Two topology diagrams of the nsp1 mixed parallel/antiparallel six-stranded β-barrel (see text). The numbering indicates the first and last residues of each β-strand.

(a) 2D ¹⁵N,¹H heteronuclear single-quantum coherence (HSQC) spectrum of nsp1(13-128). (b) 2D ¹⁵N,¹H HSQC spectrum of full-length nsp1(1-179). (c) 2D TROSY-based ¹⁵N{¹H} NOE experiment with full-length nsp1(1-179), with negative peaks shown in red. The spectra were recorded at a ¹H frequency of 600 MHz at 298 K.

Plot of the ¹⁵N{¹H} NOE intensities versus the sequence of nsp1(13-128). The data were collected at a ¹H frequency of 600 MHz at 298 K. The positions of the regular secondary structure elements are indicated. Each point represents the mean of three measurements, and the error bars represent the standard deviations of the three measurements.

Two stereo views of the globular domain of nsp1. (a) Ribbon presentation of the closest conformer of nsp1 to the mean coordinates of the bundle in Fig. 1a, shown in the same orientation as in Fig. 1a. The organization of the β-strands in the barrel is indicated by the labels. (b) Same as panel a after rotation about a horizontal axis, so that one looks at one side of the β-barrel; the axes of the β-barrel and the helix α1 are nearly perpendicular to each other, and those of the barrel and the 3₁₀-helix are nearly parallel to each other.

(a) Bundle of 20 energy-minimized CYANA conformers of nsp1(13-128). In this stereo view, the polypeptide backbone is shown as a gray spline function through the C^α positions. Selected sequence positions are identified by numerals. (b) Ribbon representation of the closest conformer to the mean coordinates of the bundle of 20 conformers used to represent the NMR structure. The β-strands are cyan, the helices are red, and polypeptide segments with nonregular secondary structure are gray. The regular secondary structures are further identified by lettering. The polypeptide segments shown in green represent the additional, structurally disordered polypeptide segments of the full-length nsp1.

Stereo view of nsp1(13-128) in the same orientation as in Fig. 2b. The side chains in the interior of the barrel are differently colored to visualize their arrangement in three layers, as discussed in the text. The polypeptide backbone is shown as a gray spline function through the C^α positions. Amino acid side chains are shown as stick drawings. Color code: red, residues of layer 1 at the barrel opening opposite to helix α1, where the four hydrophilic residues are in solvent contact; green, residues in the central layer 2; blue, residues of the third layer, which make hydrophobic contacts to the residues shown in magenta at the top, where V36, A39, L40, A43, and L47 originate from the amphipathic helix α1.

(a) Amino acid sequence of nsp1, with solvent-exposed residues highlighted in green. A residue is considered to be exposed if at least one atom of its side chain has more than 50% surface accessibility to the solvent. For glycines, the CO and H^N exposure is considered. (b) Surface views of nsp1 in a space-filling representation. In the surface view shown on the left, the structure has the same orientation as in Fig. 2b. Some of the surface-exposed side chains discussed in the text are identified with the one-letter amino acid code and the residue number. Color code: gray, hydrophobic and polar residues; red, negatively charged; blue, positively charged. (c) Sequence alignment between SARS-CoV nsp1 and MHV nsp1 identified with the FFAS server. Identical residues are shown in red. Arrows indicate single-amino-acid replacements in MHV p28 that were generated and studied by Brockway et al. (7). Mutations that are detrimental to the viral replication are identified by boldface, while those that are not detrimental are in italic. Residues removed in the truncated variant protein MHV1 nsp1ΔC are shown in lowercase (see text). Residues in β-strands and in helical secondary structures are underlined with solid and dashed lines, respectively.