Structural and Functional Analyses of the Severe Acute Respiratory Syndrome Coronavirus Endoribonuclease Nsp15
2007; Elsevier BV; Volume: 283; Issue: 6 Linguagem: Inglês
10.1074/jbc.m708375200
ISSN1083-351X
AutoresKanchan Bhardwaj, S.K. Palaninathan, Joanna María Ortiz-Alcántara, Lillian Li Yi, Linda A. Guarino, James C. Sacchettini, C. Cheng Kao,
Tópico(s)Protein Structure and Dynamics
ResumoThe severe acute respiratory syndrome (SARS) coronavirus encodes several RNA-processing enzymes that are unusual for RNA viruses, including Nsp15 (nonstructural protein 15), a hexameric endoribonuclease that preferentially cleaves 3′ of uridines. We solved the structure of a catalytically inactive mutant version of Nsp15, which was crystallized as a hexamer. The structure contains unreported flexibility in the active site of each subunit. Substitutions in the active site residues serine 293 and proline 343 allowed Nsp15 to cleave at cytidylate, whereas mutation of leucine 345 rendered Nsp15 able to cleave at purines as well as pyrimidines. Mutations that targeted the residues involved in subunit interactions generally resulted in the formation of catalytically inactive monomers. The RNA-binding residues were mapped by a method linking reversible cross-linking, RNA affinity purification, and peptide fingerprinting. Alanine substitution of several residues in the RNA-contacting portion of Nsp15 did not affect hexamer formation but decreased the affinity of RNA binding and reduced endonuclease activity. This suggests a model for Nsp15 hexamer interaction with RNA. The severe acute respiratory syndrome (SARS) coronavirus encodes several RNA-processing enzymes that are unusual for RNA viruses, including Nsp15 (nonstructural protein 15), a hexameric endoribonuclease that preferentially cleaves 3′ of uridines. We solved the structure of a catalytically inactive mutant version of Nsp15, which was crystallized as a hexamer. The structure contains unreported flexibility in the active site of each subunit. Substitutions in the active site residues serine 293 and proline 343 allowed Nsp15 to cleave at cytidylate, whereas mutation of leucine 345 rendered Nsp15 able to cleave at purines as well as pyrimidines. Mutations that targeted the residues involved in subunit interactions generally resulted in the formation of catalytically inactive monomers. The RNA-binding residues were mapped by a method linking reversible cross-linking, RNA affinity purification, and peptide fingerprinting. Alanine substitution of several residues in the RNA-contacting portion of Nsp15 did not affect hexamer formation but decreased the affinity of RNA binding and reduced endonuclease activity. This suggests a model for Nsp15 hexamer interaction with RNA. The Nidoviruses contain three families of viruses, including the Coronaviridae that cause numerous diseases in humans (1Gorbalenya A.E. Enjuanes L. Ziebuhr J. Snijder E.J. Virus Res. 2006; 117: 17-37Crossref PubMed Scopus (605) Google Scholar). Severe acute respiratory syndrome coronavirus (SARS-CoV) 3The abbreviations used are: SARSsevere acute respiratory syndromeCoVcoronavirusWTwild typeMALDI-ToFmatrix-assisted laser desorption ionization time-of-flight. is a member of the Coronavirus genus (2Lai M.M. Cavanagh D. Adv. Virus Res. 1997; 48: 1-100Crossref PubMed Google Scholar, 3Spaan W. Cavanagh D. Horzinek M.C. J. Gen. Virol. 1988; 69: 2939-2952Crossref PubMed Scopus (394) Google Scholar). It originated from animals but spread to humans, causing severe respiratory distress with a fatality rate of ∼10% (as shown by the World Health Organization, www.who.int/csr/sars/country/en/country2003_08_15.pdf). In addition to their medical importance, coronaviruses are of interest for their large ∼30-kb positive-strand genome and novel mechanisms that have evolved to replicate and transcribe this large RNA (5Sawicki S.G. Sawicki D.L. Adv. Exp. Med. Biol. 1995; 380: 499-506Crossref PubMed Scopus (146) Google Scholar, 6Lai M.M. Annu. Rev. Microbiol. 1990; 44: 303-333Crossref PubMed Google Scholar). In keeping with the novel strategies used, coronaviruses encode several unusual RNA-processing enzymes, including an RNA endoribonuclease, an RNA methyltransferase, a second RNA-dependent RNA polymerase that generates primers for coronavirus replication (7Snijder E.J. Bredenbeek P.J. Dobbe J.C. Thiel V. Ziebuhr J. Poon L.L. Guan Y. Rozanov M. Spaan W.J. Gorbalenya A.E. J. Mol. Biol. 2003; 331: 991-1004Crossref PubMed Scopus (957) Google Scholar), and a mechanism to decrease replication errors (8Eckerle L.D. Lu X. Sperry S.M. Choi L. Denison M.R. J. Virol. 2007; 81: 12135-12144Crossref PubMed Scopus (215) Google Scholar). severe acute respiratory syndrome coronavirus wild type matrix-assisted laser desorption ionization time-of-flight. Coronavirus subgenomic RNAs are particularly interesting in that they all have the same 5′ leader sequence derived from the 5′ end of the genomic RNA. This organization requires recombination as part of transcription. Various mechanisms have been proposed for subgenomic RNA production, but a discontinuous transcription mechanism is increasingly favored (5Sawicki S.G. Sawicki D.L. Adv. Exp. Med. Biol. 1995; 380: 499-506Crossref PubMed Scopus (146) Google Scholar). This model proposes that transcription regulatory sequences in the minus-strand RNA direct translocation of the ternary complex to the 5′ leader sequence, where transcription resumes. The minus-strand RNAs serves as the template for subgenomic RNA transcription. Ribonucleases that process the RNA intermediates for transcription have been proposed (9An S. Maeda A. Makino S. J. Virol. 1998; 72: 8517-8524Crossref PubMed Google Scholar, 10Pasternak A.O. Spaan W.J. Snijder E.J. J. Gen. Virol. 2006; 87: 1403-1421Crossref PubMed Scopus (255) Google Scholar, 11Brian D.A. Baric R.S. Curr. Top. Microbiol. Immunol. 2005; 287: 1-30PubMed Google Scholar), but the mechanism for the process is still not completely understood. Nsp15 (nonstructural protein 15) was predicted to be an RNA endoribonuclease as part of a bioinformatics analysis of the SARS-CoV genome (7Snijder E.J. Bredenbeek P.J. Dobbe J.C. Thiel V. Ziebuhr J. Poon L.L. Guan Y. Rozanov M. Spaan W.J. Gorbalenya A.E. J. Mol. Biol. 2003; 331: 991-1004Crossref PubMed Scopus (957) Google Scholar). Substitutions of critical residues built into infectious clones of the human Coronavirus resulted in decreased infectivity and reduced genome RNA levels (12Ivanov K.A. Hertzig T. Rozanov M. Bayer S. Thiel V. Gorbalenya A.E. Ziebuhr J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12694-12699Crossref PubMed Scopus (211) Google Scholar). A more thorough analysis of mutations in the Nsp15 ortholog of the related Arterivirus revealed that several residues, including those in the putative active site, reduced viral plaque size and decreased viral titers by up to five logs (13Posthuma C.C. Nedialkova D.D. Zevenhoven-Dobbe J.C. Blokhuis J.H. Gorbalenya A.E. Snijder E.J. J. Virol. 2006; 80: 1653-1661Crossref PubMed Scopus (65) Google Scholar). Similar results were observed with Nsp15 of mouse hepatitis virus A59 (14Kang H. Bhardwaj K. Li Y. Palaninathan S. Sacchettini J. Guarino L. Leibowitz J.L. Kao C.C. J. Virol. 2007; 81: 13587-13597Crossref PubMed Scopus (72) Google Scholar). In both arterivirus and mouse hepatitis virus, reduced subgenomic RNA levels were associated with mutations in Nsp15. Other properties of virus infection were also affected. Therefore, it is likely that Nsp15 and its orthologs play multiple roles in viral infection (14Kang H. Bhardwaj K. Li Y. Palaninathan S. Sacchettini J. Guarino L. Leibowitz J.L. Kao C.C. J. Virol. 2007; 81: 13587-13597Crossref PubMed Scopus (72) Google Scholar). To better understand how the activity of Nsp15 affects processing of viral RNAs, Bhardwaj et al. (15Bhardwaj K. Guarino L. Kao C.C. J. Virol. 2004; 78: 12218-12224Crossref PubMed Scopus (148) Google Scholar) and Ivanov et al. (12Ivanov K.A. Hertzig T. Rozanov M. Bayer S. Thiel V. Gorbalenya A.E. Ziebuhr J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12694-12699Crossref PubMed Scopus (211) Google Scholar) independently produced recombinant Nsp15 of the SARS-CoV in Escherichia coli and demonstrated endoribonuclease activity that preferentially cleaves RNAs at uridylates. This activity was stimulated by Mn2+ but not by other divalent metals such as Mg2+ (15Bhardwaj K. Guarino L. Kao C.C. J. Virol. 2004; 78: 12218-12224Crossref PubMed Scopus (148) Google Scholar). More recently, Bhardwaj et al. (16Bhardwaj K. Sun J. Holzenburg A. Guarino L.A. Kao C.C. J. Mol. Biol. 2006; 361: 243-2563Crossref PubMed Scopus (97) Google Scholar) demonstrated that Nsp15 cleaves 3′ of uridylates through the formation of a 2′-3′ cyclic phosphodiester product. Ribonucleases are generally grouped on the basis of whether they cleave 5′ or 3′ of the cognate phosphodiester (17Saida F. Uzan M. Bontems F. Nucleic Acids Res. 2003; 31: 2751-2758Crossref PubMed Scopus (24) Google Scholar). The former includes RNase H and RNase III, whereas the latter includes members of the RNase A family and RNase T1 (18Deshpande R.A. Shankar V. Crit. Rev. Microbiol. 2002; 28: 79-122Crossref PubMed Scopus (106) Google Scholar). The two types of enzymes also differ in their metal ion dependence. The RNase H and III enzymes are metal-dependent, whereas RNase A and T1 are metal-independent (19Gioia U. Laneve P. Dlakic M. Arceci M. Bozzoni I. Caffarelli E. J. Biol. Chem. 2005; 280: 18996-190026Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Thus, Nsp15 does not obviously belong to either group. Its cleavage is similar to RNase A, but its activity is enhanced by metal ion. This combination of properties is shared by XendoU, an endoribonuclease involved in small nucleolar RNA biosynthesis (19Gioia U. Laneve P. Dlakic M. Arceci M. Bozzoni I. Caffarelli E. J. Biol. Chem. 2005; 280: 18996-190026Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). An unusual feature of Nsp15 is that it assembles into a hexamer in solution and that disruption of hexamer formation reduces endoribonuclease activity (20Guarino L.A. Bhardwaj K. Dong W. Sun J. Holzenburg A. Kao C. J. Mol. Biol. 2005; 353: 1106-1117Crossref PubMed Scopus (68) Google Scholar). Transmission electron microscopy analysis showed that Nsp15 consists of a dimer of trimers that interact in an end-to-end fashion (16Bhardwaj K. Sun J. Holzenburg A. Guarino L.A. Kao C.C. J. Mol. Biol. 2006; 361: 243-2563Crossref PubMed Scopus (97) Google Scholar). A low resolution co-crystal of Nsp15 and RNA suggested that the RNA binds to the outside of the hexamer and interacts with both trimers (16Bhardwaj K. Sun J. Holzenburg A. Guarino L.A. Kao C.C. J. Mol. Biol. 2006; 361: 243-2563Crossref PubMed Scopus (97) Google Scholar). Two Nsp15 structures have been determined. Ricagno et al. (21Ricagno S. Egloff M.P. Ulferts R. Coutard B. Nurizzo D. Campanacci V. Cambillau C. Ziebuhr J. Canard B. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 11892-11897Crossref PubMed Scopus (132) Google Scholar) determined the x-ray structure of the SARS-CoV Nsp15 protein from crystals with one protomer in an asymmetric subunit. The crystal structure of mouse hepatitis virus Nsp15 was solved by Xu et al. (22Xu X. Zhai Y. Sun F. Lou Z. Su D. Xu Y. Zhang R. Joachimiak A. Zhang X.C. Bartlam M. Rao Z. J. Virol. 2006; 80: 7909-7917Crossref PubMed Scopus (67) Google Scholar), also with one subunit in the asymmetric unit. When arranged into a hexamer, the six active sites are exposed at the surface of the hexameric structure. Furthermore, the active site of Nsp15 can be modeled according to the residues of RNase A that share a common mechanism of catalysis (21Ricagno S. Egloff M.P. Ulferts R. Coutard B. Nurizzo D. Campanacci V. Cambillau C. Ziebuhr J. Canard B. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 11892-11897Crossref PubMed Scopus (132) Google Scholar, 22Xu X. Zhai Y. Sun F. Lou Z. Su D. Xu Y. Zhang R. Joachimiak A. Zhang X.C. Bartlam M. Rao Z. J. Virol. 2006; 80: 7909-7917Crossref PubMed Scopus (67) Google Scholar). In this structure, the C-terminal tail of Nsp15 folds back on the active site, suggesting that it may play a role in specific recognition of the cognate uridylate. These crystal structures, however, lack RNA, and the details of RNA interaction and many requirements of the subunit interactions remain unclear. Using the coordinates of Ricagno et al. (21Ricagno S. Egloff M.P. Ulferts R. Coutard B. Nurizzo D. Campanacci V. Cambillau C. Ziebuhr J. Canard B. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 11892-11897Crossref PubMed Scopus (132) Google Scholar), we solved the structure of an active site mutant of the SARS-CoV Nsp15 by molecular replacement. Notably, this structure has six subunits within the asymmetric unit. Predictions of the structure were examined, leading to elucidation of the requirements for specificity in RNA cleavage, subunit interaction, and RNA binding. Crystallization and Data Collection of Nsp15-H234AInitial crystallization trials were performed in sitting drops using 15 mg/ml of the Nsp15 mutant, H234A. The Hydra 96 robotic system (Robbins Scientific Co.) was used for Hampton and Wizard (Emerald Biostructures) crystallization screens. Three different conditions, all containing 2-methyl-2,4,-pentanediol as the precipitant, were judged promising for further optimization by the hanging drop vapor diffusion method of crystallization. Diffraction quality crystals were obtained at 10-15 mg/ml of H234A in 0.05-0.1 m MgCl2, 3-5% 2-methyl-2,4,-pentanediol, and 0.1 m tri-sodium citrate dehydrate (pH 5.6-5.9). The best crystal diffracted up to 2.64 Å at the synchrotron beam line 14-ID of Advance Photon Source, Chicago (Table 1). The crystal was mounted in cryoloop and flash-cooled in a liquid N2 stream after a brief soak in 30% ethylene glycol. The diffraction data set was indexed with unambiguous solution and processed using HKL2000 (23Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38573) Google Scholar).TABLE 1Data collection and refinement statisticsData collectionCell dimensions (Å)a = b = 305.757 and c = 88.74Space groupR3Number of molecules per asymmetric unit (Z)6Resolution (Å)50-2.8Completeness (%)aThe values in parenthesis represent values for the highest resolution shell.92.0 (78.2)Rsym (%)bRsym = ∑h∑i|Ih,i- |/∑h∑iIh,i, where Ih,i is the ith observation of the reflection h2 whereas is the means intensity of reflection h.6.5 (37.2)I/sigma(I)17.5 (2.6)Refinement statisticsResolution (Å)50-2.8Number of reflections in working data set66521Number of reflections in the test data set3507Rcryst (%)cRcryst = ∑|Fo|-|Fc|/|Fo|. Rfree was calculated with a fraction (5%) of randomly selected reflections excluded from refinement.19.7Rfree (%)25.3Ramachandran statisticsMost favored (%)90Allowed (%)10Root mean square deviationFrom ideal bond lengths (Å)0.015From ideal bond angle (°)0.8a The values in parenthesis represent values for the highest resolution shell.b Rsym = ∑h∑i|Ih,i- |/∑h∑iIh,i, where Ih,i is the ith observation of the reflection h2 whereas is the means intensity of reflection h.c Rcryst = ∑|Fo|-|Fc|/|Fo|. Rfree was calculated with a fraction (5%) of randomly selected reflections excluded from refinement. Open table in a new tab Structure Determination and RefinementThe crystal unit cell indicated the presence of six molecules in the R3 space group per asymmetric unit. The positions of each molecule were solved by molecular replacement using the program Phaser v.1.3 (24Storoni L.C. McCoy A.J. Read R.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 432-438Crossref PubMed Scopus (1099) Google Scholar) with the previously published catalytically active structure of Nsp15 (Protein Data Bank code 2H85) (21Ricagno S. Egloff M.P. Ulferts R. Coutard B. Nurizzo D. Campanacci V. Cambillau C. Ziebuhr J. Canard B. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 11892-11897Crossref PubMed Scopus (132) Google Scholar) as the search model. Model building was accomplished using Xtalview (25McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2022) Google Scholar) to examine the 2Fo - Fc, Fo - Fc, and composite omit electron density maps and refinement using maximum likelihood restrained refinement method in CCP4-REFMAC (26The CCP4 Suite (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 760-763Google Scholar, 27Murshudov G.N. Vagin A.A. Dodson E.D. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13870) Google Scholar) were performed. After the model reached R factors of less than 30%, water molecules were added to the structure using automated water picking in Xtalview. Medium or tight noncrystallographic averaging increased the R factors, and therefore only loose noncrystallographic averaging restraints were applied during the REFMAC refinement. The final structure has an R factor of 19.7% and an Rfree value of 25.3% with good stereochemistry as analyzed by PROCHECK (28Laskowski R.A. McArthur M.W. Moss D.S. Thornton J.M. J. Appl. Cryst. 1993; 26: 283-291Crossref Google Scholar). The final refinement statistics are in Table 1. The figures were prepared using Spock (quorum.tamu.edu) and Chimera (30Pettersen E.F. Goddard T.D. Huang C.C. Couch G.S. Greenblatt D.M. Meng E.C. Ferrin T.E. J. Compu. Chem. 2004; 25: 1605-1612Crossref PubMed Scopus (28368) Google Scholar). The atomic coordinates of the final model have been deposited in the Protein Data Bank with the code 2RHB. Molecular DockingDock 6.0 and its accessory programs (31Kuntz I.D. Blaney J.M. Oatley S.J. Langridge R. Ferrin T.E. J. Mol. Biol. 1982; 161: 269-288Crossref PubMed Scopus (1879) Google Scholar) were used to carry out the flexible docking of 3′-UMP or CMP (from Protein Data Bank code 4RSK-RNASE A:3′UMP complex structure) into the Nsp15 crystal structure (Protein Data Bank code 2H85), which was kept rigid. DMS (32Ferrin T.E. Huang C.C. Jarvis L.E. Langridge R. J. Mol. Graph. 1988; 6: 13-27Crossref Scopus (929) Google Scholar), a software that computes molecular surface, was used to create the molecular surface of the receptor. The negative image of the binding site was defined using Sphgen (31Kuntz I.D. Blaney J.M. Oatley S.J. Langridge R. Ferrin T.E. J. Mol. Biol. 1982; 161: 269-288Crossref PubMed Scopus (1879) Google Scholar) within the 10 Å radius of the catalytic site residues His234, His249, Ser293, Lys289, and Tyr342 to adopt the sphere-matching algorithm of Dock 6.0. An incremental construction (Anchor-and-Grow method) was used to allow for flexibility of the ligand. Automatic matching mode was used with the 20 configurations/ligand building cycle. Interaction between the ligand and the catalytic site was evaluated by the grid score (33Shoichet B.K. Bodian D.L. Kuntz I.D. J. Comp. Chem. 1992; 13: 380-397Crossref Scopus (370) Google Scholar, 34Meng E.C. Shoichet B.K. Kuntz I.D. J. Comp. Chem. 1992; 13: 505-524Crossref Scopus (859) Google Scholar) followed by visual inspection. Constructing the Nsp15-S293A ModelThe atomic coordinates of Nsp15 crystal structure (Protein Data Bank code 2H85) were used to construct a model of Nsp15-S293A using Modeler (Insight II, www.accelrys.com). Residues within a 10 Å radius of A293 were subjected to a medium level simulated annealing optimization with respect to the variable target function of Modeler. Site-directed Mutagenesis and Protein PurificationAmino acid substitutions were introduced in the Nsp15 expression plasmid using the QuikChange site-directed mutagenesis protocol as recommended by Stratagene. The entire open reading frame was sequenced to confirm the presence of directed mutation and absence of unintended mutations. WT and mutant proteins with a His tag at their N termini were expressed in E. coli and purified by metal ion affinity chromatography, followed by Mono Q ion exchange chromatography as described previously (15Bhardwaj K. Guarino L. Kao C.C. J. Virol. 2004; 78: 12218-12224Crossref PubMed Scopus (148) Google Scholar). The purified proteins were stored in 50 mm Tris (pH 7.9), 300 mm NaCl, 1 mm dithiothreitol, 50% (v/v) glycerol at -20 °C. The protein concentrations were determined by absorbance at 280 nm. Size Exclusion ChromatographyWT or mutant protein samples (∼100 μg) were filtered through a Superdex 200 column using a Pharmacia fast protein liquid chromatography system calibrated with the following molecular mass standards (kDa in parentheses): blue dextran (2000); thyroglobulin (660); ferritin (440); catalase (232); aldolase (140); bovine serum albumin (67); and chymotrypsinogen (25McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2022) Google Scholar) according to the protocol described in Guarino et al. (20Guarino L.A. Bhardwaj K. Dong W. Sun J. Holzenburg A. Kao C. J. Mol. Biol. 2005; 353: 1106-1117Crossref PubMed Scopus (68) Google Scholar). Reversible Protein-RNA Cross-linking and Peptide MappingH249A (10 μm) was incubated with or without 20 μm biotinylated RNA, Bio-rUC10 in 20 mm Hepes (pH 7.5), 5 mm MnCl2, and 1 mm dithiothreitol at room temperature for 5 min followed by the addition of formaldehyde to a final concentration of 0.1% in a 25-μl reaction. The cross-linking reaction was stopped by the addition of glycine at 0.2 m final concentration. After 5 min, sequencing grade trypsin (Trypsin Gold; Promega, Madison, WI) was added at a protease:substrate ratio 1:50 (w/w) in 100 mm NH4HCO3 (pH 7.8) at 37 °C and allowed to digest overnight. Streptavidin magnetic beads (New England Biolab, Beverly, MA) were used to capture biotinylated RNA and RNA-peptide conjugates as described previously by Kim et al. (4Kim Y.C. Russell W.K. Ranjith-Kumar C.T. Thomson M. Russell D.H. Kao C.C. J. Biol. Chem. 2005; 280: 38011-38019Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). RNA-peptide conjugates were reversed by incubating the samples at 70 °C for 1 h. The samples were centrifuged at 3000 × g for 5 min, and the supernatants containing the peptides were desalted using a Ziptip (Millipore, Bedford, MA). The bound peptides were eluted in 2.5 μl of 70% acetonitrile and 0.1% trifluoroacetic acid. The eluted samples were analyzed by MALDI-ToF. Affinity Chromatography10 μg of WT or mutant proteins were incubated on ice with ∼2 mg polyU-agarose in buffer A (50 mm Tris, pH 7.5, 5% glycerol, 1 mm β-mercaptoethanol, 10 mm MnCl2) containing 30 mm NaCl. After 90 min, the agarose beads were washed by centrifugation at 6,000 rpm for 10 min to remove unbound protein. Bound protein was eluted with buffer A containing increasing concentrations of 50, 100, 200, 300, 400, and 500 mm NaCl. The fractions were analyzed by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Brilliant Blue. Protein was quantitated using the Coomassie Plus Protein Assay kit from Pierce. Endoribonuclease AssaysThe real time endoribonuclease assay used a substrate purchased from Integrated DNA Technologies, Inc. (Coralville, Iowa). The substrate has a 5′ carboxyl fluorescein fluorophore at the 5′ end and tetramethylrhodamine at the 3′ end, which quenches 5′ carboxyl fluorescein fluorescence. Of the four nucleotides, only the cognate cleavage nucleotide is a ribonucleotide, whereas the others are deoxyribonucleotides. Protein and RNA substrate were incubated in 20 mm Hepes (pH 7.5), 5 mm MnCl2, 1 mm dithiothreitol, as indicated. Substrate cleavage was monitored with an excitation wavelength of 492 nm and an emission wavelength of 518 nm (16Bhardwaj K. Sun J. Holzenburg A. Guarino L.A. Kao C.C. J. Mol. Biol. 2006; 361: 243-2563Crossref PubMed Scopus (97) Google Scholar). Changes in fluorescence were measured over time using an LS55 spectrometer (PerkinElmer Life Sciences). The Crystal Structure of Nsp15 Mutant H234AA mutant version of Nsp15 protein with an alanine substitution for one of the active site histidines, H234A, was deemed suitable for crystallization because it could be expressed and purified at a level more than 10-fold higher levels than the WT Nsp15. Furthermore, it, like WT Nsp15, purified as a hexamer (20Guarino L.A. Bhardwaj K. Dong W. Sun J. Holzenburg A. Kao C. J. Mol. Biol. 2005; 353: 1106-1117Crossref PubMed Scopus (68) Google Scholar) and was competent for RNA binding (16Bhardwaj K. Sun J. Holzenburg A. Guarino L.A. Kao C.C. J. Mol. Biol. 2006; 361: 243-2563Crossref PubMed Scopus (97) Google Scholar). A single crystal of H234A protein, hereafter named Nsp15H, diffracted up to 2.64 Å with unit cell parameters a = b = 305.757 and c = 88.740 in the R3 space group. The asymmetric unit contained six molecules, and the positions of each subunit of Nsp15H were solved by molecular replacement using the structure of the catalytically active Nsp15 V291G-D300G double mutant (Protein Data Bank code 2H85) (21Ricagno S. Egloff M.P. Ulferts R. Coutard B. Nurizzo D. Campanacci V. Cambillau C. Ziebuhr J. Canard B. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 11892-11897Crossref PubMed Scopus (132) Google Scholar) as the search model. The Ricagno et al. (21Ricagno S. Egloff M.P. Ulferts R. Coutard B. Nurizzo D. Campanacci V. Cambillau C. Ziebuhr J. Canard B. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 11892-11897Crossref PubMed Scopus (132) Google Scholar) model, hereafter named Nsp15VD, has only one molecule/asymmetric unit. The asymmetric unit of Nsp15H provides additional insight into the heterogeneity among individual subunits of the hexamer and allows comparison with the WT active site in Nsp15VD. The overall structure of Nsp15H is very similar to that of Nsp15VD, with a root mean square deviation difference of 0.53-0.73 Å (344Cα atoms of each subunit of Nsp15 H were super-imposed on Nsp15VD structure using the LSQKAB routine of CCP4 (26The CCP4 Suite (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 760-763Google Scholar)). Both Nsp15H and Nsp15VD consist of a dimer of trimers, which is consistent with the solution state and with single Nsp15 particles analyzed by transmission electron microscopy and cryoelectron microscopy (16Bhardwaj K. Sun J. Holzenburg A. Guarino L.A. Kao C.C. J. Mol. Biol. 2006; 361: 243-2563Crossref PubMed Scopus (97) Google Scholar). Each subunit contains nine α-helices and twenty-one β-strands. A subunit is further organized into three domains: an N-terminal domain (residues 1-61), a middle domain (residues 62-181), and a C-terminal domain (residues 182-345) (Fig. 1A). Alanine substitutions of highly conserved residues have demonstrated that the C-terminal domain contains the active site (20Guarino L.A. Bhardwaj K. Dong W. Sun J. Holzenburg A. Kao C. J. Mol. Biol. 2005; 353: 1106-1117Crossref PubMed Scopus (68) Google Scholar), which faces away from the center of the hexamer and contains the extreme C-terminal residues, consistent with the observations of Ricagno et al. (21Ricagno S. Egloff M.P. Ulferts R. Coutard B. Nurizzo D. Campanacci V. Cambillau C. Ziebuhr J. Canard B. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 11892-11897Crossref PubMed Scopus (132) Google Scholar). When viewed from the top of the hexamer, a pore through the trimer is evident, with the N-terminal domains of the trimer lining the bottom of the pore (Fig. 1B). This pore has an inner diameter of ∼12 Å and does not interact with the substrate RNA according to the cryoelectron microscopy structure (16Bhardwaj K. Sun J. Holzenburg A. Guarino L.A. Kao C.C. J. Mol. Biol. 2006; 361: 243-2563Crossref PubMed Scopus (97) Google Scholar). The middle and the N-terminal domains have extensive contacts with the other subunits of the hexamer. These interactions are evident in the side view of the Nsp15 hexamer (Fig. 1C). Heterogeneity in the Six Subunits in Nsp15There are significant differences among the subunits of Nsp15H that are not observed in Nsp15VD. To better describe these differences, the three subunits of the upper trimer, as oriented in Fig. 1 (B-D), are labeled T1a, T1b, and T1c, and the lower trimer subunits are labeled T2a, T2b, and T2c. The T2 subunits have B factors of 49.3, whereas the T1 subunits have B factors of 69.7 Å2, respectively. The increased flexibility is shown by the worm diagram in Fig. 1D. In addition, flexibility is significantly higher in the C-terminal domains of each subunit than for the rest of that subunit (Fig. 1D). Within subunits T1 and T2, the root mean square deviation difference in the N-terminal domain is 0.27-0.34 Å, whereas the C-terminal domain had a root mean square deviation difference of 0.51-0.57 Å. The crystal packing may have some effect on the observed variations. This is insufficient, however, to affect the entire domain of each subunit. Moreover, the trimers showed significant differences between each other despite being in a similar environment. Heterogeneity is most obvious in the subunit catalytic pockets formed by residues His234, His249, Trp332, Tyr342, and Lys289. Superposition of the active site residues showed the degree of conformational flexibility between the subunits (Fig. 1E). Although the positions of Lys289 and His234 are highly similar in each of the subunits, Tyr342 is especially flexible and adopted different rotomer conformations in the different subunits (Fig. 1E). In addition, Trp332 is disordered in subunit T2c and not visible in the electron density map. The distance between the side chain oxygen atom of Tyr342 to ϵ1-nitrogen atom of Trp332 varied between 3.0 and 4.1 Å in Nsp15H. Subunit InteractionsBased on the examination of a panel of mutants, Guarino et al. (16Bhardwaj K. Sun J. Holzenburg A. Guarino L.A. Kao C.C. J. Mol. Biol. 2006; 361: 243-2563Crossref PubMed Scopus (97) Google Scholar, 20Guarino L.A. Bhardwaj K. Dong W. Sun J. Holzenburg A. Kao C. J. Mol. Biol. 2005; 353: 1106-1117Crossref PubMed Scopus (68) Google Scholar) proposed that hexamer formation is a prerequisite for enzymatic activity and RNA binding. A key mutant in this analysis was E3A, located in the N-terminal domain of Nsp15. It was inactive at lower concentrations but recovered some catalytic function at higher concentrations that should favor oligomerization (20Guarino L.A. Bhardwaj K. Dong W. Sun J. Holzenburg A. Kao C. J. Mol. Biol. 2005; 353: 1106-1117C
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