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J. Biol. Chem., Vol. 279, Issue 20, 20836-20849, May 14, 2004

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Structural Characterization of the SARS-Coronavirus Spike S Fusion Protein Core^*

Brian Tripet, Megan W. Howard, Michael Jobling, Randall K. Holmes, Kathryn V. Holmes, and Robert S. Hodges¶

From the Department of Biochemistry and Molecular Genetics and the Department of Microbiology, University of Colorado Health Sciences Center, Denver, Colorado 80262

The spike (S) glycoprotein of coronaviruses mediates viral entry into host cells. It is a type 1 viral fusion protein that characteristically contains two heptad repeat regions, denoted HR-N and HR-C, that form coiled-coil structures within the ectodomain of the protein. Previous studies have shown that the two heptad repeat regions can undergo a conformational change from their native state to a 6-helix bundle (trimer of dimers), which mediates fusion of viral and host cell membranes. Here we describe the biophysical analysis of the two predicted heptad repeat regions within the severe acute respiratory syndrome coronavirus S protein. Our results show that in isolation the HR-N region forms a stable -helical coiled coil that associates in a tetrameric state. The HR-C region in isolation formed a weakly stable trimeric coiled coil. When mixed together, the two peptide regions (HR-N and HR-C) associated to form a very stable -helical 6-stranded structure (trimer of heterodimers). Systematic peptide mapping showed that the site of interaction between the HR-N and HR-C regions is between residues 916–950 of HR-N and residues 1151–1185 of HR-C. Additionally, interchain disulfide bridge experiments showed that the relative orientation of the HR-N and HR-C helices in the complex was antiparallel. Overall, the structure of the hetero-stranded complex is consistent with the structures observed for other type 1 viral fusion proteins in their fusion-competent state.

Received for publication, January 23, 2004 , and in revised form, March 1, 2004.

^* This work was supported by National Institutes of Health Grants R01AI48717 (to R. S. H.), 940 (to R. K. H.), and RO1AI25231 (to K. V. H.) and the John Stewart Chair in Peptide Chemistry (to R. S. H.). 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.

^¶ To whom correspondence should be addressed. Tel.: 303-315-8837; Fax: 303-315-1153; E-mail: robert.hodges{at}uchsc.edu.

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