Novel {beta}-Barrel Fold in the Nuclear Magnetic Resonance Structure of the Replicase Nonstructural Protein 1 from the Severe Acute Respiratory Syndrome Coronavirus

The nonstructural protein 1 (nsp1) of the severe acute respiratorysyndrome coronavirus has 179 residues and is the N-terminalcleavage product of the viral replicase polyprotein that mediatesRNA replication and processing. The specific function of nsp1is not known. Here we report the nuclear magnetic resonancestructure of the nsp1 segment from residue 13 to 128, whichrepresents a novel ${alpha}$ /ß-fold formed by a mixed parallel/antiparallelsix-stranded ß-barrel, an ${alpha}$ -helix covering one openingof the barrel, and a 3₁₀-helix alongside the barrel. We furthercharacterized the full-length 179-residue protein and show thatthe polypeptide segments of residues 1 to 12 and 129 to 179are flexibly disordered. The structure is analyzed in a searchfor possible correlations with the recently reported activityof nsp1 in the degradation of mRNA.

INTRODUCTION

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Introduction

After the major outbreak of severe acute respiratory syndrome(SARS) in the beginning of 2003, the SARS coronavirus (SARS-CoV)became a major topic of coronavirus research. The coronavirusgenome is composed of a single plus-strand RNA of about 30 kb,which is the largest nonsegmented genome among known RNA viruses.About two-thirds of the coronavirus genome is devoted to encodingthe replicase that mediates viral RNA synthesis (64). The replicasegene comprises two large open reading frames (ORFs) locatedat the 5' end of the genome. The first one, ORF1a, encodes apolyprotein of 450 to 500 kDa (polyprotein 1a), and the secondone, ORF1b, is translated together with ORF1a after a –1ribosomal frameshift, leading to the expression of the "polyproteinlab," which has a size of 750 to 800 kDa. The replicase polyproteinis processed by ORF1a-encoded viral proteinases, which leadsto about 16 nonstructural proteins (nsp), which are numberedconsecutively from the N terminus to the C terminus of the polyprotein(14, 53).

The large number of mature proteins produced from the polyproteinindicates a high level of complexity of the viral replicationprocess. Some of the enzymatic activities that were detectedor predicted in SARS-CoV to date include the main protease (nsp5),a papain-like proteinase (nsp3d; PLpro), an RNA-dependent RNApolymerase (nsp12), an RNA helicase (nsp13), an endoribonuclease(nsp15), an ADP-ribose-1"-phosphatase (nsp3b), a deubiquitinase(nsp3d), a 3' $->$ 5' exoribonuclease (nsp14), and a ribose-2'-O-methyltransferase(nsp16) (3, 5, 18, 21, 22, 44, 59, 70). For the two proteases,the endoribonuclease, and the ADP-ribose-1"-phosphatase, three-dimensionalstructures have been solved, which together with biochemicaldata have revealed some aspects of the enzyme mechanisms (27,54, 56, 58, 63, 67; reviewed in references 4, 35, and 65). Thephysiological functions of several of the other nonstructuralproteins remain to be determined, but high-resolution structuredeterminations have allowed the identification of possible functionalsites and provided the basis for further biochemical studies(15, 30, 52, 61, 62, 68). Other replicase proteins still remainto be characterized.

Nsp1 is the N-terminal cleavage product of the replicase polyproteinand is produced by the action of PLpro. It is among the leastwell-understood nsps, and other than in coronaviruses, no viralor cellular homologs are known. Levels of sequence conservationamong the different coronaviruses are highest at the 3' endof the genome, and the sequences are very divergent at the 5'end, especially in nsp1 to nsp3, which are products of PLprocleavage. nsp1 has been proposed to be useful as a group-specificmarker (59). In the group 1 coronaviruses, nsp1 (also knownas p9) is a protein of about 110 residues, with 20 to 50% sequenceidentity among all group 1 CoVs. The viruses of subgroup 2a,such as murine hepatitis virus (MHV) and human coronavirus OC43,encode an nsp1 protein of about 245 residues, also known asp28, while the group 3 viruses (avian) do not encode an nsp1.The nsp1 of SARS-CoV, which has been classified as the onlymember to date of the subgroup 2b (19, 20, 59), comprises 180residues, with a molecular mass of 20 kDa. nsp1 sequences aredivergent between groups 2a and 2b, and no sequence similaritybetween SARS-CoV nsp1 and group 2a nsp1 proteins could be identifiedusing standard searching tools such as BLAST.

Biochemical experiments demonstrated interactions between MHVnsp1 and two other replication proteins (nsp7 and nsp10) andcolocalization with nonstructural proteins and the nucleocapsidprotein at viral replication complexes in the cytoplasm duringthe early stages of infection (6). In contrast, during the laterstages of infection, MHV nsp1 was found to colocalize with structuralproteins at virion assembly sites (6). Mutations at the nsp1/nsp2cleavage site of MHV that prevented the cleavage of nsp1 fromthe polyprotein caused slower growth and reduced RNA synthesisrelative to wild-type viruses (13). Deletion of the nsp1-codingregion in infectious clones of MHV yielded viruses that wereunable to productively infect cultured cells (7). Furthermore,exogenous expression of MHV nsp1 in mammalian cells arrestedthe cell cycle in the G₀/G₁ phase and inhibited cell proliferation(8). A point mutation in the proteolytic cleavage site betweennsp1 and nsp2 in the full-length genome, and in minigenomesof the group 1 CoV porcine transmissible gastroenteritis virus,blocked the release of nsp1 from the nascent polyprotein andcaused a dramatic reduction in virus viability (17). SARS-CoVnsp1 was shown to specifically accelerate the degradation ofmRNA and thus lead to a reduction in cellular protein synthesis,which may provide a survival advantage for the virus (31). Overall,these observations indicate that nsp1 might participate in multiplestages of the coronavirus life cycle, and they implicate thisprotein as a potentially important virulence factor.

This paper describes the nuclear magnetic resonance (NMR) structureof SARS-CoV nsp1. Before this study, no information about thethree-dimensional structure of nsp1 was available, and SARS-CoVnsp1 does not have significant amino acid sequence similaritywith any protein with known three-dimensional structure. SARS-CoVnsp1 was therefore selected for NMR structure determinationby the Consortium for Functional and Structural Proteomics ofSARS-CoV-Related Proteins (http://sars.scripps.edu). The availabilityof a high-resolution solution structure will help to guide furtherinvestigations of the biochemical and physiological functionsof nsp1.

MATERIALS AND METHODS

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Materials and Methods

Target optimization strategy. Six constructs truncated at different positions were createdbased on secondary structure prediction, using the amino acidsequence as input for the software Jpred (12). The truncatednsp1 variants were cloned in an Escherichia coli expressionplasmid derived from pET-28 under the control of the T7 promoterand in frame with 5' coding sequences for a His₆ tag and followedby a spacer sequence which ends with ENLYFQG. This strategyallows the preparation of proteins that have only one extraglycine at the N terminus after proteolysis with tobacco etchvirus protease. This expression strategy was selected aftertests of different E. coli strains and different temperaturesduring the induction in 10-ml cultures. The samples were expressedin a microexpression device (M. S. Almeida, M. Geralt, R. Horst,and K. Wüthrich, unpublished), purified using Ni²⁺ affinitychromatography, concentrated with ultrafiltration centrifugaldevices, and subjected to one-dimensional (1D) ¹H NMR screeningusing a Bruker DRX700 spectrometer with a 1-mm TXI HCN z-gradientmicroprobe. Based on the high-quality 1D ¹H NMR spectrum, theconstruct consisting of nsp1 residues 13 to 128 [nsp1(13-128)]was selected for a NMR structure determination. In an attemptto further improve this sample, variant constructs of nsp1(13-128)with Cys 52 replaced by Ala, Ser, Arg, or Asp were preparedby site-directed mutagenesis using the QuikChange kit (Stratagene)according to the manufacturer's instructions. The variants wereevaluated by 1D ¹H NMR screening for a globular fold and bycircular dichroism spectroscopy to determine their stability.

Protein preparation. Large-scale expression of uniformly ¹⁵N-labeled or ¹³C- and¹⁵N-labeled nsp1(13-128) in E. coli BL21(DE3) cells was carriedout at 18°C in 500 ml of M9 minimal medium containing either0.5 g ¹⁵NH₄Cl or 0.5 g ¹⁵NH₄Cl and 2 g [¹³C₆]-D-glucose as thesole nitrogen and carbon sources, respectively. For the proteinpurification, the cells were disrupted by sonication in thepresence of 25 mM HEPES at pH 8.0, 250 mM NaCl, 2 mM dithiothreitol,0.03% NaN₃, and EDTA-free Complete protease inhibitor tablets(Roche). The cell lysate was loaded onto a 10-ml HisTrap FFcolumn equilibrated with 50 mM imidazole in the same buffersystem as mentioned above. The retained proteins were elutedwith a 50 to 500 mM imidazole gradient and incubated with recombinanttobacco etch virus protease at 22°C for 2 days. The resultingsolution was loaded onto a 300-ml Superdex 75 column equilibratedwith 25 mM sodium phosphate at pH 7.0, 250 mM NaCl, and 0.03%NaN₃. The protein eluted with a retention volume equivalentto about 13 kDa. The solution was concentrated with ultrafiltrationcentrifugal devices and supplemented with 10% D₂O to a finalsample volume of about 300 µl.

NMR spectroscopy and structure calculation. The NMR samples contained 2 mM of nsp1(13-128). NMR spectrawere collected at 298 K with Bruker Avance 600-MHz and Avance800-MHz spectrometers equipped with TXI HCN z-gradient probes.The sequence-specific resonance assignment (66) has been describedelsewhere (1). The input for the structure calculation consistedof the chemical shift list obtained from the resonance assignment,a 3D ¹⁵N-resolved ¹H,¹H nuclear Overhauser effect spectroscopy(NOESY) spectrum, and two 3D ¹³C-resolved ¹H,¹H NOESY spectraoptimized for the aliphatic and aromatic ¹³C regions. The nuclearOverhauser effect (NOE) data were measured at 800 MHz with amixing time of 60 ms. For the peak picking of the NOESY spectra,NOE assignment, and structure calculation, the stand-alone ATNOS/CANDIDprogram (24, 25) was used in conjunction with the CYANA torsionangle dynamics algorithm (23). The standard protocol with sevencycles of peak picking, NOE assignment, and 3D structure calculationwith simulated annealing in torsion angle space (24, 25) wasapplied. Backbone ${varphi}$ and ${psi}$ dihedral angle constraints derived fromthe C chemical shifts (40, 60) were used as supplementary datain the structure calculation. The 20 conformers with the lowestresidual CYANA target function values obtained from cycle 7of the ATNOS/CANDID/CYANA calculation were energy minimizedin a water shell with the program OPALp (34, 39), using theAMBER force field (9). The program MOLMOL (33) was used to analyzethe protein structure and to prepare the figures showing theNMR structures. Analysis of the stereochemical quality of themodels was accomplished using the Joint Center for StructuralGenomics validation central suite (http://www.jcsg.org) andthe Protein Data Bank validation server (http://deposit.pdb.org/validate).

Steady-state ¹⁵N{¹H} NOEs were measured with transverse relaxation-optimizedspectroscopy (TROSY)-based experiments (55, 69) on a BrukerAvance 600-MHz spectrometer, using a saturation period of 3s and an interscan delay of 5 s.

Accession numbers. The chemical shifts have been deposited in the BioMagResBank(http://www.bmrb.wisc.edu) under accession number 7014. Theatomic coordinates of the bundle of 20 conformers used to representthe nsp1 structure have been deposited in the Protein Data Bank(http://www.rcsb.org/pdb) with the code 2GDT, and those of theconformer closest to the mean coordinates have the code 2HSX.

RESULTS AND DISCUSSION

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Results and Discussion

The 179-residue nsp1 of SARS-CoV was included in a high-throughputproteomics characterization strategy by the consortium "Functionaland Structural Proteomics of the SARS-CoV" (unpublished). Thefull-length protein and the fragment consisting of residues1 to 159 were cloned, expressed and purified by the ProteinProduction Core, and given to us for NMR screening (50). The1D ¹H NMR spectra of both constructs showed characteristicsof a globular fold as well as of disordered regions (data notshown). Based on these results, the protein was transferredto us for an NMR structure determination.

Since nsp1 has no identifiable sequence similarity with proteinswith known three-dimensional structures, it was not possibleto predict the domain structure of this protein based on sequencecomparisons. However, the presence of flexibly disordered regionsin the protein identified by ¹H NMR spectroscopy (see "Characterizationof the full-length SARS-CoV nsp1" below) was consistent withthe results of secondary structure prediction, which indicatedthat a few residues at the N terminus, as well as a greaternumber of residues in the C-terminal one-third of the protein,would not adopt regular secondary structure. To investigatethe boundaries of the globular domain and to optimize conditionsfor protein expression, sample preparation, and NMR structuredetermination, we designed a set of truncated variants of nsp1,bearing in mind the results of secondary structure predictionsfor this protein. The variant constructs have molecular massesof 12.7 kDa to 19.5 kDa, not including the N-terminal tag of3.5 kDa (Table 1). These constructs were used to transform E.coli strains Rosetta(DE3), BL21(DE3) RIL, and BL21(DE3), andthe recombinant proteins were expressed in a microshaker at37°C, 27°C, and 18°C. The best growth rates andexpression levels were obtained with the strain BL21(DE3). Table1 provides a survey of the expression results with six differentnsp1 constructs. Most of the protein in the samples expressedat 37°C was insoluble for all six variants. For two constructs,higher yields of soluble protein were obtained at 27°C,but the best results were achieved with expression at 18°C,where most of the expressed protein was in the soluble fraction.

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TABLE 1. Summary of the recombinant production of nsp1 variants in BL21(DE3) E. coli cells

Based on the results in Table 1, BL21(DE3) E. coli cells incultures at 18°C were used for the protein production. Theproteins were purified by Ni²⁺ affinity chromatography and gelfiltration chromatography. The final protein recovery in 10ml of culture was in the range of 2 to 4 mg, which representsexpression levels in the range of 11 to 29 µM (Table 1).Samples were concentrated for 1D ¹H NMR screening with a microcoilprobe (Almeida et al., unpublished). The two shortest constructs,nsp1(1-128) and nsp1(13-128), exhibited the highest expressionlevels. The nsp1(13-128) construct was selected for the structuredetermination, based on its high-quality ¹H NMR spectrum andon its greater stability in comparison to the other five constructsof Table 1.

Since cysteine residues are susceptible to oxidation and formationof intermolecular disulfide bonds, which can lead to unstableand heterogeneous protein samples, we also investigated variantconstructs of nsp1(13-128) with Cys 52 replaced by Ala, Ser,Arg, or Asp as part of our initial target optimization strategy,using 1D ¹H NMR and circular dichroism spectroscopy to evaluatetheir foldedness and stability. The variants with Ser 52, Asp52, or Arg 52 were thus found to be unstable. The variant withCys 52 replaced by Ala led to a stable, folded protein. However,after observing excellent sample stability of the wild-typeprotein despite the single Cys residue, we chose the wild-typeprotein for the structure determination.

The parameters in Table 2 show that a well-defined NMR structureof nsp1(13-128) was obtained. Above-average local disorder islimited to the C-terminal heptapeptide segment of residues 122to 128 and to a disordered loop of residues 77 to 86 (Fig. 1a).The structure of intact nsp1 includes a globular domain of residues13 to 121 and the disordered regions of residues 1 to 12 and122 to 179 (Fig. 1b).

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TABLE 2. Input for the structure calculation and characterization of the bundle of 20 energy-minimized CYANA conformers representing the NMR structure of nsp1(13-128)

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FIG. 1. (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 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 ß-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.

The structure of nsp1(13-128) represents a new fold. The sequential arrangement of the regular secondary structuresin the globular domain of nsp1 is ß1- ${alpha}$ 1-ß2-3₁₀-ß3-ß4-ß5-ß6.There is a mixed parallel/antiparallel six-stranded ß-barrel,where the spatial arrangement of the ß-strands isß1-ß2-ß5-ß3-ß4-ß6,and ß1 makes contact with ß6 (Fig. 2 and3). The ß-strands consist of residues 15 to 21, 52to 56, 69 to 73, 87 to 92, 104 to 110, and 117 to 124. The helix ${alpha}$ 1 with residues 36 to 49 is located across one barrel opening,and the 3₁₀-helix of residues 62 to 64 is positioned alongsidethe barrel. A search of the Protein Data Bank using the structureof nsp1 as input for the DALI server (26) did not indicate statisticallysignificant structural similarity to any other protein describedto date.

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FIG. 2. 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 ß-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 ß-barrel; the axes of the ß-barrel and the helix ${alpha}$ 1 are nearly perpendicular to each other, and those of the barrel and the 3₁₀-helix are nearly parallel to each other.

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FIG. 3. Two topology diagrams of the nsp1 mixed parallel/antiparallel six-stranded ß-barrel (see text). The numbering indicates the first and last residues of each ß-strand.

For the continued discussion it is helpful to adopt a systematicanalysis of ß-barrels, using the number of strands(n); the shear number (S), which measures the stagger of thestrands; and the tilt angle ( ${alpha}$ ) of each strand, which is theangle between the barrel axis and the line adjusted for bestfit to the N, C, and C' atoms of each strand (43, 46, 47). Smust be an even integer because of the hydrogen bonding patternbetween the ß-strands (46). Using standard valuesfor the mean C-C distance along the strands (a = 3.3 Å)and between the strands (b = 4.4 Å), the following geometricrelations characteristic of ß-barrel structures inproteins have been proposed (43):

Formula 1 (1)

Formula 2 (2)
Ris the barrel radius, which is defined as the average of thedistances between the C atoms of the three residues in oppositestrands that are closest to the central part of the barrel (47).

The ß-barrel in nsp1 contains six strands and hasa shear number (S) of 10. The measured tilt of the strands tothe barrel axis ( ${alpha}$ ) ranges from 38^o (ß2) to 78^o (ß4),with an average value of 60^o. The wide variation among the tiltangles of the individual strands reflects that the ß-barrelof nsp1 is pronouncedly irregular (Fig. 2a and b).

The residues used to calculate the radius of the nsp1 barrelare 18 to 20, 53 to 55, 71 to 73, 86 to 88, 106 to 108, and122 to 124, which give a mean barrel radius (R) of 7 ±1 Å. Overall, we thus have for nsp1 that the theoreticaltilt angle value of 51^o calculated from equation 1 shows a discrepancywith the observed average of 60^o, whereas the theoretical valueof the mean barrel radius of 7 Å, as calculated from equation2, is in close agreement with the observed value of 7 ±1 Å.

The interior of the nsp1 barrel and the interfaces between thetwo helices and the barrel surface consist primarily of hydrophobicresidues. The arrangement of the side chains inside the barrelis highly compact, as expected for a barrel of six strands,but the inspection of space-filling models suggests that thereis a tight cavity along the center of the barrel, with a radiusof about 1.2 Å (not shown). The inside of the barrel consistsof 17 side chains, which are contributed by all six strandsand which are arranged in three layers. One layer contains L105and the four hydrophilic residues E56, R74, K85, and R120. Thefour peripheral hydrophilic groups mediate the contacts withthe solvent at the barrel opening opposite to helix ${alpha}$ 1 (Fig.4) (in the orientation of Fig. 2b, these residues would be atthe bottom of the structure). The charged groups of the sidechains of these residues are fully solvent exposed, and E56makes a salt bridge with R120. The side chain of V21 in thesecond layer and the ßCH₂- ${gamma}$ CH₂ fragment of R120 arelocated between this first layer and the other side chains ofthe second layer, which is in the narrowest portion of the barreland includes the all-hydrophobic side chains of residues L54,I72, V87, and V122. A third layer consists of the side chainsof residues L17, L19, V70, L89, A91, L108, and L124, which makehydrophobic contacts with the side chains of residues V36, A39,L40, A43, and L47 from the amphipathic helix ${alpha}$ 1, and the sidechains of residues C52, F32, P110, and P68. The second and thirdlayers of ß-strand side chains thus combine with theinner side of the helix ${alpha}$ 1 to form a large hydrophobic core (Fig.4). It is worth noting that the variant proteins with Cys 52replaced by Ser, Asp, or Arg were unstable, which is consistentwith a disruption of the ß-barrel core, as one wouldpredict from the NMR structure.

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FIG. 4. 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 positions. Amino acid side chains are shown as stick drawings. Color code: red, residues of layer 1 at the barrel opening opposite to helix ${alpha}$ 1, 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 ${alpha}$ 1.

In the SCOP database (48), there are 13 folds with ß-barrelsof n = 6 and S = 10. Nine of these folds contain antiparallelß-barrels, i.e., the QueA-like fold (41), the hypotheticalprotein HI1480 (37), the ferredoxin reductase-like fold (10),the phage tail protein (42), the flavin mononucleotide-bindingsplit barrel (36), the reductase/isomerase/elongation factorcommon domain (10), the elongation factor/aminomethyltransferasecommon domain (32), the core binding factor ß (28),and the surface presentation of antigens fold (16). Four foldshave mixed parallel/antiparallel ß-barrels similarto the nsp1 fold, but none has the topology observed in nsp1(Fig. 3). These include the ribosomal protein L25-like (57),the ß and ß' subunits of DNA-dependent RNApolymerase (11), the double- ${psi}$ ß-barrel (38), and theacid protease fold (51). In addition to the apparently uniqueß-strand topology and the irregular ß-barrelgeometry, another interesting feature of the nsp1 fold is thatthe polypeptide chains connecting the ß-strands runalong the side of the barrel, except for the loop between ß3and ß4 (Fig. 2 and 3). This is a rare feature forbarrels with n = 6 and S = 10, and besides nsp1, it has beenobserved only between two ß-strands in the ribosomalprotein L25-like fold.

It is intriguing that none of the aforementioned folds are quiteas irregular as that of nsp1. The distortion of the nsp1 structureseems to be related to the polypeptide segments connecting theß-strands across the side of the barrel. Interestingly,although the adjoining ends of strands ß5 and ß6are the furthest apart in space of all strand combinations innsp1 (approximately 15 Å between P110 and I117), theyare connected by the shortest polypeptide segment across theside of the barrel (Fig. 2 and 3). This imposes a lower limiton the shear between these strands. The shear number of 10 seemsto be the result of a balance between tight hydrophobic packinginside the nsp1 barrel, which is favored by lower shear numbers,and unstrained arrangement of the linker polypeptide segmentson the outside the barrel, which is favored by larger shearnumbers. We discuss the ß-barrel topology in muchdetail in order to advance the hypothesis that the outstandingirregularity of the nsp1 ß-barrel might be relatedto a so-far-unknown, possibly entirely novel physiological functionof nsp1.

The arrangement of the linker polypeptide segments on the outsideof the barrel is puzzling also with regard to the folding pathwayof nsp1. For example, if the strand ß1 formed hydrogenbonds with ß2 early during translation, this wouldalso fix the first linker across the barrel, which might limitthe ease with which ß6 could make hydrogen bonds withß4 and ß1. Schemes representing the topologyof the ß-barrel (Fig. 3) would intuitively suggestthat folding starts midway during translation with the formationof a ß-hairpin of the strands ß3 and ß4.In the folded protein, this pair of ß-strands formsthe least distorted part of the ß-barrel, with highlyregular hydrogen bonds, and the loop between ß3 andß4 is the only one that does not run along the barrelsurface. In subsequent folding steps the two-stranded sheetsof ß2 and ß5 and of ß1 and ß6,respectively, might be formed, which also have quite regularhydrogen bonding in the nsp1 structure. The linkers betweenß2 and ß3 and between ß4 and ß5have almost the same lengths, which should support to positionß5 close to ß2 if ß4 is arrangedclose to ß3. The three regular two-stranded ß-sheets(Fig. 3a) are connected in the barrel by the formation of irregularhydrogen bonding patterns.

Characterization of the full-length SARS-CoV nsp1. The full-length nsp1 was characterized by comparison of thenumbers of backbone ¹⁵N-¹H correlation peaks and the H^N and¹⁵N chemical shifts with those of nsp1(13-128) and by heteronuclearNOE measurements of the truncated and full-length nsp1. Thetruncated construct nsp1(13-128) has an NMR spectrum with large¹H and ¹⁵N chemical shift dispersion (Fig. 5a), which is typicalfor a well-folded globular domain, where the atoms of differentindividual amino acid residues experience different local microsusceptibilitiesdue to the nonperiodic nature of the interiors of globular proteins.The spectrum of the full-length protein, nsp1(1-179) (Fig. 5b),contains a set of peaks that overlays very closely with thoseof nsp1(13-128), showing that the globular domain is containedin both constructs. All the additional peaks have H^N chemicalshifts of 7.9 to 8.5 ppm, which is the region characteristicof "random-coil" polypeptide chains (66).

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FIG. 5. (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.

A direct measure of intramolecular mobility is provided by theheteronuclear 2D ¹⁵N{¹H} NOE experiment, which is routinelyused to access protein dynamics on the picosecond to nanosecondtimescale (55, 69). Positive signals with intensities of $~$ 0.8identify residues in the folded cores of small and medium-sizeglobular proteins, with mobility of the individual ¹⁵N-¹H moietiesrestricted to the overall rotational tumbling of the molecule.This is illustrated with the ¹⁵N{¹H} NOE data for nsp1(13-128)(Fig. 6), which also serve as a reference for assessing thestate of the additional chain segments in nsp1(1-179). ¹⁵N{¹H}NOE values of about 0.8 are seen for most of the residues inthe regular secondary structure elements (Fig. 6). Increasedflexibility of the polypeptide chain that causes reduced NOEintensities is found in the disordered loop between residues75 and 87 and in the region of residues 94 to 103, which formsnonregular secondary structure with one ${gamma}$ -turn of residues 97to 99 and a type II ß-turn of residues 98 to 101 (Fig.1). Most of the resonances in full-length nsp1 that are notpresent in nsp1(13-128) have either small positive or negative¹⁵N{¹H} NOEs (Fig. 5c), showing that the polypeptide segmentsof residues 1 to 12 and 129 to 179 are best described as a shortN-terminal and a long C-terminal flexibly disordered tail, respectively(Fig. 1b). Interestingly, it has been determined that the carboxy-terminalhalf of the related protein MHV nsp1 is not needed for viralreplication in culture but is important for efficient proteolyticcleavage between nsp1 and nsp2 and for optimal viral replication(7).

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FIG. 6. 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.

Structure-based search for nsp1 functions. In an initial attempt to identify leads to possible nsp1 functions,Fig. 7a identifies the solvent-exposed residues of nsp1, whichwould be sterically accessible for intermolecular contacts withreaction partners. These residues give rise to an uneven electrostaticsurface charge distribution (Fig. 7b), with a large negativesurface on one face and hydrophobic, polar, and positively chargedresidues forming the opposite surface. The large contiguouspatches of positive and negative surface charge could mediatespecific as well as nonspecific intermolecular interactionsby electrostatic forces. For example, considering that it hasbeen shown that nsp1 promotes mRNA degradation (31), the areaof positive charge on the molecular surface formed by K48, R125,and K126 (Fig. 7b) is of interest as a potential site for adirect interaction with mRNA. Alternatively, the positivelyand negatively charged areas of the protein surface might beinvolved in protein-protein interactions, and nsp1 might thenexert its biological effect not by direct interactions withthe mRNA but by interacting with other proteins involved inthe regulation of cellular mRNA stability (49). In this contextit seems worth mentioning that the NMR structure determinationof nsp1(13-128) was performed in 250 mM NaCl because the proteinprecipitated at lower ionic strengths, which might be due toself-aggregation of nsp1 caused by the uneven charge distribution.

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FIG. 7. (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 nsp1 ${Delta}$ C are shown in lowercase (see text). Residues in ß-strands and in helical secondary structures are underlined with solid and dashed lines, respectively.

For comparisons with the nsp1 proteins of other coronaviruses,data are available for the p9 proteins of group 1 CoVs and forthe p28 proteins of group 2a CoVs. Using database searches suchas BLAST or PSI-BLAST (2), no significant sequence similaritybetween nsp1 of SARS-CoV and proteins of group 2a coronavirusescould be identified. However, pairwise alignment with the FFASserver (29), employing a profile/profile-based method that isable to detect distant relationships, identified 20% sequenceidentity between SARS-CoV nsp1 and MHV nsp1 (p28) over 174 alignedresidues (Fig. 7c). The FFAS score of –9.6 indicates thatthese two proteins might share the novel nsp1 three-dimensionalfold, but the remaining sequence divergence leaves open thepossibility that these proteins might perform different functionseven if they had a common fold. To our knowledge, it has notyet been determined whether the p28 proteins of group 2a coronavirusesalso promote mRNA degradation, but MHV p28 expression has beenshown to cause cell cycle arrest in cultured cells.

The most striking result of the alignment of SARS-CoV nsp1 withthe polypeptide fragment consisting of residues 46 to 247 ofMHV p28 is the observation of a consensus sequence, LRKxGxKG,positioned at the end of strand ß6 of the globulardomain of SARS-CoV nsp1, which is conserved not only in MHVp28 (Fig. 7c) but also in human CoV OC43 p28. It includes thetwo residues R125 and K126, which contribute to the positivelycharged patch on the nsp1 molecular surface (Fig. 7b). If futurestudies of the p28 proteins of group 2a CoVs should show thatthese proteins share mRNA degradation activity with SARS-CoVnsp1, this conserved region could be a candidate for mRNA interaction.

Analysis of the nsp1 structure also provides indications forfunctional differences between the p28 proteins and SARS-CoVnsp1. For example, the motif K109-R110-L111 in MHV p28 was identifiedby Chen et al. as a potential cyclin-binding motif (8), andSARS-CoV nsp1 lacks residues corresponding to R110 and L111.In addition, Chen et al. identified residues 30 to 33 (S/NPER)of p28 as a potential site for phosphorylation by cyclin-dependentkinases (8). These residues occur in an N-terminal 45-residuesegment of p28 that appears not to be homologous to SARS-CoVnsp1. The propensity to induce cell cycle arrest may thereforebe unique to MHV p28, or possibly to the group 2a p28 proteinsin general, and it might not be shared by SARS-CoV nsp1 evenif it turned out that these proteins all share a similar fold.

In other comparisons, no significant sequence identity betweenSARS-CoV nsp1 and the nsp1 (p9) proteins of the group 1 CoVscould be detected. These results are consistent with the analysisby Snijder et al. (59), who described nsp1 as a specific markerof group 2 CoVs. The p9 proteins of group 1 CoVs most likelydiffer from those of group 2 CoVs in both structure and function.

The MHV1 p28 protein was subjected to a mutagenesis study byBrockway et al. (7), who generated single-amino-acid replacementsand truncated versions of this protein and studied their impacton viral replication in cultured cells. Among the mutationsfound to affect viral replication, only some occur in residuesconserved between MHV1 p28 and SARS-CoV nsp1 (Fig. 7c). Deletionof the entire p28 protein or of the polypeptide segment fromresidue 87 to 164 of MHV p28 prevented the virus from productivelyinfecting cultured cells (7). If MHV p28 and SARS-CoV nsp1 didindeed share a similar fold, the latter construct would lackmost of the globular domain. In contrast, the carboxy-terminalhalf of MHV p28 (residues 124 to 241) has been shown to be dispensablefor replication in culture, but it is important for efficientproteolytic cleavage of the protein and for optimal viral replication.If MHV p28 were to contain regular secondary structures similarto those of SARS-CoV nsp1, removal of the polypeptide segmentfrom residue 124 to 241 would correspond to the loss of thestrands ß3, ß4, ß5, and ß6,as well as of the flexibly disordered C-terminal tail, whichwould appear to entail a considerable disruption of the proteinfold. The following considerations might help to resolve theapparent ensuing discrepancies. First, the increased flexibilityand lack of a globular fold in the C-terminal region of theprotein may ensure accessibility of the protease recognitionsite between nsp1 and nsp2 but may not be directly involvedwith the activity exerted by the protein. Second, it appearsthat the strands ß1 and ß2 and the helix ${alpha}$ 1 might provide for a sufficiently stable fold to maintain theso-far-unidentified biological activity, in particular if oneassumes that the additional N-terminal 45-residue segment ofMHV p28, which is not homologous to SARS-CoV nsp1, could participatein a globular fold and help to stabilize the shortened protein.

In conclusion, this paper shows that the SARS-CoV protein nsp1,which is encoded at the 5' terminus of the genome, forms a previouslyunknown complex ß-barrel fold with several uniquestructural features. We hypothesize that the uniqueness of theirregular ß-barrel fold may be related to a so-far-unknown,unique biological function of nsp1. The definition of the globularregion of nsp1 and the identification of residues on the molecularsurface likely to contribute to mRNA degradation activity mayprovide a platform for continued research on the role of thisprotein in SARS-CoV and in other coronaviruses.

ACKNOWLEDGMENTS

We thank Kumar Saikatendu, Jeremiah Joseph, Vanitha Subramanian,Benjamin W. Neuman, Michael J. Buchmeier, Raymond C. Stevens,and Peter Kuhn of the Consortium for Functional and StructuralProteomics of the SARS-CoV for providing us with samples ofnsp1(1-179) and nsp1(1-159) for the initial NMR screening. Theuse of the high-performance computing facility at The ScrippsResearch Institute is gratefully acknowledged.

This study was supported by NIAID/NIH contract no. HHSN266200400058C"Functional and Structural Proteomics of the SARS-CoV" to P.Kuhn and M. J. Buchmeier and by the Joint Center for StructuralGenomics through NIH/NIGMS grant no. U54-GM074898. Additionalsupport was obtained for M.S.A. through the Pew Latin AmericanFellows Program in the Biological Sciences and the Skaggs Institutefor Chemical Biology and for M.A.J. through a fellowship fromthe Canadian Institutes of Health Research and the Skaggs Institutefor Chemical Biology. Kurt Wüthrich is the Cecil H. andIda M. Green Professor of Structural Biology at TSRI and a memberof the Skaggs Institute for Chemical Biology.

FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular Biology, MB-44, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Phone: (858) 784-8011. Fax: (858) 784-8014. E-mail: wuthrich{at}scripps.edu.

Published ahead of print on 3 January 2007.

REFERENCES

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