC5 (Murshudov et al, 1997) and COOT (Emsley & Cowtan, 2004), respectively. Further refinement Gene cloning protein production and purification was performed using Phenix (Adams et al, 2010). Final statistics for data collection and structure refinement are presented in Table 1. SARS-CoV-2 N-NTD (amino acid residues 44–174, GISAID accession ID: EPI_ISL_402119) fused at its N-terminus with a hexa-histidine tag Biochemical characterization of N proteins was cloned into the pET-21a vector (Novagen) with NdeIand XhoI restriction sites (Li et al, 2019). Transformed E. coli strain BL21 (DE3) The purified protein was analysed with an analytical gel filtration clones were grown in LB medium containing 100 lg/ml ampicillin to assay with a calibrated Superdex 75 10/300 GL column (GE an OD of 0.6–0.8 at 37°C. Expression of the recombinant proteins Healthcare). The sample was further analysed with SDS–PAGE. was induced by the addition of 0.5 mM isopropyl-b-D-1-thiogalacto- The analytical ultracentrifugation assay was performed according pyranoside (IPTG), and incubation was continued for a further 16 h to a previously reported method (Wang et al, 2017). The proteins at 16°C. Cells were harvested by centrifugation at 7,000 g for 15 min were prepared in 20 mM Tris (pH 8.0) and 150 mM NaCl at a at 4°C and then resuspended in lysis buffer [20 mM Tris–HCl (pH 8.0) concentration of 0.8 mg/ml. The assay was performed on an opti- and 150 mM NaCl] and further homogenized with a low-temperature mal ProteomeLab XL-I analytical ultracentrifuge (Beckman Coulter) ultra-high pressure cell disrupter (JNBIO, China). The lysate was clari- at a speed of 228,000 g. The molecular mass analysis was performed fied by centrifugation at 20,000 g for 60 min at 4°C. The supernatant with XL-I data analysis software. was purified by metal affinity chromatography using a HisTrap HP 5 ml column (GE Healthcare). Proteins were eluted using lysis buffer supplemented with 300 mM imidazole. The proteins were further Data availability purified by gel filtration chromatography using a HiLoad 16/600 Superdex 75 PG (GE Healthcare) with a running buffer of 20 mM Atomic coordinates and structure factors have been deposited in the Tris–HCl (pH 8.0) and 50 mM NaCl, and the collected protein frac- Protein Data Bank with accession codes 7CDZ (https://www.rcsb.org/ tions were concentrated to 15 mg/ml using a membrane concentrator structure/7CDZ) and 7CE0 (https://www.rcsb.org/structure/7CE0). with a molecular weight cut-off of 10 kDa (Millipore). SARS-CoV-2 N-CTD (amino acid residues 255–364) was Expanded View for this article is available online. constructed and expressed in the same way as N-NTD. During metal affinity chromatography, N-CTDs were eluted using lysis buffer Acknowledgements supplemented with 500 mM imidazole. The proteins were further We thank the staff of BL19U1 beamlines at Shanghai Synchrotron Radiation purified by gel filtration chromatography using a HiLoad 16/600 Facility. We thank Qian Wang (Institute of Microbiology CAS) for help with Superdex 75 PG (GE Healthcare) with a running buffer of 20 mM analytical ultracentrifugation experiment. This work was supported by the Tris–HCl (pH 8.0) and 150 mM NaCl, and the collected protein frac- Ministry of Science and Technology of the People’s Republic of China tions were concentrated to 10 mg/ml using a membrane concentra- (2020YFC0845900), the National Science and Technology Major Project tor with a molecular weight cut-off of 10 kDa (Millipore). (2018ZX10733403), and the National Natural Science Foundation of China (NSFC) (81702015). H.S. is supported by the Youth Innovation Promotion Asso- Crystallization, data collection and structure determination ciation CAS (2017117). G.F.G. is supported partly by the COVID-19 Emergency Project of CAS and the External Cooperation Program of CAS Crystallization trials were set up with commercial crystallization kits (153211KYSB20160001). (Hampton Research) using the sitting drop vapour diffusion method. The resultant drop was then sealed, equilibrating against 100 ll reser- Author contributions voir solution at 18°C. Diffractable crystals of SARS-CoV-2 N-NTD GFG and HS designed and supervised the study. YP, ND, YL and SD conducted were obtained in 0.1 M SPG (succinic acid, sodium phosphate the experiments. JQ collected the data sets and solved the structures. HS, TL monobasic monohydrate and glycine mix buffer pH 6.0) (Molecular and GFG analysed the data and wrote the manuscript. Dimensions, MD2-59) with 25% w/v polyethylene glycol 1,500 at 18°C. SARS-CoV-2 N-CTD crystals were obtained in 4 M potassium Conflict of interest formate, 0.1 M Bis-Tris propane (pH 9.0) and 2% w/v polyethylene The authors declare that they have no conflict of interest. glycol monomethyl ether 2,000 at 18°C. Crystals were flash-cooled in liquid nitrogen after a brief soaking in reservoir solution with the addi- tion of 17% (v/v) glycerol. The X-ray diffraction data were collected References under cryogenic conditions (173°C) at Shanghai Synchrotron Radia- tion Facility (SSRF) beam line BL19U1 and indexed, integrated and Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, scaled with HKL3000 (Minor et al, 2006). Hung LW, Kapral GJ, Grosse-Kunstleve RW et al (2010) PHENIX: a comprehensive Python-based system for macromolecular structure SARS-CoV-2 N-NTD and N-CTD structures were solved by the molecular replacement method using Phaser (Read, 2001) from the solution. Acta Crystallogr D Biol Crystallogr 66: 213 – 221 CCP4 programme suite (Winn et al, 2011), with the structures of Ashkenazy H, Abadi S, Martz E, Chay O, Mayrose I, Pupko T, Ben-Tal N (2016) ConSurf 2016: an improved methodology to estimate and visualize SARS-CoV N-NTD (PDB: 2OFZ) (Saikatendu et al, 2007) and N-CTD (PDB: 2CJR) (Chen et al, 2007) as the search models, respectively. evolutionary conservation in macromolecules. Nucleic Acids Res 44: Initial restrained rigid-body refinement and manual model building W344 – W350 10 of 12 The EMBO Journal 39:e105938 | 2020 ª 2020 The Authors Ya Peng et al The EMBO Journal Bárcena M, Oostergetel GT, Bartelink W, Faas FG, Verkleij A, Rottier PJ, Koster Hörber S, Soldo J, Relker L, Jürgens S, Guther J, Peter S, Lehmann R, Peter A AJ, Bosch BJ (2009) Cryo-electron tomography of mouse hepatitis virus: (2020) Evaluation of three fully-automated SARS-CoV-2 antibody assays. insights into the structure of the coronavirion. Proc Natl Acad Sci USA 106: Clin Chem Lab Med https://doi.org/10.1515/cclm-2020-0975 582 – 587 Jiang S, Shi Z, Shu Y, Song J, Gao GF, Tan W, Guo D (2020) A distinct name is Beigel JH, Tomashek KM, Dodd LE, Mehta AK, Zingman BS, Kalil AC, Hohmann needed for the new coronavirus. Lancet 395: 949 E, Chu HY, Luetkemeyer A, Kline S et al (2020) Remdesivir for the Kang S, Yang M, Hong Z, Zhang L, Huang Z, Chen X, He S, Zhou Z, Zhou Z, treatment of Covid-19 – preliminary report. N Engl J Med https://doi.org/ Chen Q et al (2020) Crystal structure of SARS-CoV-2 nucleocapsid protein 10.1056/NEJMoa2007764 RNA binding domain reveals potential unique drug targeting sites. Acta Burbelo PD, Riedo FX, Morishima C, Rawlings S, Smith D, Das S, Strich JR, Pharm Sin B 10: 1228 – 1238 Chertow DS, Davey RT, Cohen JI (2020) Sensitivity in detection of Klein S, Cortese M, Winter SL, Wachsmuth-Melm M, Neufeldt CJ, Cerikan B, antibodies to nucleocapsid and spike proteins of severe acute respiratory Stanifer ML, Boulant S, Bartenschlager R, Chlanda P (2020) SARS-CoV-2 syndrome Coronavirus 2 in patients with coronavirus disease 2019. J Infect structure and replication characterized by in situ cryo-electron Dis 222: 206 – 213 tomography. bioRxiv https://doi.org/10.1101/2020.06.23.167064 [PREPRINT] Cao B, Wang Y, Wen D, Liu W, Wang J, Fan G, Ruan L, Song B, Cai Y, Wei M Li C, Chai Y, Song H, Weng C, Qi J, Sun Y, Gao GF (2019) Crystal structure of et al (2020) A trial of lopinavir-ritonavir in adults hospitalized with severe african swine fever virus dUTPase reveals a potential drug target. MBio 10: Covid-19. N Engl J Med 382: 1787 – 1799 e02483-19 Cavalcanti AB, Zampieri FG, Rosa RG, Azevedo LCP, Veiga VC, Avezum A, Li Q, Guan X, Wu P, Wang X, Zhou L, Tong Y, Ren R, Leung KSM, Lau EHY, Damiani LP, Marcadenti A, Kawano-Dourado L, Lisboa T et al (2020) Wong JY et al (2020) Early transmission dynamics in Wuhan, China, of Hydroxychloroquine with or without Azithromycin in mild-to-moderate novel coronavirus-infected pneumonia. N Engl J Med 382: 1199 – 1207 Covid-19. N Engl J Med https://doi.org/10.1056/NEJMoa2019014 Lin SY, Liu CL, Chang YM, Zhao J, Perlman S, Hou MH (2014) Structural basis Chang CK, Hou MH, Chang CF, Hsiao CD, Huang TH (2014) The SARS coronavirus for the identification of the N-terminal domain of coronavirus nucleocapsid protein–forms and functions. Antiviral Res 103: 39 – 50 nucleocapsid protein as an antiviral target. J Med Chem 57: 2247 – 2257 Chang CK, Jeyachandran S, Hu NJ, Liu CL, Lin SY, Wang YS, Chang YM, Hou Lin SM, Lin SC, Hsu JN, Chang CK, Chien CM, Wang YS, Wu HY, Jeng US, MH (2016) Structure-based virtual screening and experimental validation Kehn-Hall K, Hou MH (2020) Structure-based stabilization of non-native of the discovery of inhibitors targeted towards the human coronavirus protein-protein interactions of coronavirus nucleocapsid proteins in nucleocapsid protein. Mol BioSyst 12: 59 – 66 antiviral drug design. J Med Chem 63: 3131 – 3141 Che XY, Qiu LW, Pan YX, Wen K, Hao W, Zhang LY, Wang YD, Liao ZY, Hua X, Lu X, Pan J, Tao J, Guo D (2011) SARS-CoV nucleocapsid protein antagonizes Cheng VC et al (2004) Sensitive and specific monoclonal antibody-based IFN-b response by targeting initial step of IFN-b induction pathway, capture enzyme immunoassay for detection of nucleocapsid antigen in and its C-terminal region is critical for the antagonism. Virus Genes 42: sera from patients with severe acute respiratory syndrome. J Clin Microbiol 37 – 45 42: 2629 – 2635 Lu R, Zhao X, Li J, Niu P, Yang B, Wu H, Wang W, Song H, Huang B, Zhu N Chen CY, Chang CK, Chang YW, Sue SC, Bai HI, Riang L, Hsiao CD, Huang TH et al (2020) Genomic characterisation and epidemiology of 2019 novel (2007) Structure of the SARS coronavirus nucleocapsid protein RNA- coronavirus: implications for virus origins and receptor binding. Lancet binding dimerization domain suggests a mechanism for helical packaging 395: 565 – 574 of viral RNA. J Mol Biol 368: 1075 – 1086 Maisonnasse P, Guedj J, Contreras V, Behillil S, Solas C, Marlin R, Naninck T, Cong Y, Kriegenburg F, de Haan CAM, Reggiori F (2017) Coronavirus Pizzorno A, Lemaitre J, Goncalves A et al (2020) Hydroxychloroquine use nucleocapsid proteins assemble constitutively in high molecular against SARS-CoV-2 infection in non-human primates. Nature https://doi. oligomers. Sci Rep 7: 5740 org/10.1038/s41586-020-2558-4 Cong Y, Ulasli M, Schepers H, Mauthe M, V’Kovski P, Kriegenburg F, Thiel V, Minor W, Cymborowski M, Otwinowski Z, Chruszcz M (2006) HKL-3000: the de Haan CAM, Reggiori F (2020) Nucleocapsid protein recruitment to integration of data reduction and structure solution–from diffraction replication-transcription complexes plays a crucial role in coronaviral life images to an initial model in minutes. Acta Crystallogr D Biol Crystallogr cycle. J Virol 94:e01925-19 62: 859 – 866 Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Mu J, Xu J, Zhang L, Shu T, Wu D, Huang M, Ren Y, Li X, Geng Q, Xu Y et al Acta Crystallogr D Biol Crystallogr 60: 2126 – 2132 (2020) SARS-CoV-2-encoded nucleocapsid protein acts as a viral Gorbalenya AE, Baker SC, Baric RS, de Groot RJ, Drosten C, Gulyaeva AA, suppressor of RNA interference in cells. Sci China Life Sci 63: 1 – 4 Haagmans BL, Lauber C, Leontovich AM, Neuman BW et al.(2020)The Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular species severe acute respiratory syndrome-related coronavirus: structures by the maximum-likelihood method. Acta Crystallogr D Biol classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol 5: Crystallogr 53: 240 – 255 536 – 544. Neuman BW, Adair BD, Yoshioka C, Quispe JD, Orca G, Kuhn P, Milligan RA, Gui M, Liu X, Guo D, Zhang Z, Yin CC, Chen Y, Xiang Y (2017) Electron Yeager M, Buchmeier MJ (2006) Supramolecular architecture of severe microscopy studies of the coronavirus ribonucleoprotein complex. Protein acute respiratory syndrome coronavirus revealed by electron Cell 8: 219 – 224 cryomicroscopy. J Virol 80: 7918 – 7928 Hoffmann M, Mosbauer K, Hofmann-Winkler H, Kaul A, Kleine-Weber H, Kruger Nguyen THV, Lichiere J, Canard B, Papageorgiou N, Attoumani S, Ferron F, N, Gassen NC, Muller MA, Drosten C, Pohlmann S (2020) Chloroquine does Coutard B (2019) Structure and oligomerization state of the C-terminal not inhibit infection of human lung cells with SARS-CoV-2. Nature https:// region of the Middle East respiratory syndrome coronavirus nucleoprotein. doi.org/10.1038/s41586-020-2575-3 Acta Crystallogr D Struct Biol 75: 8 – 15 ª 2020 The Authors The EMBO Journal 39:e105938 | 2020 11 of 12 The EMBO Journal Ya Peng et al Ni L, Ye F, Cheng ML, Feng Y, Deng YQ, Zhao H, Wei P, Ge J, Gou M, Li X et al Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan (2020) Detection of SARS-CoV-2-specific humoral and cellular immunity in RM, Krissinel EB, Leslie AG, McCoy A et al (2011) Overview of the CCP4 COVID-19 convalescent individuals. Immunity 52: 971 – 977 suite and current developments. Acta Crystallogr D Biol Crystallogr 67: Papageorgiou N, Lichiere J, Baklouti A, Ferron F, Sevajol M, Canard B, Coutard 235 – 242 B(2016) Structural characterization of the N-terminal part of the MERS- Wu F, Zhao S, Yu B, Chen YM, Wang W, Song ZG, Hu Y, Tao ZW, Tian JH, Pei CoV nucleocapsid by X-ray diffraction and small-angle X-ray scattering. YY et al (2020a) A new coronavirus associated with human respiratory Acta Crystallogr D Struct Biol 72: 192 – 202 disease in China. Nature 579: 265 – 269 Peng TY, Lee KR, Tarn WY (2008) Phosphorylation of the arginine/serine Wu Y, Li C, Xia S, Tian X, Kong Y, Wang Z, Gu C, Zhang R, Tu C, Xie Y et al dipeptide-rich motif of the severe acute respiratory syndrome coronavirus (2020b) Identification of human single-domain antibodies against SARS- nucleocapsid protein modulates its multimerization, translation inhibitory CoV-2. Cell Host Microbe 27: 891 – 898 activity and cellular localization. FEBS J 275: 4152 – 4163 Wu Y, Wang F, Shen C, Peng W, Li D, Zhao C, Li Z, Li S, Bi Y, Yang Y et al Read RJ (2001) Pushing the boundaries of molecular replacement with (2020c) A noncompeting pair of human neutralizing antibodies maximum likelihood. Acta Crystallogr D Biol Crystallogr 57: 1373 – 1382 block COVID-19 virus binding to its receptor ACE2. Science 368: Saikatendu KS, Joseph JS, Subramanian V, Neuman BW, Buchmeier MJ, 1274 – 1278 Stevens RC, Kuhn P (2007) Ribonucleocapsid formation of severe acute Xiang F, Wang X, He X, Peng Z, Yang B, Zhang J, Zhou Q, Ye H, Ma Y, Li H respiratory syndrome coronavirus through molecular action of the N- et al (2020) Antibody detection and dynamic characteristics in patients terminal domain of N protein. J Virol 81: 3913 – 3921 with COVID-19. Clin Infect Dis https://doi.org/10.1093/cid/ciaa461 Shi R, Shan C, Duan X, Chen Z, Liu P, Song J, Song T, Bi X, Han C, Wu L et al Ye Q, West AMV, Silletti S, Corbett KD (2020) Architecture and self-assembly (2020) A human neutralizing antibody targets the receptor-binding site of of the SARS-CoV-2 nucleocapsid protein. Protein Sci https://doi.org/10. SARS-CoV-2. Nature 584: 120 – 124 1002/pro.3909 Surjit M, Liu B, Jameel S, Chow VT, Lal SK (2004) The SARS coronavirus Yu IM, Oldham ML, Zhang J, Chen J (2006) Crystal structure of the severe nucleocapsid protein induces actin reorganization and apoptosis in COS-1 acute respiratory syndrome (SARS) coronavirus nucleocapsid protein cells in the absence of growth factors. Biochem J 383: 13 – 18 dimerization domain reveals evolutionary linkage between corona- and Surjit M, Liu B, Chow VT, Lal SK (2006) The nucleocapsid protein of severe arteriviridae. J Biol Chem 281: 17134 – 17139 acute respiratory syndrome-coronavirus inhibits the activity of cyclin- Zhang L, Lin D, Sun X, Curth U, Drosten C, Sauerhering L, Becker S, Rox K, cyclin-dependent kinase complex and blocks S phase progression in Hilgenfeld R (2020a) Crystal structure of SARS-CoV-2 main protease mammalian cells. J Biol Chem 281: 10669 – 10681 provides a basis for design of improved alpha-ketoamide inhibitors. Szelazek B, Kabala W, Kus K, Zdzalik M, Twarda-Clapa A, Golik P, Burmistrz Science 368: 409 – 412 M, Florek D, Wladyka B, Pyrc K et al (2017) Structural characterization of Zhang X, Tan Y, Ling Y, Lu G, Liu F, Yi Z, Jia X, Wu M, Shi B, Xu S et al human coronavirus NL63 N protein. J Virol 91:e02503-16 (2020b) Viral and host factors related to the clinical outcome of COVID-19. Tan W, Zhao X, Ma X, Wang W, Niu P, Xu W, Gao GF, Wu G (2020) A novel Nature 583: 437 – 440 coronavirus genome identified in a cluster of pneumonia cases - Wuhan, Zhou H, Chen X, Hu T, Li J, Song H, Liu Y, Wang P, Liu D, Yang J, Holmes EC China 2019–2020. China CDC Wkly 2: 61 – 62 et al (2020a) A novel bat coronavirus closely related to SARS-CoV-2 Wang H, Han M, Qi J, Hilgenfeld R, Luo T, Shi Y, Gao GF, Song H (2017) contains natural insertions at the S1/S2 cleavage site of the spike protein. Crystal structure of the C-terminal fragment of NS1 protein from yellow Curr Biol 30: 2196 – 2203 fever virus. Sci China Life Sci 60: 1403 – 1406 Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, Si HR, Zhu Y, Li B, Wang C, Horby PW, Hayden FG, Gao GF (2020a) A novel coronavirus outbreak Huang CL et al (2020b) A pneumonia outbreak associated with a new of global health concern. Lancet 395: 470 – 473 coronavirus of probable bat origin. Nature 579: 270 – 273 Wang J, Peng Y, Xu H, Cui Z, Williams RO (2020b) The COVID-19 vaccine race: Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, Zhao X, Huang B, Shi W, Lu R challenges and opportunities in vaccine formulation. AAPS PharmSciTech et al (2020) A novel coronavirus from patients with pneumonia in China, 21: 225 2019. N Engl J Med 382: 727 – 733 Wang M, Cao R, Zhang L, Yang X, Liu J, Xu M, Shi Z, Hu Z, Zhong W, Xiao G Zuniga S, Cruz JL, Sola I, Mateos-Gomez PA, Palacio L, Enjuanes L (2010) (2020c) Remdesivir and chloroquine effectively inhibit the recently Coronavirus nucleocapsid protein facilitates template switching and is emerged novel coronavirus (2019-nCoV) in vitro. Cell Res 30: 269 – 271 required for efficient transcription. J Virol 84: 2169 – 2175 12 of 12 The EMBO Journal 39:e105938 | 2020 ª 2020 The Authors

Journal

The EMBO Journal – Springer Journals

Published: Oct 15, 2020

Keywords: 2019‐nCoV; antivirals; dimerization; nucleocapsid; RNA binding

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