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J. Biol. Chem., Vol. 283, Issue 6, 3655-3664, February 8, 2008

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Structural and Functional Analyses of the Severe Acute Respiratory Syndrome Coronavirus Endoribonuclease Nsp15^*

Kanchan Bhardwaj¹, Satheesh Palaninathan¹, Joanna Maria Ortiz Alcantara, Lillian Li Yi, Linda Guarino, James C. Sacchettini, and C. Cheng Kao²

From the Departments of Biochemistry and Biophysics and Entomology, Texas A & M University, College Station, Texas 77843-2128

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.

Received for publication, October 9, 2007 , and in revised form, November 15, 2007.

The atomic coordinates and structure factors (code 2RHB) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Institutes of Health Grant 1R56AI06164 and a Small Grant for Exploratory Research from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and supplemental Figs. S1-S3.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 979-458-2235; Fax: 979-845-9274; E-mail: ckao{at}tamu.edu.

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