Nuclear magnetic resonance structure shows that the severe acute respiratory syndrome coronavirus-unique domain contains a macrodomain fold

Abstract

The nuclear magnetic resonance (NMR) structure of a central segment of the previously annotated severe acute respiratory syndrome (SARS)-unique domain (SUD-M, for "middle of the SARS-unique domain") in SARS coronavirus (SARS-CoV) nonstructural protein 3 (nsp3) has been determined. SUD-M(513-651) exhibits a macrodomain fold containing the nsp3 residues 528 to 648, and there is a flexibly extended N-terminal tail with the residues 513 to 527 and a C-terminal flexible tail of residues 649 to 651. As a follow-up to this initial result, we also solved the structure of a construct representing only the globular domain of residues 527 to 651 [SUD-M(527-651)]. NMR chemical shift perturbation experiments showed that SUD-M(527-651) binds single-stranded poly(A) and identified the contact area with this RNA on the protein surface, and electrophoretic mobility shift assays then confirmed that SUD-M has higher affinity for purine bases than for pyrimidine bases. In a further search for clues to the function, we found that SUD-M(527-651) has the closest three-dimensional structure homology with another domain of nsp3, the ADP-ribose-1"-phosphatase nsp3b, although the two proteins share only 5% sequence identity in the homologous sequence regions. SUD-M(527-651) also shows three-dimensional structure homology with several helicases and nucleoside triphosphate-binding proteins, but it does not contain the motifs of catalytic residues found in these structural homologues. The combined results from NMR screening of potential substrates and the structure-based homology studies now form a basis for more focused investigations on the role of the SARS-unique domain in viral infection.

FIG. 1.

Superposition of the 2D [¹⁵N,¹H]-HSQC spectra of SUD-M(513-651) (red) and SUD-M(527-651) (blue). The protein concentrations were 1.2 mM and 1.4 mM for SUD-M(513-651) and SUD-M(527-651), respectively. The solvent contained 25 mM sodium phosphate buffer at pH 6.5, 150 mM NaCl, and 2 mM NaN₃. The spectra were recorded at a ¹H frequency of 600 MHz and a temperature of 25°C, with 256 increments in the ¹⁵N dimension and 4 scans/increment. The resonance assignments for SUD-M(527-651) are marked in blue, where the assignments for the crowded central region are shown as an insert in the lower right corner. Residue −1 indicates the methionine residue of the tetrapeptide segment ⁻⁴GSHM⁻¹ that is left after thrombin cleavage (see the text). The side-chain amide resonances of asparagine and glutamine are connected by blue horizontal lines.

FIG. 2.

NMR structure of SUD-M(527-651). (a) Stereo view of the polypeptide backbone of a bundle of 20 energy-minimized conformers superimposed for the minimal RMSD value of the backbone atoms of residues 528 to 648. The helical regular secondary structures are red, the β-strands are green, and the polypeptide segments with no regular secondary structure are gray. Selected sequence positions are identified by numerals. (b) Stereo view in the same orientation as described above (a), of a ribbon presentation of the closest conformer of SUD-M(527-651) to the mean coordinates of the bundle above (a). The regular secondary structures are identified. (c) Same as above (b) after a 90° rotation about a horizontal axis. (d) Topology of the regular secondary structures in SUD-M(527-651). β-Strands are shown as gray arrows, helices in the front of the β-sheet are in black, and helices behind the β-sheet are represented by white rectangles. The numbers represent the starts and the ends of the individual regular secondary structure elements.

FIG. 3.

(a) NMR structure of SUD-M(513-651). The polypeptide backbone of a bundle of 20 energy-minimized conformers has been superimposed for the minimal RMSD value calculated for the backbone atoms of residues 528 to 648. The flexibly extended N-terminal tail of residues 513 to 527 and the C-terminal flexible tail of residues 649 to 651 are red. (b) Relative ¹⁵N{¹H}-NOE intensities plotted versus the sequence of SUD-M(513-651). Diamonds represent the experimental measurements, which are linked in sequential order by straight lines. Gaps represent either proline residues or residues for which the ¹⁵N-¹H correlation peak could not be integrated because of spectral overlap. The experiment was recorded at a ¹H frequency of 600 MHz using a saturation period of 3.0 s and a total interscan delay of 5.0 s. The red line represents a cutoff at 0.6; residues with values below this cutoff value are identified as having high-frequency intramolecular mobility. Positions of the regular secondary structures are indicated at the bottom of the figure.

FIG. 4.

Superposition of pairs of 2D [¹⁵N,¹H]-HSQC spectra of 0.4 mM SUD-M(527-651) (solvent composed of 25 mM sodium phosphate buffer at pH 6.5, 150 mM NaCl, and 2 mM NaN₃) recorded in the absence (red peaks) and presence (blue peaks) of 0.4 mM of three different ligands: poly(A₁₀) ssRNA (a), poly(U₁₀) ssRNA (b), and ADP-ribose (c). (a and b) The peaks that show chemical shift changes after the addition of the ligand are identified. The spectra were recorded on a Bruker DRX 700 spectrometer with a 1.7-mm TXI HCN z-gradient probehead at a temperature of 25°C; 256 increments in the ¹⁵N dimension were accumulated, with 32 scans per increment.

FIG. 5.

Space-filling models of the NMR structure of SUD-M(527-651). (a) Regions affected by poly(A₁₀) ssRNA binding (data from Fig. 4a) are highlighted in magenta. (b) The residues in positions structurally corresponding to those that contact the ADP-ribose ligand in nsp3b are highlighted. (c) Display of the electrostatic surface potential, with positive and negative electrostatic charges represented in blue and red, respectively. (d) Nsp3b (PDB accession number 2ACF). Shown is the same presentation of the electrostatic surface potential as that in panel c. (c and d) The black circle surrounds the ligand-binding clefts discussed in the text. Selected residues within the cleft of SUD-M(527-651) and in the active site of nsp3b are identified.

FIG. 6.

Investigation of RNA binding by electrophoretic mobility shift assay (see the text). Data are given for poly(A₁₅), poly(U₁₀), poly(A₁₀), (ACUG)₅, TRS(+), TRS(-), and 5′-CCCGAUACCC-3′ (GAUA). These single-stranded oligonucleotides were incubated with various concentrations of SUD-M either at room temperature (a and d) or at 37°C (b and c) before analysis by native polyacrylamide gel electrophoresis. Lane designations indicate the final concentration of protein and RNA or the presence of a double-stranded DNA marker (DNA). The binding assays in a and b were carried out in low-salt buffer (50 mM phosphate at pH 6.5 containing 56 mM NaCl, 7% glycerol, and 4 mM MgCl₂), and those in c and d were carried out using buffer containing physiological salt concentrations (50 mM phosphate at pH 6.5 with 150 mM NaCl, 7% glycerol, and 4 mM MgCl₂). Nucleic acid was detected by SYBR gold staining (left), and protein was detected by SYPRO ruby staining (right). White arrowheads indicate the electrophoretic mobility of SUD-M, and black arrowheads indicate free nucleic acid. Complexes of intermediate mobility are indicated by a gray filled bracket.

FIG. 7.

Stereo view of a ribbon presentation showing a superposition of the NMR structure of SUD-M(527-651) (red) and the X-ray structure of nsp3b (33) (gray). The following residues for the superposition were identified with the software DALI (15), yielding an RMSD value of 2.9 Å for the C^α atoms of these residues: residues 527 to 541, 544 to 552, 553 to 556, 557 to 566, 567 to 570, 572 to 575, 576 to 579, 580 to 586, 588 to 597, 599 to 625, and 626 to 649 in SUD-M(527-651) and residues 199 to 213, 214 to 222, 227 to 230, 232 to 241, 244 to 247, 263 to 266, 268 to 271, 274 to 280, 290 to 299, 300 to 326, and 328 to 351 in nsp3b. The insertions in the sequence of nsp3b are highlighted in yellow (see the text). Selected sequence positions are identified by black numerals for nsp3b and red numerals for SUD-M(527-651).

FIG. 8.

3D structure-based sequence alignment of the SUD-M protein with its closest structural homologues, as identified through a DALI (16) search of the PDB with SUD-M(527-651). PDB accession numbers are given in parentheses. Above the sequence, the locations of the regular secondary structures in SUD-M are indicated by cylinders for helices and by arrows for β-strands. The aligned residues are highlighted in red, and conserved sequence motifs described in the text are indicated in boldface type. The residues that form the adenosine-binding cleft in nsp3b and the corresponding residues in SUD-M are boxed. The loops that form the ribose-binding cleft in nsp3b (see the text) and the corresponding regions in SUD-M are underlined (SUD-M has a three-residue deletion at the location corresponding to loop 3). The residues that show chemical shift changes upon the addition of poly(A₁₀) ssRNA (see the text and Fig. 4a) are marked by green lines above the sequence.

FIG. 9.

Conservation of SUD-M in bat coronavirus lineages. (a) Multiple-sequence alignment of domains homologous to SARS-CoV SUD-M. Homologies are highlighted with clustalx conservation coloring, and sequences are numbered from the first residue of nsp3. (b) Schematic representation of the homology between macrodomains found in coronavirus nsp3 and eukaryotic and prokaryotic organisms. Genetic homology (BLAST) (blue), structural homology (DALI) (red), and combined homology (violet) are indicated. Coronavirus subgroup nomenclature was taken from http://www.ncbi.nlm.nih.gov/Taxonomy/Browser. PEDV, porcine epidemic diarrhea virus; TGEV, transmissible gastroenteritis virus; FCoV, feline coronavirus.

FIG. 10.

Structural coverage of the N-terminal half of the 1,922-residue nsp3. Initially annotated domains are marked above the thick line, and the numbers below this line represent the residues that bound the individual domains. Circles indicate globular folds, with the NMR structures in green and the crystal structures in blue. The curved thick lines represent flexibly disordered segments that were characterized by NMR ¹⁵N{¹H}-NOE measurements either as an N-terminal attachment of the nearest globular domain (red) or as a C-terminal tail (black). The white boxes indicate domains that are currently being investigated by NMR. The residues binding the individual structural entities are indicated below the circles and the boxes. The dotted line represents SUD-M(513-651), and the broken line represents SUD-M(527-651). UB1 and UB2 are ubiquitin-like folds. AC is a region rich in acidic residues. ADRP is an ADP-ribose-1"-phosphatase. SUD-N (MBD) (27), SUD-M, and SUD-C represent three structural regions of the SARS-unique domain. PL2^pro is a papain-like protease.