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CORD-19:58c67994f47dc6c8b552cff7de2892719259ec52 JSONTXT 9 Projects

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Id Subject Object Predicate Lexical cue
T1 0-127 Sentence denotes Solution Structure of the C-terminal Dimerization Domain of SARS Coronavirus Nucleocapsid Protein Solved by the SAIL-NMR Method
T2 129-137 Sentence denotes Abstract
T3 138-418 Sentence denotes The C-terminal domain (CTD) of the severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (NP) contains a potential RNA-binding region in its N-terminal portion and also serves as a dimerization domain by forming a homodimer with a molecular mass of 28 kDa.
T4 419-578 Sentence denotes So far, the structure determination of the SARS-CoV NP CTD in solution has been impeded by the poor quality of NMR spectra, especially for aromatic resonances.
T5 579-819 Sentence denotes We have recently developed the stereo-array isotope labeling (SAIL) method to overcome the size problem of NMR structure determination by utilizing a protein exclusively composed of stereo-and regio-specifically isotope-labeled amino acids.
T6 820-933 Sentence denotes Here, we employed the SAIL method to determine the high-quality solution structure of the SARS-CoV NP CTD by NMR.
T7 934-1117 Sentence denotes The SAIL protein yielded less crowded and better resolved spectra than uniform 13 C and 15 N labeling, and enabled the homodimeric solution structure of this protein to be determined.
T8 1118-1272 Sentence denotes The NMR structure is almost identical with the previously solved crystal structure, except for a disordered putative RNA-binding domain at the N-terminus.
T9 1273-1484 Sentence denotes Studies of the chemical shift perturbations caused by the binding of single-stranded DNA and mutational analyses have identified the disordered region at the N-termini as the prime site for nucleic acid binding.
T10 1485-1567 Sentence denotes In addition, residues in the β-sheet region also showed significant perturbations.
T11 1568-1838 Sentence denotes Mapping of the locations of these residues onto the helical model observed in the crystal revealed that these two regions are parts of the interior lining of the positively charged helical groove, supporting the hypothesis that the helical oligomer may form in solution.
T12 1840-1959 Sentence denotes Severe acute respiratory syndrome (SARS) is a recently emergent disease caused by the SARSassociated coronavirus (CoV).
T13 1960-2130 Sentence denotes 1, 2 The SARS-CoV nucleocapsid protein (NP) packages the viral genomic RNA into a ribonucleoprotein complex and is crucial for the assembly of infectious virus particles.
T14 2131-2448 Sentence denotes Based on comparative NMR studies of SARS-CoV NP deletion constructs, the protein contains two structural domains: the N-terminal domain (NTD; containing residues 45-181) and the C-terminal domain (CTD; containing residues 248-365), 3 flanked by the long, disordered N-and C-termini and the linker sequence (Fig. 1a) .
T15 2449-2554 Sentence denotes The NTD reportedly acts as a putative RNA-binding domain, and the CTD functions as a dimerization domain.
T16 2555-2639 Sentence denotes 3, 4 Recently, the CTD has also been shown to bind nucleic acids with high affinity.
T17 2640-2753 Sentence denotes 5 Structural investigations of the isolated SARS-CoV NP CTD have been performed by NMR and X-ray crystallography.
T18 2754-2948 Sentence denotes In a previous NMR study, the topological structure of the isolated SARS-CoV NP CTD as a homodimer was elucidated based on limited intersubunit and intrasubunit nuclear Overhauser effects (NOEs).
T19 2949-3103 Sentence denotes 6 This topology was confirmed by the subsequently reported crystal structures of the SARS-CoV NP CTD and of a shorter construct spanning residues 270-370.
T20 3104-3262 Sentence denotes 5, 7 The crystal structure of the CTD revealed that residues 248-280 form a positively charged patch, which acts as a putative oligonucleotide-binding region.
T21 3263-3420 Sentence denotes The patch also participates in intermolecular and intramolecular interactions within the crystal, resulting in the formation of an octameric asymmetric unit.
T22 3421-3536 Sentence denotes However, previous biochemical and biophysical studies have shown that the CTD exists solely as a dimer in solution.
T23 3537-3616 Sentence denotes 6 These findings motivated us to investigate the nature of the CTD in solution.
T24 3617-3784 Sentence denotes Initial attempts at the complete structure elucidation of the SARS-CoV NP CTD through NMR were impeded by its short T 2 relaxation times and significant peak overlaps.
T25 3785-3961 Sentence denotes 6 Recently, we have developed the stereo-array isotope labeling (SAIL) method, which utilizes proteins exclusively composed of stereo-and regiospecifically labeled amino acids.
T26 3962-4178 Sentence denotes 8 Compared to conventional uniform 13 C, 15 N isotopic labeling, the quality of the spectra of the SAIL sample was sufficiently improved, so that a high-resolution solution structure determination could be performed.
T27 4179-4318 Sentence denotes The sharpened resonance lines and reduced peak overlap with the SAIL method are due to the selective deuteration of many nonlabile protons.
T28 4319-4445 Sentence denotes The remaining protons with known stereo-specific assignments provide plentiful information about the structure of the protein.
T29 4446-4543 Sentence denotes In this study, we performed an NMR study of the SARS-CoV NP CTD with the help of the SAIL method.
T30 4544-4757 Sentence denotes Compared to the protein uniformly labeled with 13 C and 15 N, the SAIL sample significantly improved the quality of the NMR spectra, to the extent that a high-resolution structure determination could be performed.
T31 4758-4923 Sentence denotes The tertiary structure obtained by NMR is almost identical with that of the crystal structure, except for a disordered putative RNA-binding domain at the N-terminus.
T32 4924-5059 Sentence denotes We further applied NMR, mutation analyses, and electrophoretic mobility shift assays (EMSAs) to pinpoint the nucleic-acid-binding site.
T33 5060-5180 Sentence denotes The active site thus identified agrees well with the helical ribonucleoprotein model suggested by the crystal structure.
T34 5181-5234 Sentence denotes Preparation of the SAIL sample of the SARS-CoV NP CTD
T35 5235-5455 Sentence denotes The preparation of proteins composed of SAIL amino acids requires cell-free expression to efficiently incorporate the SAIL amino acids into the protein without them being affected by metabolic scrambling in living cells.
T36 5456-5554 Sentence denotes The expression of the SARS-CoV NP CTD was initially examined in a small-scale reaction (Fig. 1b) .
T37 5555-5751 Sentence denotes Subsequently, the 1 H-15 N heteronuclear single-quantum coherence (HSQC) of the 15 N-labeled SARS-CoV NP CTD produced by cell-free expression was compared with that produced by in vivo expression.
T38 5752-5911 Sentence denotes These spectra were identical, thus confirming that the structures of the proteins produced by cell-free and in vivo expressions are identical (data not shown).
T39 5912-6097 Sentence denotes The final sample of the SAIL SARS-CoV NP CTD was produced by using the Escherichia coli cell-free protein synthesis system, with optimizations for the production of labeled NMR samples.
T40 6098-6156 Sentence denotes 9 The SAIL method improves the quality of the NMR spectrum
T41 6157-6313 Sentence denotes The 1 H-13 C constant time (CT) HSQC spectra of an aliphatic region were compared between uniformly labeled (UL) and SAIL samples under the same conditions.
T42 6314-6501 Sentence denotes In the case of the UL sample, the signals were prone to overlap between diastereotopic pairs, and some signals were severely broadened beyond detection in the methylene region (Fig. 2a) .
T43 6502-6627 Sentence denotes In contrast, the corresponding spectrum from the SAIL sample had much better quality than that from the UL sample (Fig. 2b) .
T44 6628-6835 Sentence denotes The signal/noise ratios for the SAIL sample were several times higher than those for the corresponding UL sample, consistent with previous results for calmodulin-and maltodextrin-binding proteins (Fig. 2c) .
T45 6836-7103 Sentence denotes 8 For the SARS-CoV NP CTD, the number of peaks to be observed theoretically in the 1 H-13 C CT HSQC spectra, including methyl, methylene, and methane protons, decreased from 517 for the UL sample to 343 for the SAIL sample, greatly simplifying the analytical process.
T46 7104-7198 Sentence denotes For the aromatic region, improvement due to the use of the SAIL method was even more striking.
T47 7199-7298 Sentence denotes The aromatic rings of UL Phe and Tyr contain four and five 13 C-1 H pairs, respectively (Fig. 3a) .
T48 7299-7485 Sentence denotes In the case of SAIL, the six-membered aromatic rings are labeled by alternating 13 C-1 H and 12 C-2 H moieties ( 13 C at the ε and γ positions; 12 C at the δ and ζ positions) (Fig. 3b) .
T49 7486-7717 Sentence denotes 10 In the UL sample, signals for the 1 H-13 C moieties at the δ, ε, and ζ positions of Phe, and at the δ position of Tyr, are overlapped around 131 ppm in the carbon dimension, thus resulting in severe spectral crowding (Fig. 3c ).
T50 7718-7911 Sentence denotes In contrast, the corresponding region for the SAIL sample was much simpler due to the presence of Comparisons of NMR spectra for the SARS-CoV NP CTD between UL and SAIL in the methylene region.
T51 7912-7989 Sentence denotes Aliphatic region of 1 H-13 C CT-HSQC for UL (a) and SAIL (b) SARS-CoV NP CTD.
T52 7990-8043 Sentence denotes Both spectra were acquired under the same conditions.
T53 8044-8080 Sentence denotes The sample concentration was 0.5 mM.
T54 8081-8183 Sentence denotes In (b), assignments for the SAIL sample are labeled. (c) Crosssections from (a) (red) and (b) (black).
T55 8184-8319 Sentence denotes The peak scales are identical between the UL and SAIL spectra. signals exclusively from the 1 H-13 C moieties at the ε position of Phe.
T56 8320-8458 Sentence denotes The short relaxation times led to the detection of resonances that were severely broadened beyond detection with the UL sample (Fig. 3d ).
T57 8459-8693 Sentence denotes Since one-bond 13 C-13 C couplings in the UL protein did not exist in the SAIL protein, as shown in the Tyr Hε-Cε region ( Fig. 3e and f) , the CT technique with a long evolution time (∼17 ms) is thus not required for the SAIL sample.
T58 8694-8889 Sentence denotes 10 With the benefits mentioned above, we were able to acquire a set of NMR spectra with high sensitivity, and the expected chemical shifts were assigned to 91.2% completeness for the SAIL sample.
T59 8890-9052 Sentence denotes Even though the SARS-CoV NP CTD existed as an octamer in the asymmetric unit in the crystal, abundant evidence suggests that it exists as a homodimer in solution.
T60 9053-9166 Sentence denotes 3, 6 We manually assigned some of the intersubunit NOE peaks from the SARS-CoV NP CTD based on previous NMR work.
T61 9167-9334 Sentence denotes 6 These manually assigned intersubunit distance restraints were included in the combined automated assignments of the NOE peaks and the structure calculation by CYANA.
T62 9335-9399 Sentence denotes 11 NOE-derived distance restraints totalling 2615 were obtained.
T63 9400-9571 Sentence denotes Out of 100 structures calculated, the 20 structures with the lowest target function values were selected and energy-refined as the final structures of the SARS-CoV NP CTD.
T64 9572-9647 Sentence denotes The structural statistics for the NMR structure are summarized in Table 1 .
T65 9648-9840 Sentence denotes Consistent with the previously reported topological structure of the SARS-CoV NP CTD, the NMR structure of the SARS-CoV NP CTD adopts a domain-swapped homodimer conformation ( Fig. 4a and b) .
T66 9841-10024 Sentence denotes The resulting structures exhibited good convergence for both the backbone and the side chains when the regions between residues 260 and 365 of each dimer were superimposed (Fig. 4a) .
T67 10025-10259 Sentence denotes The model has a disordered region spanning residues 248-259 and protruding from the dimer core, which appears to be the result of internal dynamics and prevents the detection of long-range NOEs based on heteronuclear NOE measurements.
T68 10260-10549 Sentence denotes 15 N-{ 1 H} NOE experiments recorded for the SARS-CoV NP CTD showed that the hetero- To demonstrate the absence of the 1 J cc coupling of aromatic rings for SAIL phenylalanine and tyrosine residues, all 1 H-13 C HSQC spectra for the aromatic regions were recorded without the CT technique.
T69 10550-10711 Sentence denotes nuclear NOE values for this region are smaller than those for the structured region, indicating that the two N-termini are flexible in solution (data not shown).
T70 10712-10905 Sentence denotes The secondary structural elements of the CTD in solution were defined based on the DSSP algorithm, 12 and they corresponded well to those identified in the previous NMR study ( Fig. 4b and c ).
T71 10906-11088 Sentence denotes 6 Prior to our NMR investigation, the crystal structures of constructs spanning residues 270-370 [Protein Data Bank (PDB) ID 2gib] and residues 248-365 (PDB ID 2cjr) were elucidated.
T72 11089-11237 Sentence denotes 5, 7 The overall folds and secondary structure arrangements are very similar between the crystal structures and the NMR structure ( Fig. 4a and c) .
T73 11238-11426 Sentence denotes The backbone RMSD between the protomers of the mean NMR structure and the crystal structure of the CTD spanning residues 248-365 is 1.45 Å if residues 260-319 and 333-358 are superimposed.
T74 11427-11621 Sentence denotes If the NMR structure is superimposed with the crystal structure spanning residues 270-370, then the backbone RMSD between the protomers is 1.26 Å for the regions of residues 274-319 and 333-358.
T75 11622-11812 Sentence denotes There are two differences, however: first, in both crystal structures, the β-sheet is distorted around residues 320-332 compared to those of the NMR structure (upper left of Fig. 5a and b) .
T76 11813-12061 Sentence denotes Second, the two N-termini (residues 248-259) protruding from the dimer core are disordered in the NMR structure, whereas, in the crystal structure, they are involved in a number of intramonomer and intradimer contacts and are more rigid (Fig. 4a) .
T77 12062-12192 Sentence denotes The disorder of the N-termini in solution was further supported by the analysis of backbone amide-exchange rates (data not shown).
T78 12193-12358 Sentence denotes We suspect that at least some of these differences are likely to result from the different solvent conditions used for crystallization and/or crystalpacking effects.
T79 12359-12482 Sentence denotes Deletion studies revealed that residues 248-280 are essential for the nucleic-acid-binding activity of the SARS-CoV NP CTD.
T80 12483-12561 Sentence denotes 5 However, the exact residues involved in the binding had not been identified.
T81 12562-12850 Sentence denotes To identify these residues, we conducted chemical shift displacement (CSD) studies by titrating 10-mer (dT 10 ) or 20-mer (dT 20 ) poly-deoxythymine (poly-dT) single-stranded DNA (ssDNA) into the protein samples. ssDNA were used throughout this study as surrogates of single-stranded RNA.
T82 12851-13155 Sentence denotes Titration of dT 10 or dT 20 into the protein sample caused a concentration-dependent gradual shift of some resonances, instead of the appearance of a new set of resonances, suggesting that the binding occurs in the fast-exchange regime, which is indicative of a low-affinity nucleic-acid-binding protein.
T83 13156-13395 Sentence denotes 13 For dT 10 , significant chemical shift changes were localized primarily in the N-terminal region, particularly K250, E253, A254, S256, K257, and K258 ( Fig. 6a and b) , while the majority of the other resonances were scarcely affected.
T84 13396-13554 Sentence denotes This result suggests that dT 10 binds to the SARS-CoV NP at the N-terminal flexible segment, without affecting the overall structure of the protein (Fig. 4) .
T85 13555-13623 Sentence denotes The binding constant estimated from the CSD studies at various dT 10
T86 13625-13871 Sentence denotes Similarly, dT 20 also generated significant chemical shift changes in the same set of N-terminal residues; however, it also induced CSD of the resonances of R320, H335, and A337, which are located in the βsheet of the CTD dimer ( Fig. 6b and d) .
T87 13872-14096 Sentence denotes Since these residues are not affected when the SARS-CoV NP CTD is bound to dT 10 ( Fig. 6a and c) , we can rule out the effect of long-range structural alterations induced by the binding of oligonucleotides to the N-termini.
T88 14097-14221 Sentence denotes Our results suggest that R320, H335, and A337 also contribute to nucleic acid binding and could be part of the binding site.
T89 14222-14341 Sentence denotes This observation is unexpected, as these residues are sequentially and structurally distant from the N-terminal region.
T90 14342-14434 Sentence denotes It should be noted that we could only add dT 20 up to a [dT 20 ]/[CTD monomer] ratio of 1:4.
T91 14435-14478 Sentence denotes A higher ratio of DNA caused precipitation.
T92 14479-14702 Sentence denotes As such, we could not obtain a reliable dissociation constant for the complex, and the chemical shift perturbation shown in Fig. 6b may not be the maximum change expected at a saturating dT 20 concentration for the complex.
T93 14703-15010 Sentence denotes To further quantify the relative contribution of positively charged residues to oligonucleotide binding by the SARS-CoV NP CTD, we produced double mutants targeting K257/K258 and measured the effect of the mutations on the apparent dissociation constant (K d ) with fluorescently labeled dT 20 through EMSA.
T94 15011-15221 Sentence denotes 14 Since the mutation sites are located in the inherently flexible regions, the mutations did not cause any structural perturbations to other parts of the CTD dimer, as monitored by 15 N HSQC spectra (Fig. 7) .
T95 15222-15266 Sentence denotes The EMSA results are summarized in Table 2 .
T96 15267-15540 Sentence denotes We found that the chargepreserving K257R/K258R mutant did not affect the apparent binding affinity, compared to the wildtype construct, whereas the K257Q/K258Q mutant showed a 5-fold reduction in affinity, although binding was not completely abolished (Fig. 8 , Table 2 ).
T97 15541-15713 Sentence denotes Our results suggest that the positive charges at positions 257 and 258 of the SARS-CoV NP CTD are significant determinants of its binding affinity towards oligonucleotides.
T98 15714-15771 Sentence denotes Similarly, we also generated the R320A and H335A mutants.
T99 15772-15963 Sentence denotes The 15 N HSQC spectra of these mutants revealed that, in both cases, the structural perturbations are mostly limited to the regions adjacent to the Arg320 and His335 mutation sites (Fig. 9) .
T100 15964-16144 Sentence denotes However, the R320A mutation had a chemical shift perturbation larger than that of the H335A mutation, probably because the Arg side chain made more contacts with adjacent residues.
T101 16145-16229 Sentence denotes The changes in the respective apparent dissociation constants were measured by EMSA.
T102 16230-16494 Sentence denotes Mutations at this secondary binding site lowered the apparent 5 The two subunits in each structure are in orange and magenta for the NMR structure, and in red and green for the crystal structure. (b) Ribbon diagram of the solution structure of the SARS-CoV NP CTD.
T103 16495-16589 Sentence denotes Secondary structure elements are labeled for one subunit. (c) Sequence of the SARS-CoV NP CTD.
T104 16590-16850 Sentence denotes The secondary structures for the NMR structure and for the crystal structure spanning residues 248-365 are shown above the sequence, with red cylinders for α-helices and yellow arrows for β-strands. affinity towards dT 20 by about twofold (Fig. 8 , Table 2 ).
T105 16851-16992 Sentence denotes Although the effects were not as remarkable as those of the mutations near the N-terminus, the loss of binding affinity was still measurable.
T106 16993-17130 Sentence denotes Our results are in agreement with the hypothesis that the β-sheet region of the SARS-CoV NP CTD is part of the nucleic-acid-binding site.
T107 17131-17315 Sentence denotes The SAIL method is characterized by a sophisticated labeling pattern that is highly optimized for structure determination, in contrast to other preexisting isotope labeling techniques.
T108 17316-17527 Sentence denotes Conventional strategies utilizing selective protonation under a predeuterated background lead to a compromise between increased intensity of the labeled protons and loss of information about the deuterated ones.
T109 17528-17764 Sentence denotes For instance, while the selective protonation of the methyl protons of Ile, Leu, and Val in a deuterated background is very effective for the observation of methyl protons, one cannot obtain any information on the remaining side chains.
T110 17765-17969 Sentence denotes 15 The SAIL method, on the other hand, combines the merits of increased intensity through the deuteration of redundant protons and preservation of the structural information by leaving key protons intact.
T111 17970-18076 Sentence denotes Of particular interest is the use of SAIL aromatic residues, which was first demonstrated with calmodulin.
T112 18077-18285 Sentence denotes 10 Assignments of chemical shift and NOE peaks involving aromatic signals are indispensable for the highquality structure determination of proteins, since aromatic residues are often part of the folding core.
T113 18286-18461 Sentence denotes In the case of the SARS-CoV NP CTD, the aromatic resonances of the UL protein were severely overlapped, making their assignment difficult, if not impossible ( Fig. 3b and e) .
T114 18462-18642 Sentence denotes The introduction of aromatic SAIL amino acids in the sample resolved this problem (Fig. 3d and f) , ultimately leading to the elucidation of the SARS-CoV NP CTD solution structure.
T115 18643-18816 Sentence denotes This study is the first case to have demonstrated that the use of the SAIL phenylalanine and tyrosine residues was effective in the NMR spectral analysis of a large protein.
T116 18817-18925 Sentence denotes This is also the first instance of a homodimeric protein structure to have been solved by the SAIL approach.
T117 18926-19094 Sentence denotes The SAIL method becomes more effective with increasing molecular weight and allows for the structures of larger proteins with even more intricate features to be solved.
T118 19095-19096 Sentence denotes 8
T119 19097-19245 Sentence denotes There are two distinct differences between the structure of the SARS-CoV NP CTD in solution and the structure of the SARS-CoV NP CTD in the crystal.
T120 19246-19486 Sentence denotes The first is the orientation of the short turn in the β-sheet: in the crystal, the short turn is closer to the N-terminal residues of the same protomer than in the solution structure, resulting in a more compact crystal structure (Fig. 5) .
T121 19487-19624 Sentence denotes This is observed in all of the crystal structures of the SARS-CoV NP CTD solved to date, regardless of the space group and the construct.
T122 19625-19701 Sentence denotes 5, 7 It is possible that crystal packing is responsible for the compactness.
T123 19702-19874 Sentence denotes On the other hand, the less compact solution structure of the short hairpin turn could be the result of the The second difference lies in the conformation of the N-termini.
T124 19875-20087 Sentence denotes The N-termini in the crystal structure of the SARS-CoV NP CTD, spanning residues 248-365, form an ordered conformation anchored by intramolecular and intermolecular interactions between various adjacent residues.
T125 20088-20268 Sentence denotes 5 However, the N-termini in solution are disordered and, in agreement with our previous studies, 6 lack a short helix formed by residues 259-263 in the crystal structure (Fig. 4) .
T126 20269-20466 Sentence denotes The CTD is arranged as an octamer within the unit cell of the crystal, and this short helix could be the result of different solvent conditions, crystal packing, and/or the oligomerization process.
T127 20467-20646 Sentence denotes 16 Residues within and adjacent to the short helix participate in intramolecular and intermolecular interactions within the crystal and contribute to the formation of the octamer.
T128 20647-20880 Sentence denotes 5 It is possible that the short helix is selectively stabilized by the formation of new protein-protein contacts in the crystal octamer, similar to those observed for the binding of intrinsically disordered proteins to their targets.
T129 20881-21002 Sentence denotes 17 Transient formation of the short helix is also observed in a few of the conformers within the NMR structural ensemble.
T130 21003-21108 Sentence denotes In the crystal structure of SARS-CoV NP 248-365 , we have previously found that the CTD forms an octamer.
T131 21109-21225 Sentence denotes 5 The packaging of the octamers in the asymmetric unit of the crystal results in two parallel basic helical grooves.
T132 21226-21329 Sentence denotes Residues 248-280 form a positively charged patch similar to that in the infectious bronchitis virus NP.
T133 21330-21427 Sentence denotes 18 These patches form a large part of the basic helical groove observed in the crystal structure.
T134 21428-21587 Sentence denotes We postulate that the basic helical groove may serve as the RNA attachment site, and the structure suggests a mechanism for helical RNA packaging in the virus.
T135 21588-21643 Sentence denotes However, the octamer has not been observed in solution.
T136 21644-21873 Sentence denotes In our DNA titration study, we found that the spin-spin relaxation time, T 2 , of the amide resonances decreases, upon the addition of the DNA oligomer, at a rate faster than that expected for sheer increases in molecular weight.
T137 21874-21957 Sentence denotes We suspect that the DNA complex forms transient higher-order multimers in solution.
T138 21958-22062 Sentence denotes Within the helical oligomer model, one expects nucleic acid binding to stabilize the oligomer structure.
T139 22063-22304 Sentence denotes To investigate the consistency of our NMR chemical shift perturbation data with the proposed helical model, we mapped the spatial locations of the residues perturbed by dT 20 binding onto the helical model proposed by Chen et al. (Fig. 10 ).
T140 22305-22500 Sentence denotes 5 It clearly shows that both the N-terminal residues and the additional perturbed residues in the β-sheet region, namely, R320 and H335, form the interior lining of the positively charged groove.
T141 22501-22623 Sentence denotes The two N-termini of the dimer reside on the outside edge Table 2 . and in the innermost part of the groove, respectively.
T142 22624-22730 Sentence denotes The arrangement of the helical structure is such that the interior N-termini are still solvent-accessible.
T143 22731-22819 Sentence denotes On the other hand, the perturbed β-sheet residues reside in the midregion of the groove.
T144 22820-22982 Sentence denotes Thus, while the current NMR titration results do not prove the validity of the helical model, they can still be satisfactorily accommodated within this framework.
T145 22983-23142 Sentence denotes More rigorous data, such as the determination of the structure of the CTD/nucleic acid complex, are necessary to provide definitive proof of the helical model.
T146 23143-23414 Sentence denotes We have previously shown that the SARS-CoV NP is a modular protein comprising two independent structural domains connected by a 66-residue linker and flanked on each end by the long, disordered Nand C-termini, which comprise 44 and 57 residues, respectively 3 (Fig. 1a) .
T147 23415-23482 Sentence denotes The NTD, comprising residues 45-181, has been shown to bind to RNA.
T148 23483-23572 Sentence denotes 4 Here we showed that the CTD also binds to DNA with similar affinity to that of the NTD.
T149 23573-23716 Sentence denotes We have shown previously that the di-domain fragment NP45-365 binds to DNA and RNA with higher affinity than that of the respective NTD or CTD.
T150 23717-23951 Sentence denotes 15 Taken together, all of these observations suggest that the SARS-CoV NP binds to RNA at multiple sites, and the binding strength is enhanced by the multivalency effect, as multiple binding sites are in contact with the RNA molecule.
T151 23952-24131 Sentence denotes 19 This charge-based nonspecific binding mode works in conjunction with intrinsic disorder to confer two main advantages to the nonspecific binding of oligonucleotides to the CTD.
T152 24132-24405 Sentence denotes First, because the disordered region is not locked into a single conformation, binding to a variety of partners can occur regardless of the structural features of the partner, as long as the electrostatic interaction provides enough free energy to maintain the bound state.
T153 24406-24511 Sentence denotes This property allows the CTD to bind to oligonucleotides with different sequences or tertiary structures.
T154 24512-24677 Sentence denotes This is an important feature of RNA chaperones, of which the SARS-CoV NP is a member, and hints at the possibility that residues 248-270 are involved in the process.
T155 24678-25053 Sentence denotes 20, 21 Second, the unstructured protein molecule can have a greater capture radius for a specific binding site than that of the folded state with its restricted conformational freedom, the so-called "fly-casting mechanism." 22 In this binding scenario, the unfolded state binds weakly at a relatively large distance, and then folds as the protein approaches the binding site.
T156 25054-25264 Sentence denotes These two advantages could act together to ensure that the CTD is able to bind to a variety of nucleotide sequences with enough affinity to carry out its function, namely, the encapsulation of the viral genome.
T157 25265-25416 Sentence denotes We envision the extended conformation of the NP molecule as a whole to facilitate its initial contact with the RNA molecule in a fly-casting mechanism.
T158 25417-25549 Sentence denotes Subsequent rearrangement of the NP molecule in the RNA framework then results in favorable packing of the complex in a helical form.
T159 25550-25641 Sentence denotes The SARS-CoV NP CTD was cloned from SARS-CoV TW1 strain sequencing vectors (a gift from Dr.
T160 25642-25647 Sentence denotes P.-J.
T161 25648-25715 Sentence denotes Chen, National Taiwan University Hospital) as previously described.
T162 25716-25903 Sentence denotes 6 Mutants of the SARS-CoV NP CTD were produced with a QuickChange II kit (Stratagene, La Jolla, CA) on a RoboCycler 96 (Stratagene), in accordance with the manufacturer's recommendations.
T163 25904-25978 Sentence denotes Primers used for mutagenesis were purchased from Mission Biotech (Taiwan).
T164 25979-26027 Sentence denotes Mutations were confirmed through DNA sequencing.
T165 26028-26306 Sentence denotes The SARS-CoV NP CTD, encompassing residues 248-365 including an extra MHHHHHHAMG sequence at the N-terminus, was expressed in the E. coli BL21 (DE3) strain for nonlabeled and uniformly labeled samples, as described previously, 6 and in a cell-free reaction for the SAIL samples.
T166 26307-26427 Sentence denotes The production of nonlabeled and uniformly labeled samples by in vivo expression was performed in a conventional manner.
T167 26428-26529 Sentence denotes The proteins expressed in E. coli were purified in accordance with our previously described protocol.
T168 26530-26618 Sentence denotes 6 The cell-free expression of the SARS-CoV NP CTD was performed as described previously.
T169 26619-26715 Sentence denotes 9 The S30 extract containing minimal residual amino acids was used for the cell-free expression.
T170 26716-26924 Sentence denotes In the cell-free synthesis of the SARS-CoV NP CTD, the concentration of each SAIL amino acid was set to 0.5 mM, and 2.3 mg of the SAIL-SARS-CoV NP CTD was obtained from a total of 70 mg of SAIL amino mixture.
T171 26925-27053 Sentence denotes SAIL amino acids were obtained from SAIL Technologies, Inc. ‡ The SAIL-SARS-CoV NP CTD thus produced was mainly in soluble form.
T172 27054-27314 Sentence denotes The SAIL protein was purified by Ni-NTA affinity chromatography in 50 mM sodium phosphate (pH 7.4) and 150 mM NaCl, followed by gel filtration in a buffer containing 50 mM sodium phosphate (pH 7.4), 150 mM NaCl, and 1 mM ethylenediaminetetraacetic acid (EDTA).
T173 27315-27403 Sentence denotes The eluted SAIL-SARS-CoV NP CTD was then concentrated and exchanged with the NMR buffer.
T174 27404-27720 Sentence denotes The SAIL-SARS-CoV NP CTD sample contained 0.5 mM (10% D 2 O buffer) and 0.5 mM (100% D 2 O buffer) of the SAIL SARS-CoV NP CTD in NMR buffer [10 mM sodium phosphate pH 6.0, 50 mM NaCl, 1 mM EDTA, 1 mM 2,2-dimethyl-2-silapentane-5-sulfonate, 0.01% NaN 3 , 10% D 2 O, and Complete Mini protease inhibitor mix (Roche)].
T175 27721-27925 Sentence denotes SAIL-adapted NMR experiments for the structure determination were performed at 30°C with Bruker 600-MHz or 800-MHz spectrometers equipped with a TXI triple resonance room-temperature probe or a cryoprobe.
T176 27926-28130 Sentence denotes 1 H-15 N HSQC spectra were obtained with a 1-mM 15 N-labeled sample in NMR buffer on a Bruker Avance 500-MHz spectrometer equipped with a TXI cryoprobe, using an in-house adaptation of the pulse sequence.
T177 28131-28436 Sentence denotes For mutant characterization and protein-ssDNA-binding studies in NMR buffer, 15 N-labeled samples were prepared in NMR buffer, and spectra were recorded on Bruker Avance 600-MHz or 800-MHz ‡ www.Sail-technologies.com (legend on next page) spectrometers equipped with QXI quadruple resonance or TXI probes.
T178 28437-28601 Sentence denotes The acquired data were processed with the XwinNMR suite (Bruker Biospin, Germany) or with iNMR (Nucleomatica, Italy), and chemical shift assignments were performed.
T179 28602-28711 Sentence denotes The chemical shifts were referenced to 2,2-dimethyl-2-silapentane-5-sulfonate and deposited in BioMagResBank.
T180 28712-28920 Sentence denotes The NMR structure calculation of the SARS-CoV NP CTD was started using 28 independent pairs of intersubunit distance restraints obtained from isotope-filtered NOE spectroscopy experiments reported previously.
T181 28921-29542 Sentence denotes 6 Automated NOE cross-peak assignments 15 and structure calculations with torsion-angle dynamics 16 were performed using a modified version of the program CYANA 2.1, which incorporates SAIL labeling patterns, 4 takes the homodimer symmetry explicitly into account for the network anchoring of NOE assignments, 15 ensures an identical conformation of the two monomers by imposing torsion-angle difference restraints on all corresponding torsion angles, and maintains a symmetric relative orientation of the two monomers by applying distance difference restraints between symmetry-related intermolecular C α -C α distances.
T182 29543-29685 Sentence denotes Backbone torsion-angle restraints obtained from database searches with the program TALOS 17 were incorporated into the structural calculation.
T183 29686-29725 Sentence denotes Hydrogen-bond restraints were not used.
T184 29726-29897 Sentence denotes CYANA structure calculations were started from 100 randomized conformers, and simulated annealing with 20,000 torsionangle dynamics time steps per conformer was performed.
T185 29898-30064 Sentence denotes The 20 conformers with the lowest final CYANA target function values were subjected to restrained energy refinement in explicit solvent against the AMBER force field.
T186 30065-30067 Sentence denotes 18
T187 30069-30254 Sentence denotes A series of 2D 15 N-edited HSQC spectra of uniformly 15 N-labeled SARS-CoV NP CTD protein (0.5 mM) was in NMR buffer by titrating in different amounts of poly-dT ssDNA (Purigo, Taiwan).
T188 30255-30330 Sentence denotes The affected amide correlations experienced CSD upon the addition of ssDNA.
T189 30331-30632 Sentence denotes The unaffected and shifted resonances in uncrowded regions were easily assigned, whereas the shifted resonances in crowded regions were assigned by stepwise titration of the protein with small amounts of ssDNA and by tracing of the changes in the CSD until the desired final concentration is achieved.
T190 30633-30702 Sentence denotes The final protein/ ssDNA ratio was 1:1 for 10-mer and 4:1 for 20-mer.
T191 30703-30820 Sentence denotes Protein/ssDNA ratios higher than the one presented here resulted in the formation of a precipitate within the sample.
T192 30821-31042 Sentence denotes The weighted CSD for each residue was calculated with the formula: CSD = (1/2((Δδ 1 HN ) 2 + (Δδ 15 N /5) 2 ) 1/2 , where Δδ denotes the chemical shift difference between the final complex and the free protein resonances.
T193 31043-31268 Sentence denotes The experimental error in the weighted CSD from the spectral resolution was calculated as: (1/2((SW 1 HN /points in 1 H) 2 + ((SW 15 N /points in 15 N)/5) 2 )) 1/2 , where SW denotes the total spectral width of the dimension.
T194 31269-31393 Sentence denotes Amides with CSD values larger than the average shift of all the peaks plus the experimental error were selected as affected.
T195 31394-31490 Sentence denotes All experiments were conducted in NMR buffer with 6aminohexylfluorescein-labeled ssDNA (Purigo).
T196 31491-31591 Sentence denotes Reactions were set up in 20-μl aliquots each containing 50 nM 6-aminohexylfluorescein-labeled ssDNA.
T197 31592-31744 Sentence denotes Protein was added to the aliquots starting at a concentration of 500 μM, with each following aliquot containing a 2-fold serial dilution of the protein.
T198 31745-31814 Sentence denotes A control reaction was set up where only ssDNA and buffer were added.
T199 31815-31985 Sentence denotes The aliquots were allowed to react at room temperature for 30 min, and then were loaded on a 0.5 × Tris-borate EDTA buffer DNA retardation gel (Invitrogen, Carlsbad, CA).
T200 31986-32139 Sentence denotes The gel was run at 30 V and 4°C for 2.5 h, and the bands were visualized with a Typhoon 9410 variable mode imager (Amersham Biosciences, Piscataway, NJ).
T201 32140-32263 Sentence denotes Quantitation of the free ssDNA band was achieved through the ImageJ software (National Institutes of Health, Bethesda, MA).
T202 32264-32372 Sentence denotes Bound ssDNA was estimated by subtracting the free ssDNA band of each reaction from that of the control lane.
T203 32373-32433 Sentence denotes The fraction of bound ssDNA was fitted against the equation:
T204 32434-32667 Sentence denotes Y = 1/(1 + (K d /X) n ), using GraphPad Prism (GraphPad Software, San Diego, CA), where Y is the fraction of ssDNA bound to the protein, X is the protein concentration, K d is the dissociation constant, and n is the Hill coefficient.
T205 32668-32704 Sentence denotes All experiments were repeated twice.
T206 32705-32859 Sentence denotes Chemical shift assignments and atomic coordinates have been deposited in BioMagResBank (accession code 15511) and PDB (accession code 2jw8), respectively.