Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Dec;83(24):12998-3008.
doi: 10.1128/JVI.01253-09. Epub 2009 Oct 14.

Nuclear magnetic resonance structure of the nucleic acid-binding domain of severe acute respiratory syndrome coronavirus nonstructural protein 3

Pedro Serrano  1 , Margaret A Johnson, Amarnath Chatterjee, Benjamin W Neuman, Jeremiah S Joseph, Michael J Buchmeier, Peter Kuhn, Kurt Wüthrich
Affiliations

Nuclear magnetic resonance structure of the nucleic acid-binding domain of severe acute respiratory syndrome coronavirus nonstructural protein 3

Pedro Serrano et al. J Virol. 2009 Dec.

Abstract

The nuclear magnetic resonance (NMR) structure of a globular domain of residues 1071 to 1178 within the previously annotated nucleic acid-binding region (NAB) of severe acute respiratory syndrome coronavirus nonstructural protein 3 (nsp3) has been determined, and N- and C-terminally adjoining polypeptide segments of 37 and 25 residues, respectively, have been shown to form flexibly extended linkers to the preceding globular domain and to the following, as yet uncharacterized domain. This extension of the structural coverage of nsp3 was obtained from NMR studies with an nsp3 construct comprising residues 1066 to 1181 [nsp3(1066-1181)] and the constructs nsp3(1066-1203) and nsp3(1035-1181). A search of the protein structure database indicates that the globular domain of the NAB represents a new fold, with a parallel four-strand beta-sheet holding two alpha-helices of three and four turns that are oriented antiparallel to the beta-strands. Two antiparallel two-strand beta-sheets and two 3(10)-helices are anchored against the surface of this barrel-like molecular core. Chemical shift changes upon the addition of single-stranded RNAs (ssRNAs) identified a group of residues that form a positively charged patch on the protein surface as the binding site responsible for the previously reported affinity for nucleic acids. This binding site is similar to the ssRNA-binding site of the sterile alpha motif domain of the Saccharomyces cerevisiae Vts1p protein, although the two proteins do not share a common globular fold.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
(a to c) Stereo views of the NMR structure of nsp3(1066-1181). (a) Bundle of 20 energy-minimized CYANA conformers. The polypeptide backbone is shown as a gray spline function through the Cα positions. Selected sequence positions in the globular domain are indicated by numerals, where the numbers 1 to 116 correspond to nsp3 residues 1066 to 1181. (b) Ribbon representation of the conformer in panel a that is closest to the mean coordinates. The regular secondary structure elements and the two chain ends at positions 6 and 112 are indicated. (c) All-heavy-atom presentation of the conformer in panel b. The backbone is represented by a gray spline function through the Cα atoms, amino acid side chains with local displacements of ≤0.6 Å are colored blue, those with local displacements of >0.6 Å are red, and the three Trp residues are highlighted in green (see the text). (d) Representation of the electrostatic potential surface showing the area that was found, by chemical shift perturbation experiments, to contain the residues involved in ssRNA binding. The locations of selected residues are identified.
FIG. 2.
FIG. 2.
Histogram of the amide proton Pf versus the amino acid sequence of nsp3(1066-1181); the numbers 1 to 116 along the horizontal axis correspond to nsp3 residues 1066 to 1181. At the top, the positions of the regular secondary structure elements are indicated. Pf is defined as log(kin/kex), where kex is the measured hydrogen/deuterium exchange rate constant and kin is the intrinsic hydrogen/deuterium exchange rate constant for the same residue type when completely exposed to the solvent water (2). Higher Pf values reflect lower amide proton exchange rates with the solvent, which result from involvement in hydrogen bonding networks and/or sequestration from the solvent by the protein's tertiary structure.
FIG. 3.
FIG. 3.
β-Sheet topology in nsp3(1066-1181), where blue lines represent hydrogen bonds that were identified by MOLMOL (26) in at least 10 conformers out of the ensemble of 20 conformers depicted in Fig. 1. Interstrand 1H-1H NOEs are indicated by double-headed black arrows. Amide groups of residues with Pf values of ≥2.0 are color coded in red.
FIG. 4.
FIG. 4.
(a) Superposition of the 2D [15N,1H]-HSQC spectra of nsp3(1066-1181) (black) and nsp3(1035-1181) (green). (b) Superposition of the 2D [15N,1H]-HSQC spectra of nsp3(1066-1181) (black) and nsp3(1066-1203) (red). (c) Plot of relative 15N{1H}-NOE intensities, Irel, versus the amino acid sequence of the nsp3 fragment comprising residues 1035 to 1203. Red squares represent the experimental measurements for the backbone amide groups in the construct nsp3(1066-1203), and green squares represent those for nsp3(1035-1181). The broken vertical line is used to indicate that the NMR signals of the residues 1035 to 1065 were assigned as a group (see Materials and Methods), and the data points to the left of this line have arbitrarily been arranged in the order of decreasing Irel values. The 15N{1H}-NOEs were recorded at a 1H frequency of 600 MHz by using a saturation period of 3.0 s and a total interscan delay of 5.0 s (46, 61).
FIG. 5.
FIG. 5.
Structural organization of the nsp3 fragment containing residues 1 to 1318. The solid line at the top indicates the initial domain annotation based on bioinformatics and phylogenetic analyses. The dashed line to the right represents the C-terminal segment of residues 1318 to 1922 of nsp3. Below, the presently known structural coverage with globular domains and flexibly disordered linker segments is shown. Ribbon representations are used for the globular domains. Flexibly disordered regions revealed by NMR spectroscopy and disordered segments implicated by X-ray crystallography are shown as blue and green lines, respectively. Gray lines and rectangles represent polypeptide segments with so far unknown structures. SUD-C, C-terminal region of SUD.
FIG. 6.
FIG. 6.
Studies of RNA binding using chemical shift perturbation experiments. Panel a shows the 2D [15N,1H]-HSQC spectra of nsp3(1066-1181) in the absence (red) and presence (blue) of a threefold excess of ssRNA1 (see the text for the sequence). Residues with chemical shift changes are indicated, and some are also shown in panels b to d in expanded plots of the superimposed 2D [15N,1H]-HSQC spectra.
FIG. 7.
FIG. 7.
Ribbon representations of residues 1071 to 1177 in the NMR structure of nsp3(1066-1181) (left) and residues 445 to 517 in the SAM of Vst1p (right). In nsp3(1066-1181), the side chains of the residues with significant chemical shift perturbations (Fig. 6) are indicated. For the Vts1p SAM (PDB code 2ese), the residues located in the ssRNA interaction site described by Oberstrass et al. (37) are indicated.
FIG. 8.
FIG. 8.
Sequence alignment of the polypeptide segment nsp3(1066-1181) that forms the globular domain of the SARS-CoV NAB with homologues from other group II coronaviruses. Protein multiple-sequence alignment was performed using ClustalW2 and included sequences from SARS-CoV Tor2 (accession no. AAP41036) and representatives of three protein clusters corresponding to three group II coronavirus lineages identified by a BLAST search: bat coronavirus HKU5-5 (BtCoV-HKU5-5; accession no. ABN10901), BtCoV-HKU9-1 (accession no. P0C6T6), and human coronavirus HKU1-N16 (HCoV-HKU1-N16; accession no. ABD75496). Above the sequences, the positions in full-length SARS-CoV nsp3, the locations of the regular secondary structures in the presently solved NMR structure of the SARS-CoV NAB globular domain, and the residue numbering in this domain are indicated. Amino acids are colored according to conservation and biochemical properties, following ClustalW conventions. Residues implicated in interactions with ssRNA are marked with inverted black triangles. In the present context, the key features are that there is only one position with conservation of K or R (red) and that there are extended sequences with conservation of hydrophobic residues (blue) (see the text).
FIG. 9.
FIG. 9.
Results from EMSA experiments demonstrating nucleic acid binding to nsp3e. In each column, pairs of gel photographs are shown: on the left, the gels are stained for RNA, and on the right, the same gels are stained for protein. The nucleic acid sequence and the protein concentrations, ranging from 0 to 495 μM, are given above each gel. To the left, the position of the protein is indicated by an open triangle, that of the RNA is indicated by a closed triangle, and the positions of protein-RNA complexes are indicated by open rectangles. The nucleic acid concentrations used were 80 μM for 20-mers, 160 μM for decamers, and 190 μM for octamers. dN20 and rN20, randomized 20-mer DNA and RNA.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Almeida, M. S., M. A. Johnson, T. Herrmann, M. Geralt, and K. Wuthrich. 2007. Novel beta-barrel fold in the nuclear magnetic resonance structure of the replicase nonstructural protein 1 from the severe acute respiratory syndrome coronavirus. J. Virol. 81:3151-3161. - PMC - PubMed
    1. Bai, Y., J. S. Milne, L. Mayne, and S. W. Englander. 1993. Primary structure effects on peptide group hydrogen exchange. Proteins 17:75-86. - PMC - PubMed
    1. Baker, S. C., K. Yokomori, S. Dong, R. Carlisle, A. E. Gorbalenya, E. V. Koonin, and M. M. Lai. 1993. Identification of the catalytic sites of a papain-like cysteine proteinase of murine coronavirus. J. Virol. 67:6056-6063. - PMC - PubMed
    1. Barretto, N., D. Jukneliene, K. Ratia, Z. Chen, A. D. Mesecar, and S. C. Baker. 2005. The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity. J. Virol. 79:15189-15198. - PMC - PubMed
    1. Chatterjee, A., M. A. Johnson, P. Serrano, B. Pedrini, J. S. Joseph, B. W. Neuman, K. Saikatendu, M. J. Buchmeier, P. Kuhn, and K. Wuthrich. 2009. Nuclear magnetic resonance structure shows that the severe acute respiratory syndrome coronavirus-unique domain contains a macrodomain fold. J. Virol. 83:1823-1836. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources

-