Id |
Subject |
Object |
Predicate |
Lexical cue |
T1 |
0-127 |
Epistemic_statement |
denotes |
Solution Structure of the C-terminal Dimerization Domain of SARS Coronavirus Nucleocapsid Protein Solved by the SAIL-NMR Method |
T2 |
138-418 |
Epistemic_statement |
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. |
T3 |
1568-1838 |
Epistemic_statement |
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. |
T4 |
1960-2130 |
Epistemic_statement |
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. |
T5 |
2555-2639 |
Epistemic_statement |
denotes |
3, 4 Recently, the CTD has also been shown to bind nucleic acids with high affinity. |
T6 |
3421-3536 |
Epistemic_statement |
denotes |
However, previous biochemical and biophysical studies have shown that the CTD exists solely as a dimer in solution. |
T7 |
3617-3784 |
Epistemic_statement |
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. |
T8 |
3962-4178 |
Epistemic_statement |
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. |
T9 |
4544-4757 |
Epistemic_statement |
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. |
T10 |
5060-5180 |
Epistemic_statement |
denotes |
The active site thus identified agrees well with the helical ribonucleoprotein model suggested by the crystal structure. |
T11 |
6836-7103 |
Epistemic_statement |
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. |
T12 |
8890-9052 |
Epistemic_statement |
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. |
T13 |
10025-10259 |
Epistemic_statement |
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. |
T14 |
10550-10711 |
Epistemic_statement |
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). |
T15 |
11622-11800 |
Epistemic_statement |
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. |
T16 |
12193-12358 |
Epistemic_statement |
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. |
T17 |
12483-12561 |
Epistemic_statement |
denotes |
5 However, the exact residues involved in the binding had not been identified. |
T18 |
12851-13155 |
Epistemic_statement |
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. |
T19 |
13396-13549 |
Epistemic_statement |
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. |
T20 |
13555-13624 |
Epistemic_statement |
denotes |
The binding constant estimated from the CSD studies at various dT 10 |
T21 |
13625-13859 |
Epistemic_statement |
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. |
T22 |
13960-14096 |
Epistemic_statement |
denotes |
6a and c) , we can rule out the effect of long-range structural alterations induced by the binding of oligonucleotides to the N-termini. |
T23 |
14097-14221 |
Epistemic_statement |
denotes |
Our results suggest that R320, H335, and A337 also contribute to nucleic acid binding and could be part of the binding site. |
T24 |
14222-14341 |
Epistemic_statement |
denotes |
This observation is unexpected, as these residues are sequentially and structurally distant from the N-terminal region. |
T25 |
14342-14434 |
Epistemic_statement |
denotes |
It should be noted that we could only add dT 20 up to a [dT 20 ]/[CTD monomer] ratio of 1:4. |
T26 |
14479-14607 |
Epistemic_statement |
denotes |
As such, we could not obtain a reliable dissociation constant for the complex, and the chemical shift perturbation shown in Fig. |
T27 |
14608-14702 |
Epistemic_statement |
denotes |
6b may not be the maximum change expected at a saturating dT 20 concentration for the complex. |
T28 |
15541-15713 |
Epistemic_statement |
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. |
T29 |
15772-15958 |
Epistemic_statement |
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. |
T30 |
15964-16144 |
Epistemic_statement |
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. |
T31 |
16851-16992 |
Epistemic_statement |
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. |
T32 |
16993-17130 |
Epistemic_statement |
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. |
T33 |
17131-17315 |
Epistemic_statement |
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. |
T34 |
18550-18642 |
Epistemic_statement |
denotes |
3d and f) , ultimately leading to the elucidation of the SARS-CoV NP CTD solution structure. |
T35 |
18643-18816 |
Epistemic_statement |
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. |
T36 |
19625-19701 |
Epistemic_statement |
denotes |
5, 7 It is possible that crystal packing is responsible for the compactness. |
T37 |
19702-19874 |
Epistemic_statement |
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. |
T38 |
20269-20466 |
Epistemic_statement |
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. |
T39 |
20647-20880 |
Epistemic_statement |
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. |
T40 |
21428-21587 |
Epistemic_statement |
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. |
T41 |
21588-21643 |
Epistemic_statement |
denotes |
However, the octamer has not been observed in solution. |
T42 |
21644-21873 |
Epistemic_statement |
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. |
T43 |
22820-22982 |
Epistemic_statement |
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. |
T44 |
22983-23142 |
Epistemic_statement |
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. |
T45 |
23143-23408 |
Epistemic_statement |
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. |
T46 |
23415-23482 |
Epistemic_statement |
denotes |
The NTD, comprising residues 45-181, has been shown to bind to RNA. |
T47 |
23717-23951 |
Epistemic_statement |
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. |
T48 |
24132-24405 |
Epistemic_statement |
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. |
T49 |
24512-24677 |
Epistemic_statement |
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. |
T50 |
24678-24901 |
Epistemic_statement |
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." |
T51 |
25054-25264 |
Epistemic_statement |
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. |
T52 |
25417-25549 |
Epistemic_statement |
denotes |
Subsequent rearrangement of the NP molecule in the RNA framework then results in favorable packing of the complex in a helical form. |
T53 |
25716-25903 |
Epistemic_statement |
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. |