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Id Subject Object Predicate Lexical cue
T1 0-134 Sentence denotes Structure-Based Stabilization of Non-native Protein–Protein Interactions of Coronavirus Nucleocapsid Proteins in Antiviral Drug Design
T2 136-144 Sentence denotes Abstract
T3 145-257 Sentence denotes Structure-based stabilization of protein–protein interactions (PPIs) is a promising strategy for drug discovery.
T4 258-392 Sentence denotes However, this approach has mainly focused on the stabilization of native PPIs, and non-native PPIs have received little consideration.
T5 393-570 Sentence denotes Here, we identified a non-native interaction interface on the three-dimensional dimeric structure of the N-terminal domain of the MERS-CoV nucleocapsid protein (MERS-CoV N-NTD).
T6 571-660 Sentence denotes The interface formed a conserved hydrophobic cavity suitable for targeted drug screening.
T7 661-892 Sentence denotes By considering the hydrophobic complementarity during the virtual screening step, we identified 5-benzyloxygramine as a new N protein PPI orthosteric stabilizer that exhibits both antiviral and N-NTD protein-stabilizing activities.
T8 893-1160 Sentence denotes X-ray crystallography and small-angle X-ray scattering showed that 5-benzyloxygramine stabilizes the N-NTD dimers through simultaneous hydrophobic interactions with both partners, resulting in abnormal N protein oligomerization that was further confirmed in the cell.
T9 1161-1320 Sentence denotes This unique approach based on the identification and stabilization of non-native PPIs of N protein could be applied toward drug discovery against CoV diseases.
T10 1322-1334 Sentence denotes Introduction
T11 1335-1456 Sentence denotes Small-molecule stabilization of protein–protein interactions (PPIs) is an extremely promising strategy in drug discovery.
T12 1457-1606 Sentence denotes It can be used to treat cancers and viral infections.1−3 Stabilizing PPIs with small molecules may be allosteric or direct (also called orthosteric).
T13 1607-2151 Sentence denotes This process alters the oligomerization equilibrium of the protein and enables small molecules to modulate its physiological function.4−7 The anticancer drug paclitaxel, for example, allosterically enhances microtubule structure assembly by binding to β-tubulin.8,9 On the other hand, rapamycin, another anticancer agent, binds directly to the interface between FKBP12 and mTOR and stabilizes the structure of the complex.10 The most well-characterized PPIs suitable as targets for drug development form natively under physiological conditions.
T14 2152-2295 Sentence denotes However, non-native interactions, which may form under extreme circumstances such as inside a crystal lattice, are also potential drug targets.
T15 2296-2551 Sentence denotes For example, nucleozin exerts its antiviral activity by stabilizing the non-native PPI interface between the two neighboring nucleoprotein trimers within the influenza virus, which results in abnormal protein oligomerization and loss of viral viability.11
T16 2552-2655 Sentence denotes Middle East respiratory syndrome coronavirus (MERS-CoV) belongs to the betacoronavirus (β-CoVs) family.
T17 2656-2990 Sentence denotes It causes severe respiratory distress with a high mortality rate in humans.12−14 Recently, a closely related novel coronavirus, coronavirus disease 2019 (COVID-19), caused an outbreak of pneumonia in Wuhan, which further underscored the risk of CoVs to the global public health.15,16 However, there is no effective treatment for CoVs.
T18 2991-3175 Sentence denotes Thus, there is an urgent need to develop new antiviral agents against CoVs.14,17 MERS-CoV packages its genome in a nucleocapsid (N) protein and forms a ribonucleoprotein (RNP) complex.
T19 3176-3234 Sentence denotes The RNP is essential for viral transcription and assembly.
T20 3235-3603 Sentence denotes Several studies suggested that the modulation of CoV N protein oligomerization by small molecules is a feasible antiviral drug development strategy.18,19 The CoV N protein is organized into the N-terminal domain (NTD) and the C-terminal domain (CTD), with both domains participating in RNA binding.20,21 All CoV N-NTD structures are folded in a monomeric conformation.
T21 3604-3739 Sentence denotes In contrast, the CoV N-CTDs are always dimeric and are responsible for N protein oligomerization via protein–protein interactions.22,23
T22 3740-3834 Sentence denotes Here, we report the crystal structure of MERS-CoV N-NTD in a non-native dimeric configuration.
T23 3835-3939 Sentence denotes We used the non-native dimer interface as the target in virtual screening for an orthosteric stabilizer.
T24 3940-4123 Sentence denotes To this end, we considered the binding scores and hydrophobic complementarity of the acquired poses, and further selected the potential leads P1–P3 from Acros and ZINC drug databases.
T25 4124-4226 Sentence denotes Of these, only 5-benzyloxygramine (P3) had both antiviral and stabilizing activities on the N protein.
T26 4227-4386 Sentence denotes Small-angle X-ray scattering (SAXS) and cell-based assays showed that P3 induces abnormal full-length N protein oligomerization in vitro and at the cell level.
T27 4387-4510 Sentence denotes We also described the structure of MERS-CoV N-NTD complexed with 5-benzyloxygramine and revealed its stabilizing mechanism.
T28 4511-4655 Sentence denotes Our findings provide insight into the development of a new therapeutic approach based on stabilizing a non-native protein interaction interface.
T29 4656-4751 Sentence denotes It may lead to the discovery and development of new treatments for various infectious diseases.
T30 4753-4760 Sentence denotes Results
T31 4762-4822 Sentence denotes Structure of the N-Terminal Domain of the MERS-CoV N Protein
T32 4823-4954 Sentence denotes We determined the crystal structure of MERS-CoV N-NTD by molecular replacement (MR) using the structure of HCoV-OC43 N-NTD (PDB ID:
T33 4955-5114 Sentence denotes 4J3K) as the search model.24 The final structure was refined to R-factor and R-free values of 0.26 and 0.29, respectively, at a resolution of 2.6 Å (Table S1).
T34 5115-5268 Sentence denotes Each asymmetric unit contained four N-NTD molecules assembled into two identical dimers with an overall RMSD of 0.28 Å between the dimers (Figure S1A,B).
T35 5269-5358 Sentence denotes The monomers shared a similar structural core preceded by a flexible region (Figure S1D).
T36 5359-5650 Sentence denotes The core consisted of a five-stranded antiparallel β-sheet sandwiched between loops arranged in a right-handed, fist-shaped structure conserved among the CoVs.25 In our structure, however, the loop connecting strands β2 and β3 protruding out of the core into other CoV N proteins was absent.
T37 5651-5755 Sentence denotes Unlike the reported structures that have a monomeric conformation, our structure was atypically dimeric.
T38 5756-5836 Sentence denotes Figure 1B shows the details of the interactions in the MERS-CoV N protein dimer.
T39 5837-5892 Sentence denotes We named the units monomer 1 and monomer 2 (Figure 1A).
T40 5893-6123 Sentence denotes According to the amino acid composition of the binding site on monomer 2, we divided the dimeric interface into two areas: one located on the N-terminus flexible region and the other on the loop between β4 and β5 of the N protein.
T41 6124-6331 Sentence denotes In the first area, W43, N66, N68, Y102, and F135 of monomer 1 generated a conserved hydrophobic pocket permitting the side chain of M38 of monomer 2 to enter this hole by a hydrophobic contact (Figure 1C,D).
T42 6332-6465 Sentence denotes H37 and N39 of monomer 2 were packed against W43 and F135 of monomer 1, respectively, and contributed to the hydrophobic interaction.
T43 6466-6585 Sentence denotes The side chains of N39 of monomer 2 formed one hydrogen bond with the N68 backbone in monomer 1 at a distance of 2.6 Å.
T44 6586-6634 Sentence denotes The second area was relatively more hydrophilic.
T45 6635-6823 Sentence denotes The main chain oxygens of G104, F135, and T137 of monomer 2 formed three hydrogen bonds with the side chains of Q73 and T134 of monomer 1 at distances of 3.8, 3.2, and 3.7 Å, respectively.
T46 6824-6954 Sentence denotes The side chain of N139 on monomer 2 formed a hydrogen bond with the main chain oxygen of T137 on monomer 1 at a distance of 3.6 Å.
T47 6955-7070 Sentence denotes The interactions of the first and second areas comprise buried surface areas (BSA) of 289 and 103 Å2, respectively.
T48 7071-7218 Sentence denotes The small surface area buried at the interface accounts for ∼5 kcal mol–1 binding energy,26 which translates to a dissociation constant of ∼200 mM.
T49 7219-7367 Sentence denotes Thus, the dimer described here is unique in that it is non-native and relies on vector-fusion residues (H37 and M38) to maintain its dimeric status.
T50 7368-7660 Sentence denotes This property may also explain why the present structure has an oligomeric status different from previously reported structures for the CoV N protein.24,27−30 We used cross-linking experiments to analyze the oligomeric capacity of MERS N-NTD containing the vector-fusion residues in solution.
T51 7661-7711 Sentence denotes MERS N-NTD had a dimeric conformation in solution.
T52 7712-7896 Sentence denotes Our structure indicated that W43 played an essential role in forming the hydrophobic pocket accommodating the vector-fusion residues and, therefore, mediated the N-NTD dimer formation.
T53 7897-7983 Sentence denotes The W43A mutation significantly reduced the oligomeric tendency of N-NTD (Figure S1C).
T54 7984-8114 Sentence denotes This further supports that the “exogenous residues” encoded by the vector backbone mediated the formation of the non-native dimer.
T55 8115-8197 Sentence denotes We also superimposed the previously published structure of MERS-CoV N-NTD (PDB ID:
T56 8198-8278 Sentence denotes 4ud1)27 containing a native N-terminal flexible region with our dimer structure.
T57 8279-8424 Sentence denotes The side chain of N38 in the native structure could not interact with the hydrophobic pocket as the former is hydrophilic and short (Figure S1E).
T58 8425-8570 Sentence denotes Thus, it may be possible to utilize small compounds to replace the vector-fusion residues and stabilize the PPI through hydrophobic interactions.
T59 8571-8769 Sentence denotes Figure 1 Structure and sequence of MERS-CoV N-NTD. (A) Overall structure of the MERS-CoV N-NTD dimer containing monomers 1 and 2 is depicted as a cartoon and colored yellow and green, respectively.
T60 8770-8887 Sentence denotes Residues involved in dimerization are shown as sticks and highlighted in (B). (B) Interactions among MERS-CoV N-NTDs.
T61 8888-9085 Sentence denotes Dimerization is mediated mainly by vector-fusion residues interacting with the conserved hydrophobic regions on the core structure (first area) along with the residues surrounding it (second area).
T62 9086-9171 Sentence denotes Interacting residues on monomers 1 and 2 are labeled in black and blue, respectively.
T63 9172-9259 Sentence denotes Vector-fusion and conserved hydrophobic regions are colored cyan and red, respectively.
T64 9260-9348 Sentence denotes The color of all other interacting residues is the same as that for each monomer in (A).
T65 9349-9479 Sentence denotes Polar contacts are indicated with red dashed lines. (C) Upper panel: close-up of the interacting region of vector-fusion residues.
T66 9480-9565 Sentence denotes The surface was colored according to the hydrophobicity level at the protein surface.
T67 9566-9653 Sentence denotes Vector-fusion residues (black) are shown as sticks to emphasize the hydrophobic pocket.
T68 9654-9666 Sentence denotes Lower panel:
T69 9667-9760 Sentence denotes 2D diagrams of the interaction between the hydrophobic pocket and the vector-fusion residues.
T70 9761-9793 Sentence denotes The latter are labeled in black.
T71 9794-9932 Sentence denotes The hydrophobic contacts are indicated with black dashed lines. (D) Sequence alignment of various CoV N proteins in the N-terminal region.
T72 9933-9982 Sentence denotes Red letters indicate strictly conserved residues.
T73 9983-10030 Sentence denotes Cyan indicates conservative substitution sites.
T74 10031-10117 Sentence denotes Hydrophobic regions involved in unusual dimerization are indicated by black triangles.
T75 10119-10193 Sentence denotes Direct Targeting of the Non-native Dimer Interface for Antiviral Screening
T76 10194-10317 Sentence denotes We performed a structure-based virtual screening by targeting W43 in the hydrophobic pocket of the N-NTD dimeric interface.
T77 10318-10505 Sentence denotes H37 and M38 were removed from the template to identify compounds that could replace the vector-fusion residues and, therefore, contribute to the stabilizing effect (Figures 2A and S2A,B).
T78 10506-10671 Sentence denotes We chose the highest-scoring hits, listed in Table S2, based on shape complementarity, the presence of aromatic moieties, and the ability to stack onto W43 of N-NTD.
T79 10672-11073 Sentence denotes Because the formation of the non-native dimers was primarily mediated by hydrophobic interactions in our structure (Figure 1C,D), we next considered the hydrophobic complementarity between the acquired ligands and N-NTD in the form of the lipophilic match surface (SL/L).31 We also took into account the ability of the drug to permeate cells by aiming for lower topological polar surface areas (TPSA).
T80 11074-11157 Sentence denotes Based on the above criteria, three compounds were finally chosen for further study.
T81 11158-11288 Sentence denotes Benzyl-2-(hydroxymethyl)-1-indolinecarboxylate (P1) and 5-benzyloxygramine (P3) had higher SL/L and docking scores and lower TPSA.
T82 11289-11369 Sentence denotes The clinical drug etodolac (P2) had a comparable SL/L but a lower docking score.
T83 11370-11405 Sentence denotes It too was selected as a candidate.
T84 11406-11788 Sentence denotes However, only P3 induced a comparatively larger blue shift in the intrinsic N-NTD fluorescence spectrum, indicating that the microenvironment surrounding the tryptophans of the protein increased in rigidity and hydrophobicity in the presence of P3.32 The result also indicated that P3 bound more tightly to the N protein than P1 or P2 by interacting with the W43 pocket (Figure 2B).
T85 11789-11933 Sentence denotes Fluorescent thermal stability assays disclosed that the N-NTD denaturation melting temperature had increased from 42 to 45 °C when P3 was added.
T86 11934-12011 Sentence denotes The sigmoidal melting curve for MERS-CoV N-NTD changed in the presence of P3.
T87 12012-12121 Sentence denotes The delay in protein denaturation suggests that P3 stabilized the MERS-CoV N-NTD dimer structure (Figure 2C).
T88 12122-12270 Sentence denotes We then measured the cytotoxic concentration (CC50) and effective concentration (EC50) for each compound using Vero E6 cells infected with MERS-CoV.
T89 12271-12373 Sentence denotes Table 1 shows that P3 had a favorable therapeutic index among the lead compounds tested in this study.
T90 12374-12441 Sentence denotes Therefore, P3 is an excellent candidate inhibitor against MERS-CoV.
T91 12442-12611 Sentence denotes Figure 2 Compound P3 was a potent stabilizer of the MERS-CoV N protein. (A) Schematic depicting the rationale used in designing the allosteric stabilizer of this study.
T92 12612-12974 Sentence denotes An orthosteric stabilizer may be used to bind to the non-native interaction interface of the N protein and stabilize the abnormal interaction between proteins. (B) Conformation and (C) stability analyses were performed based on the FL spectra of NTD (1 μM) incubated with P1–P3 (10 μM) for 1 h with a buffer consisting of 50 mM Tris-HCl (pH 8.3) and 150 mM NaCl.
T93 12975-13039 Sentence denotes Table 1 CC50 and EC50 and Therapeutic Indexes of Lead Compounds
T94 13040-13079 Sentence denotes quantal dose–response relationship (μM)
T95 13080-13119 Sentence denotes compound CC50a EC50b TIc (CC50/EC50)
T96 13120-13141 Sentence denotes P1 459.69 >100 NAd
T97 13142-13163 Sentence denotes P2 569.77 >100 NAd
T98 13164-13186 Sentence denotes P3 805.32 32.1 25.1
T99 13187-13195 Sentence denotes a CC50:
T100 13196-13232 Sentence denotes Half maximal toxicity concentration.
T101 13233-13241 Sentence denotes b EC50:
T102 13242-13279 Sentence denotes Half maximum effective concentration.
T103 13280-13286 Sentence denotes c TI:
T104 13287-13305 Sentence denotes Therapeutic index.
T105 13306-13312 Sentence denotes d NA:
T106 13313-13326 Sentence denotes Nonavailable.
T107 13328-13389 Sentence denotes Structural Model of P3-Induced MERS-CoV N Protein Aggregation
T108 13390-13479 Sentence denotes We used SAXS to assess the effects of P3 on the full-length MERS-CoV N protein structure.
T109 13480-13580 Sentence denotes The fitted distance distribution function of the protein with and without P3 are shown in Figure 3A.
T110 13581-13718 Sentence denotes P3 increased the maximum dimension (Dmax) and radius of gyration (Rg) of the protein from 207 to 230 Å and from 58 to 65 Å, respectively.
T111 13719-13803 Sentence denotes Thus, the size of the MERS-CoV N protein in solution was altered upon binding to P3.
T112 13804-14042 Sentence denotes Figure 3 P3-induced abnormal aggregation on the full-length MERS-CoV N protein. (A–E) SAXS analysis of the full-length MERS-CoV N protein. (A) Normalized results from GNOM showing pairwise distance distribution P(r) and maximum distance.
T113 14043-14428 Sentence denotes The radius of gyration fitted to 207 and 230 Å for the N protein and the N-P3 complex, respectively. “r” represents pairwise distances. (B, C) Scattering profiles of the N protein (B) and the N-P3 complex (C) and normalization fitting with GNOM (dashed lines). (D, E) Representative models of the N protein (D) and the N-P3 complex (E) generated by CRYSOL simulations of the SAXS data.
T114 14429-14454 Sentence denotes Only α carbons are shown.
T115 14455-14757 Sentence denotes NTD (yellow), CTD (green), and disorder region (cyan). (F, G) Conformation (F) and stability (G) analyses based on FL spectra of the MERS-CoV N protein (1 μM) incubated with P3 (10 μM) for 1 h in a buffer consisting of 50 mM Tris-HCl, 150 mM NaCl (pH 8.3). (H) Schematic of the P3 inhibition mechanism.
T116 14758-14875 Sentence denotes Left panel: in the absence of RNA, N proteins organize as a dimeric building block contributed by N-CTD dimerization.
T117 14876-15052 Sentence denotes Middle panel: P3 promoted the dimerization of N-NTDs from different building blocks, by which the distance between CTD cuboids was shortened and N protein aggregation occurred.
T118 15053-15152 Sentence denotes Right panel: octameric conformation of building blocks buried in the RNA-binding surface of N-CTDs.
T119 15153-15212 Sentence denotes It hindered the formation of filamentous ribonucleocapsids.
T120 15213-15358 Sentence denotes The presence of multiple intrinsically disordered regions in the N protein precluded the determination of its structure by X-ray crystallography.
T121 15359-15508 Sentence denotes Instead, we used rigid body modeling of the SAXS data with the N-terminal domain (NTD; solved in this study) and the C-terminal domains (CTD, PDB ID:
T122 15509-15622 Sentence denotes 6G13).23 In this way, we obtained structural models for the free N protein and its complex with P3 (Figure 3B,C).
T123 15623-15652 Sentence denotes Excellent fits were obtained.
T124 15653-15773 Sentence denotes Representative structural models for the full-length protein without and with P3 are shown in Figure 3D,E, respectively.
T125 15774-15875 Sentence denotes The free N protein formed a tetramer through CTD with the NTD freely hanging in solution (Figure 3D).
T126 15876-16076 Sentence denotes The conformation of the solution was consistent with structures previously reported for other CoV N proteins.33 The N-P3 complex formed a compact hexadecamer with a sunburst configuration (Figure 3E).
T127 16077-16175 Sentence denotes The CTDs formed a central ring and non-native NTD dimers formed “spikes” protruding from the ring.
T128 16176-16348 Sentence denotes Consistent with ligand-induced aggregation, we observed a “blue shift” in the fluorescence spectrum of the full-length MERS-CoV N protein in the presence of P3 (Figure 3F).
T129 16349-16551 Sentence denotes The addition of P3 also delayed N protein thermal denaturation and changed the shape of the denaturation curve, further suggesting that large protein aggregates formed in the presence of P3 (Figure 3G).
T130 16552-16627 Sentence denotes The structure explains how N-NTD dimerization decreased MERS-CoV viability.
T131 16628-16688 Sentence denotes The N protein packages the viral genome into an RNP complex.
T132 16689-16948 Sentence denotes Several models for N-CTD dimer assembly have been proposed for the formation of filamentous RNPs.33 All of the proposed interfaces between N-CTD dimers occurred on the side-faces of the CTD cuboid perpendicular to the proposed RNA-binding surface (Figure 3H).
T133 16949-17240 Sentence denotes Combinatorial use of any region on the side-faces of the CTD dimer cuboid may facilitate manipulation of the RNP length and curvature without obstructing the RNA-binding surface.28,34 However, the SAXS results indicated that N-CTD aggregation occurred on the β-sheet floor of the CTD cuboid.
T134 17241-17427 Sentence denotes For this reason, the RNA-binding surface of the CTD is occluded by the neighboring CTD on the ring and by the non-native NTD dimer making direct contact with the CTD (Figures 3H and S3).
T135 17428-17611 Sentence denotes In addition, the CTD cuboids in the aggregation naturally form a topologically closed octamer, leaving no open ends for further addition of CTD cuboids to form a long filamentous RNP.
T136 17612-17769 Sentence denotes Both the loss of the RNA-binding surface and the inability to incorporate further N protein molecules beyond an octamer may inhibit the formation of the RNP.
T137 17770-17853 Sentence denotes Therefore, P3 may inhibit MERS-CoV RNP formation by inducing N protein aggregation.
T138 17855-17909 Sentence denotes P3 Inhibits MERS-CoV by Inducing N Protein Aggregation
T139 17910-17990 Sentence denotes We demonstrated that P3 had the best characteristics as a therapeutic candidate.
T140 17991-18245 Sentence denotes To determine the anti-MERS-CoV activity of P3 in the cell, the effects of P3 incubation on extracellular viral titers and intracellular viral RNA levels were assessed by plaque assays on Vero E6 cells (Figure 4A) and by RT-qPCR (Figure 4B), respectively.
T141 18246-18350 Sentence denotes At 50 μM, P3 marginally affected the viral titer after 48 h but suppressed viral RNA replication by 40%.
T142 18351-18421 Sentence denotes At 100 μM, P3 halted both viral production and replication after 48 h.
T143 18422-18485 Sentence denotes This result proved the capacity of P3 as an antiviral compound.
T144 18486-18630 Sentence denotes We then examined MERS-CoV N protein distribution and expression in the infected cells with or without P3 treatment to confirm our SAXS findings.
T145 18631-18792 Sentence denotes Immunofluorescence microscopy (Figure 4C) showed condensation of the intracellular N protein fluorescence signal in infected Vero E6 cells treated with 50 μM P3.
T146 18793-18849 Sentence denotes Thus, P3 may induce intracellular N protein aggregation.
T147 18850-18910 Sentence denotes At 100 μM, P3 suppressed N protein expression in most cells.
T148 18911-18967 Sentence denotes However, a few presented with intense N protein signals.
T149 18968-19111 Sentence denotes P3 may have restrained the MERS-CoV N proteins inside the infected cells that promoted the formation of new virions that could not be released.
T150 19112-19180 Sentence denotes In this way, the adjacent cells could not be infected with MERS-CoV.
T151 19181-19305 Sentence denotes The data, therefore, suggest that P3 may inhibit MERS-CoV by inducing abnormal aggregation of the N protein inside the cell.
T152 19306-19380 Sentence denotes This finding is consistent with the results of our structure-based assays.
T153 19381-19585 Sentence denotes Figure 4 Compound P3 was a potential inhibitor against MERS-CoV. (A, B) Viral titers (A) and RNA (B) of MERS-CoV measured by plaque assay and RT-qPCR, respectively, decreased after P3 treatment for 48 h.
T154 19586-19665 Sentence denotes Relative RNA levels were determined by comparing MERS alone at each time point.
T155 19666-19701 Sentence denotes GAPDH RNA was the internal control.
T156 19702-19765 Sentence denotes All values are presented as mean ± SE (standard error of mean).
T157 19766-19911 Sentence denotes One-way Anova was used for statistics (*p < 0.05, **p < 0.01, ***p < 0.001). (C) MERS-CoV nucleocapsid protein decreased after 48 h P3 treatment.
T158 19912-20001 Sentence denotes Nucleocapsid protein expressions (red) were examined under a confocal microscope at ×680.
T159 20002-20037 Sentence denotes Nuclei were stained blue with DAPI.
T160 20039-20106 Sentence denotes Crystal Structure of MERS-CoV N-NTD Complexed with Potent Compounds
T161 20107-20237 Sentence denotes We attempted to obtain crystals of MERS-CoV N-NTD in complex with compounds P1, P2, and P3 by cocrystallization or ligand-soaking.
T162 20238-20382 Sentence denotes With the exception of P2, the complex structures of N-NTD with P1 and P3 were solved at resolutions of 3.09 and 2.77 Å, respectively (Table S1).
T163 20383-20454 Sentence denotes The overall structures of the complexes resembled that of apo-MERS-CoV.
T164 20455-20638 Sentence denotes Both complexes revealed well-defined unbiased densities in the dimer interface and permitted detailed analysis of the interactions between the compounds and MERS-CoV N-NTD (Figure 5).
T165 20639-20761 Sentence denotes The interactions between the N protein and each compound were calculated with the Discovery Studio Client (v19.1.0.18287).
T166 20762-20858 Sentence denotes Most interactions were hydrophobic contacts, which were consistent with our selection rationale.
T167 20859-21002 Sentence denotes In the P1 complex, N68, F135, and D143 on monomer 1 and V41, G106, P107, and T137 on monomer 2 packed against P1 to create a dimer (Figure 5A).
T168 21003-21086 Sentence denotes In addition, two nonbonding interactions were detected between P1 and the monomers.
T169 21087-21192 Sentence denotes There was a π-anion interaction between the benzene ring of the P1 indoline moiety and D143 of monomer 1.
T170 21193-21315 Sentence denotes There was also a π-donor hydrogen bond between the other P1 benzene ring and the T137 side chain of monomer 2 (Figure 5B).
T171 21316-21449 Sentence denotes Relative to P1, P3 bound more deeply into the dimer interface and interacted with a larger number of residues on both N-NTD monomers.
T172 21450-21611 Sentence denotes The amino acid composition of this binding region was W43, N66, N68, S69, T70, N73, and F135 on monomer 1 and V41, G104, T105, G106, A109, and T137 on monomer 2.
T173 21612-21816 Sentence denotes These residues along with P3 generated a massive hydrophobic driving force allowing the proteins and ligands to pack against each other and stabilize the dimeric conformation of the N protein (Figure 5C).
T174 21817-21891 Sentence denotes Several nonbonding interactions were also observed at the P3-binding site.
T175 21892-22035 Sentence denotes These included the interaction between the P3 benzene ring and N68 of monomer 1 and A109 of monomer 2 via π-lone pair and π-alkyl interactions.
T176 22036-22119 Sentence denotes The dimethylaminomethyl moiety of P3 was a major source of nonbonding interactions.
T177 22120-22228 Sentence denotes Three π-cation interactions formed between this moiety and the aromatic groups of W43 and F135 in monomer 1.
T178 22229-22348 Sentence denotes This moiety also formed a π-lone pair interaction with N66 and a π-sigma interaction with W43 of monomer 1 (Figure 5D).
T179 22349-22558 Sentence denotes The structural analyses explain the comparatively stronger binding of P3 to N-NTD (Figure 2B) and corroborated the thermal stabilization effects (Figure 2C) and antiviral activities (Table 1) of the compounds.
T180 22559-22630 Sentence denotes Figure 5 Structures of MERS-CoV N-NTD complexed with potent compounds.
T181 22631-23119 Sentence denotes The structures were solved using HCoV-OC43 N-NTD (PDB:4J3K) as the search model.24 Left panel: (Upper) structural superimposition of the MERS-CoV N-NTD:P1 complex (monomers 1 and 2 are in purple and pink, respectively) and the MERS-CoV N-NTD:P3 complex (monomers 1 and 2 are in brown and green, respectively) with compounds depicted as stick structures. (Lower) Interactions involving vector-fusion residues in the non-native dimer of the apoprotein shown for comparison with (A) and (B).
T182 23120-23154 Sentence denotes Color is the same as in Figure 1A.
T183 23155-23239 Sentence denotes Right panel: detailed interactions among MERS-CoV N-NTD and P1 (A, B) and P3 (C, D).
T184 23240-23350 Sentence denotes Different Fo–Fc maps were contoured at ∼2.5 σ. (A) Detailed stereoview of interactions at the P1-binding site.
T185 23351-23410 Sentence denotes The color of each monomer is the same as in the left panel.
T186 23411-23533 Sentence denotes Residues constructing the P1-binding pocket are labeled and showed as sticks. (B) Schematic of P1 bound to MERS-CoV N-NTD.
T187 23534-23613 Sentence denotes Hydrophobic contacts between P1 and each monomer are displayed as dashed lines.
T188 23614-23731 Sentence denotes Nonbonding interactions are indicated by cyan arrows. (C) Detailed stereoview of interactions at the P3-binding site.
T189 23732-23791 Sentence denotes The color of each monomer is the same as in the left panel.
T190 23792-23913 Sentence denotes Residues belonging to the P3-binding pocket are labeled and shown as sticks. (D) Schematic of P3 bound to MERS-CoV N-NTD.
T191 23914-23993 Sentence denotes Hydrophobic contacts between P3 and each monomer are displayed as dashed lines.
T192 23994-24046 Sentence denotes Nonbonding interactions are indicated by red arrows.
T193 24048-24074 Sentence denotes Discussion and Conclusions
T194 24075-24209 Sentence denotes The use of small molecules to stabilize PPI interfaces has been shown to be a viable approach for the development of new therapeutics.
T195 24210-25201 Sentence denotes Most compounds created by structure-based design to manipulate the PPI depend on detailed knowledge of the native interacting interfaces.35−37 In contrast, most of the compounds with therapeutic potential that stabilize non-native PPI interfaces were discovered by chance alone.38 The anti-influenza compound nucleozin was initially discovered using a chemical genetics approach, and its ability to stabilize non-native nucleoprotein oligomers was elucidated much later.39,40 Swinholide A, a cytotoxic marine macrolide, has been known to disrupt the actin cytoskeleton and act as an anticancer agent, but it took ten years to discover that it stabilized G-actin as a non-native homodimer complex.41−43 However, these examples did underscore the importance of hydrophobicity as a crucial factor stabilizing protein–protein and protein–ligand associations.44 Here, we demonstrated the possibility of using the hydrophobic interactions on non-native interfaces as targets for virtual screening.
T196 25202-25433 Sentence denotes Combined with suitable selection criteria focusing on both shape and hydrophobic complementarities between the ligand and the receptor, small-molecule compounds that stabilize non-native PPIs may be identified in a rational manner.
T197 25434-25637 Sentence denotes Using the above approach, we successfully identified a compound P3 that affected the biochemical activity of our target protein and showed efficacy against our target pathogen MERS-CoV (Figures 2 and 4).
T198 25638-25829 Sentence denotes P3-mediated non-native MERS-CoV N-NTD dimerization induced abnormal N protein aggregation by influencing the oligomeric behavior of N-CTD and eventually halting its function in RNP formation.
T199 25830-25973 Sentence denotes To the best of our knowledge, this structure-based strategy for targeting non-native interfaces has never been proposed for therapeutic design.
T200 25974-26075 Sentence denotes Thus, non-native interaction interfaces in proteins may comprise a new drug development target class.
T201 26076-26212 Sentence denotes For β-coronaviruses such as MERS-CoV, the amino acids comprising the non-native interaction interface on N-NTD are relatively conserved.
T202 26213-26436 Sentence denotes The hydrophobic pocket surrounding W43 and F135 on monomer 1 is shared among other β-coronaviruses.24,28 The interacting surface on monomer 2, which includes G104, T105, G106, and A109, is also highly conserved (Figure S4).
T203 26437-26594 Sentence denotes This conservation may provide certain advantages when developing compounds with broad-spectrum activity against a target pathogen family, including COVID-19.
T204 26595-26749 Sentence denotes When we tested P3 on mouse hepatitis virus (MHV), we observed a reduction in viral titer, indicating that P3 may also inhibit MHV replication (Figure S5).
T205 26750-26828 Sentence denotes On the other hand, targeting non-native interaction interfaces is not trivial.
T206 26829-27000 Sentence denotes As interface formation is induced by an external agent, computations aimed at predicting native PPI structures may not be able to identify potential non-native interfaces.
T207 27001-27114 Sentence denotes Nevertheless, several stratagems may assist in the identification of potential non-native interaction interfaces.
T208 27115-27237 Sentence denotes One strategy is to search for target protein structures with crystal packing contacts known to be biologically irrelevant.
T209 27238-27564 Sentence denotes Another approach is to identify weakly interacting sites through NMR chemical shift perturbation and hydrogen-deuterium exchange MS.45 Once these potential non-native interaction interfaces are identified, standard screening and then functional characterization may be conducted for small compounds that bind to the interface.
T210 27565-27682 Sentence denotes To summarize, we demonstrated that non-native interaction interfaces might form when proteins abnormally oligomerize.
T211 27683-27833 Sentence denotes Using the MERS-CoV N protein as an example, we showed that non-native interfaces might serve as targets for small-molecule, structure-based screening.
T212 27834-27946 Sentence denotes We also proposed several stratagems to rationally identify potential non-native interface targets for screening.
T213 27947-28114 Sentence denotes We believe that we have discovered and tested an alternative drug discovery paradigm that could help expand the repertoire of lead compounds against various pathogens.
T214 28116-28136 Sentence denotes Experimental Section
T215 28138-28147 Sentence denotes Chemicals
T216 28148-28284 Sentence denotes The compounds P1, P2, and P3 were purchased from Maybridge Chemical Company, TCI Chemicals, and Sigma-Aldrich Corporation, respectively.
T217 28285-28372 Sentence denotes The reagents used in this study were purchased from Sigma Chemical Co. (St. Louis, MO).
T218 28373-28462 Sentence denotes The purity of all compounds is higher than 95% and was used without further purification.
T219 28464-28509 Sentence denotes Cloning, Protein Expression, and Purification
T220 28510-28763 Sentence denotes The MERS-CoV N proteins were prepared according to previously described methods.46 In brief, the cDNA fragments of MERS-CoV N proteins were cloned into a pET-28a expression vector (Merck, Darmstadt, Germany) containing a histidine tag-encoding sequence.
T221 28764-28928 Sentence denotes Vectors encoding the single mutants N39A, N39G, and W43A were generated using the QuikChange site-directed mutagenesis protocol with the primers listed in Table S3.
T222 28929-29003 Sentence denotes The vectors were transformed into Escherichia coli BL21 (DE3) pLysS cells.
T223 29004-29213 Sentence denotes The cells were grown to an optical density range of 0.6–0.8 at 600 nm at 37 °C and protein expression induced with 1.0 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), followed by incubation at 10 °C for 24 h.
T224 29214-29381 Sentence denotes The cells were harvested by centrifugation (6000g, 12 min, 4 °C) and resuspended in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 15 mM imidazole, and 1 mM PMSF; pH 7.5).
T225 29382-29473 Sentence denotes The cells were lysed by sonication and centrifuged (10000g, 40 min, 4 °C) to remove debris.
T226 29474-29642 Sentence denotes The supernatant was purified by injection into a Ni–NTA column (Merck, Darmstadt, Germany) and eluted with buffer containing imidazole at a gradient range of 15–300 mM.
T227 29643-29811 Sentence denotes Pure protein fractions were collected, dialyzed with low-salt buffer, concentrated, and quantified by the Bradford method (BioShop Canada Inc., Burlington, ON, Canada).
T228 29813-29848 Sentence denotes Crystallization and Data Collection
T229 29849-30015 Sentence denotes MERS-CoV N-NTD crystals were grown as previously described:46 MERS-CoV N-NTD was crystallized at room temperature (∼25 °C) by the sitting-drop vapor-diffusion method.
T230 30016-30259 Sentence denotes A protein solution (2 μL; 10 mg mL–1) was mixed with an equal volume of crystallization solution consisting of 75 mM ammonium sulfate, 2 mM NaBr, and 29% PEG 3350 (Sigma-Aldrich Corp., St. Louis, MO) and equilibrated against a 300 μL solution.
T231 30260-30356 Sentence denotes MERS-CoV N-NTD:P3 co-crystals were obtained using a crystallization solution containing 2 mM P3.
T232 30357-30534 Sentence denotes MERS-CoV N-NTD crystals in complex with P1 were obtained by soaking native MERS-CoV N-NTD crystals for 90 s at room temperature in a crystallization solution containing 2 mM P1.
T233 30535-30743 Sentence denotes Diffraction datasets for MERS-CoV N-NTD alone and in complex with P1 were collected at beamline 13B1 of the Taiwan Light Source (TLS) of the National Synchrotron Research Center (NSRRC; Hsinchu City, Taiwan).
T234 30744-30853 Sentence denotes Diffraction of the MERS-CoV N-NTD:P3 complex was performed at the beamline SP44XU of SPring-8 (Hyogo, Japan).
T235 30855-30894 Sentence denotes Structural Determination and Refinement
T236 30895-30961 Sentence denotes Diffraction data were processed and scaled with HKL-2000 software.
T237 30962-31152 Sentence denotes The structures were solved by molecular replacement (MR) in Phenix47 using HCoV-OC43 N-NTD (PDB:4J3K) as the search model.24 The initial models were rebuilt and refined by Coot48 and Phenix.
T238 31153-31247 Sentence denotes Structures were visualized using PyMOL (The PyMOL Molecular Graphics System, version 2.3.0).49
T239 31249-31274 Sentence denotes Chemical Cross-Link Assay
T240 31275-31419 Sentence denotes Protein solutions containing 40 μM of wild-type or mutated MERS-CoV N-NTD were incubated with glutaraldehyde at a final concentration of 1% v/v.
T241 31420-31534 Sentence denotes The reaction was conducted at room temperature for 10 min and quenched with the addition of 1 M Tris-HCl (pH 7.5).
T242 31535-31656 Sentence denotes The samples were then stored on ice and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
T243 31658-31717 Sentence denotes Discovery of Orthosteric PPI Stabilizers for MERS N Protein
T244 31718-31893 Sentence denotes To screen for compounds that induce hydrophobic PPI between MERS N-NTDs, a model of dimeric MERS-CoV N-NTD without the H37 and M38 residues was used in virtual drug screening.
T245 31894-32051 Sentence denotes The Sigma-Aldrich, Acros Organics, and ZINC drug databases were screened with LIBDOCK molecular docking software to obtain compounds acting on the N protein.
T246 32052-32117 Sentence denotes The N protein binding pocket was represented by a set of spheres.
T247 32118-32186 Sentence denotes Each compound in the database was docked in a pocket comprising W43.
T248 32187-32341 Sentence denotes The hydrophobic complementarity between ligands and receptors was calculated with PLATINUM.31 Compounds with higher docking scores are listed in Table S2.
T249 32343-32368 Sentence denotes Fluorescence Measurements
T250 32369-32470 Sentence denotes Fluorescence assays were performed in a buffer consisting of 50 mM Tris-HCl (pH 8.3) and 150 mM NaCl.
T251 32471-32582 Sentence denotes One micromolar N protein was incubated either with the control buffer or each compound (10 μM) at 4 °C for 1 h.
T252 32583-32691 Sentence denotes Tryptophan fluorescence was acquired with a Jasco FP-8300 fluorescence spectrometer (JASCO International Co.
T253 32692-32797 Sentence denotes Ltd., Tokyo, Japan) at an excitation wavelength of 280 nm and an emission wavelength range of 300–400 nm.
T254 32799-32827 Sentence denotes Thermostability Measurements
T255 32828-33007 Sentence denotes Thermostability assays were conducted in a buffer consisting of 50 mM Tris-HCl (pH 7.5) and 150 mM NaCl and with a JASCO FP-8300 fluorescence spectrometer (JASCO International Co.
T256 33008-33028 Sentence denotes Ltd., Tokyo, Japan).
T257 33029-33140 Sentence denotes One micromolar N protein was incubated either with the control buffer or each compound (10 μM) at 4 °C for 2 h.
T258 33141-33302 Sentence denotes UV absorbance vs temperature profiles were acquired by ramping the temperature from 4–95 °C at a 1 °C min–1 and recording the absorbance at 280 nm every 0.5 min.
T259 33304-33346 Sentence denotes Determining CC50 and EC50 of Hit Compounds
T260 33347-33447 Sentence denotes Vero E6 cells were infected with MERS-CoV with M.O.I = 0.1 and treated with lead compounds for 48 h.
T261 33448-33510 Sentence denotes Cell viability was determined by the neutral red uptake assay.
T262 33511-33561 Sentence denotes CC50 and EC50 were determined by % cell viability.
T263 33562-33616 Sentence denotes CC50 was determined for cells treated with drugs only.
T264 33617-33683 Sentence denotes EC50 was determined for MERS-infected cells after drug treatments.
T265 33685-33732 Sentence denotes Small-Angle X-ray Scattering (SAXS) Experiments
T266 33733-33956 Sentence denotes SAXS experiments were performed at the BL23A SAXS beamline at the TLS of NSRRC, using a monochromatic X-ray beam (λ = 0.828 Å), with an integrated HPLC system of an Agilent-Bio SEC-3 300 Å column (Agilent Technologies, Inc.
T267 33957-33974 Sentence denotes Santa Clara, CA).
T268 33975-34194 Sentence denotes Protein samples (44 μM MERS-CoV N and MERS-CoV N:P3 complex prepared by incubating the 44 μM native protein with 440 μM P3) were prepared in a buffer consisting of 50 mM Tris-HCl (pH 8.5) and 150 mM NaCl on ice for 1 h.
T269 34195-34279 Sentence denotes Then, a 100 μL aliquot was injected into the column at a flow rate of 0.02 mL min–1.
T270 34280-34442 Sentence denotes After passing through the column, the sample solution was directed into a quartz capillary (2 mm dia.) for subsequent buffer and sample SAXS measurement at 288 K.
T271 34443-34542 Sentence denotes The sample-to-detector distance of 2.5 m used covered a scattering vector q range of 0.01–0.20 Å–1.
T272 34543-34612 Sentence denotes Here, q is defined as q = (4π/λ) sin θ, with the scattering angle 2θ.
T273 34613-34712 Sentence denotes Thirty-six frames were collected for each sample elution with an X-ray frame exposure time of 30 s.
T274 34713-34892 Sentence denotes Frames of good data overlapping (namely, of low radiation damage effects) were merged for improved data statistics and analyzed to determine initial Rg using PRIMUS (version 3.1).
T275 34893-35013 Sentence denotes The P(r) distance distribution and Dmax were calculated from the experimental scattering curve using GNOM (version 4.1).
T276 35014-35227 Sentence denotes An ensemble optimization method (EOM) analysis was performed through the EMBL Hamburg web interface.50 Modeling of the rigid body crystal structure was calculated and generated using CRYSOL (ATSAS Program Suite v.
T277 35228-35285 Sentence denotes 2.8.2).51 The crystal structures of MERS-CoV NTD (PDB ID:
T278 35286-35386 Sentence denotes 4UD1)27 and MERS-CoV NTD:P3 (solved in this study) and the CTD domain of MERS-CoV N protein (PDB ID:
T279 35387-35437 Sentence denotes 6G13)23 were used as rigid bodies in EOM analysis.
T280 35438-35526 Sentence denotes With the EOM analysis, 1000 models were generated in the beginning as a structural pool.
T281 35527-35687 Sentence denotes Selected from the SAXS profiles of the structural pool was an ensemble of models that could fit the experimental scattering curve with their linear combination.
T282 35688-35858 Sentence denotes Tetrameric MERS-CoV NP conformations and 16-mer MERS-CoV:P3 conformations were selected because their ensemble generated curves fit best to the experimental SAXS results.
T283 35860-35875 Sentence denotes Viral Infection
T284 35876-36041 Sentence denotes Vero E6 cells (ATCC No: CRL-1586) were seeded onto culture plates with complete Dulbecco’s modified Eagle’s medium (DMEM) and incubated overnight prior to infection.
T285 36042-36264 Sentence denotes MERS-CoV (HCoV-EMC/2012) at a multiplicity of infection (M.O.I.) of 0.1 was added to the cells and incubated at 37 °C for 1 h, followed by washing thrice with phosphate-buffered saline (PBS) to remove the unattached virus.
T286 36265-36324 Sentence denotes Fresh complete culture medium was then added to the plates.
T287 36326-36338 Sentence denotes Plaque Assay
T288 36339-36425 Sentence denotes Vero E6 cells were seeded in 12-well plates and incubated overnight before the assays.
T289 36426-36560 Sentence denotes Samples containing MERS-CoV were serially diluted 10× with MEM, added to the wells, and incubated for 1 h with agitation every 15 min.
T290 36561-36624 Sentence denotes After incubation, the inocula were removed and washed with PBS.
T291 36625-36767 Sentence denotes An overlay medium comprising 2× MEM and 1.5% (w/v) agarose (1:1) was added to the wells followed by incubation at 37 °C and 5% CO2 for 3 days.
T292 36768-36881 Sentence denotes The plates were fixed with 10% (v/v) formalin containing 0.2% (w/v) crystal violet, and the plaques were counted.
T293 36883-36890 Sentence denotes RT-qPCR
T294 36891-37037 Sentence denotes Total RNA of infected Vero E6 cells was extracted with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
T295 37038-37178 Sentence denotes Reverse transcription and PCR amplification were performed with an iTaq Universal One-Step RT-qPCR Kit (Bio-Rad Laboratories, Hercules, CA).
T296 37179-37283 Sentence denotes Real-time PCR was performed in a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA).
T297 37284-37356 Sentence denotes The primer pairs used to amplify the viral RNA were as follows: GAPDH-F:
T298 37357-37392 Sentence denotes 5′-GAAGGTGAAGGTCGGAGTC-3′; GAPDH-R:
T299 37393-37434 Sentence denotes 5′-GAAGATGGTGATGGGATTTC-3′;53 MERS-CoV-F:
T300 37435-37475 Sentence denotes 5′-CCACTACTCCCATTTCGTCAG-3′; MERS-CoV-R:
T301 37476-37631 Sentence denotes 5′-CAGTATGTGTAGTGCGCATATAAGCA-3′.54 The MERS RNA levels were normalized to that of GAPDH and compared between MERS-CoV groups at 24 h.p.i. and at 48 h.p.i.
T302 37633-37657 Sentence denotes Immunofluorescence Assay
T303 37658-37786 Sentence denotes Vero E6 cells were seeded in eight-well chamber slides and incubated overnight prior to infection with MERS-CoV at M.O.I. = 0.1.
T304 37787-37926 Sentence denotes The cells were fixed with 4% (v/v) paraformaldehyde for 20 min at 4 °C, followed by permeabilization in 0.1% (v/v) Triton X-100 for 10 min.
T305 37927-37998 Sentence denotes Then, 7.5% (v/v) BSA was used as a blocking buffer for 30 min at 37 °C.
T306 37999-38115 Sentence denotes Anti-MERS-CoV N primary antibody (1:500 dilution; Sino Biological Inc., Beijing, China) was used to stain the virus.
T307 38116-38326 Sentence denotes The cells were incubated overnight, washed thrice with PBS, and incubated with Alex Fluor 568 anti-rabbit secondary antibody (1:1000 dilution; Thermo Fisher Scientific, Waltham, MA) for 1 h at room temperature.
T308 38327-38394 Sentence denotes 4′,6-Diamidino-2-phenylindole (DAPI) was added during the PBS wash.
T309 38395-38511 Sentence denotes MERS nucleocapsid expression was examined under a confocal microscope (LSM-700; Carl Zeiss AG, Oberkochen, Germany).
T310 38513-38545 Sentence denotes Supporting Information Available
T311 38546-39336 Sentence denotes The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.9b01913.Dimeric conformation of MERS-CoV N-NTD (Figure S1); LibDock pose of dimeric MERS-CoV N-NTD with potent compounds (Figure S2); P3-induced aggregation of the MERS-CoV N protein sequesters the RNA-binding region on the N-CTD dimer (Figure S3); potential target sites for broad-spectrum antiviral compound development (Figure S4); antiviral activity of P3 against MHV (mouse hepatitis virus) (Figure S5); crystallographic data collection and refinement statistics (Table S1); pW43 docking pose with chemical structures, docking scores, and biochemical properties of 17 potential hits (Table S2); primers used for mutagenesis in this study (Table S3); molecular formula strings (PDF)
T312 39337-39381 Sentence denotes Molecular Formula Strings_JMC_revision (CSV)
T313 39383-39405 Sentence denotes Supplementary Material
T314 39406-39426 Sentence denotes jm9b01913_si_001.pdf
T315 39427-39447 Sentence denotes jm9b01913_si_002.csv
T316 39449-39464 Sentence denotes Accession Codes
T317 39465-39631 Sentence denotes For PDB codes 6KL2 (native), 6KL5 (N-P1 complex), and 6KL6 (N-P3 complex), authors will release the atomic coordinates and experimental data upon article publication.
T318 39633-39653 Sentence denotes Author Contributions
T319 39654-39728 Sentence denotes ○ S.-M.L., S.-C.L., J.-N.H., and C.-k.C. contributed equally to this work.
T320 39730-39750 Sentence denotes Author Contributions
T321 39751-39757 Sentence denotes Jia N.
T322 39758-39770 Sentence denotes Hsu, Shih C.
T323 39771-39783 Sentence denotes Lin, Yong S.
T324 39784-39801 Sentence denotes Wang, and Ming H.
T325 39802-39831 Sentence denotes Hou designed research; Jia N.
T326 39832-39844 Sentence denotes Hsu, Shih C.
T327 39845-39857 Sentence denotes Lin, Shan M.
T328 39858-39871 Sentence denotes Lin, Ching M.
T329 39872-39890 Sentence denotes Chien, and Yong S.
T330 39891-39923 Sentence denotes Wang performed research; Hung Y.
T331 39924-39932 Sentence denotes Wu, U S.
T332 39933-39941 Sentence denotes Jeng, K.
T333 39942-39964 Sentence denotes Kehn-Hall, and Ming H.
T334 39965-40020 Sentence denotes Hou contributed new reagents or analytic tools; Shih C.
T335 40021-40033 Sentence denotes Lin, Shan M.
T336 40034-40047 Sentence denotes Lin, Ching M.
T337 40048-40066 Sentence denotes Chien, and Ming H.
T338 40067-40097 Sentence denotes Hou analyzed the data; Shih C.
T339 40098-40110 Sentence denotes Lin, Shan M.
T340 40111-40124 Sentence denotes Lin, Chung K.
T341 40125-40140 Sentence denotes Chang, Ching M.
T342 40141-40159 Sentence denotes Chien, and Ming H.
T343 40160-40180 Sentence denotes Hou wrote the paper.
T344 40182-40241 Sentence denotes This work was supported by the MOST 106-2628-M-005-001-MY3.
T345 40243-40295 Sentence denotes The authors declare no competing financial interest.
T346 40297-40312 Sentence denotes Acknowledgments
T347 40313-40476 Sentence denotes We thank the National Synchrotron Radiation Research Center (Taiwan) and the synchrotron radiation facility SPring-8 (Japan) for X-ray diffraction data collection.
T348 40477-40490 Sentence denotes Abbreviations
T349 40491-40524 Sentence denotes PPIs protein–protein interactions
T350 40525-40578 Sentence denotes MERS-CoV Middle East Respiratory Syndrome Coronavirus
T351 40579-40600 Sentence denotes NTD N-terminal domain
T352 40601-40622 Sentence denotes CTD C-terminal domain
T353 40623-40637 Sentence denotes N nucleocapsid
T354 40638-40660 Sentence denotes β-CoVs betacoronavirus
T355 40661-40682 Sentence denotes RNP ribonucleoprotein
T356 40683-40716 Sentence denotes SAXS small-angle X-ray scattering
T357 40717-40741 Sentence denotes MR molecular replacement
T358 40742-40766 Sentence denotes BSA buried surface areas
T359 40767-40803 Sentence denotes TPSA topological polar surface areas
T360 40804-40832 Sentence denotes CC50 cytotoxic concentration
T361 40833-40861 Sentence denotes EC50 effective concentration
T362 40862-40886 Sentence denotes D max maximum dimension
T363 40887-40910 Sentence denotes R g radius of gyration
T364 40911-40936 Sentence denotes MHV mouse hepatitis virus