A Fluorogenic Peptide Containing the Processing Site of Human SARS Corona Virus S-Protein: Kinetic Evaluation and NMR Structure Elucidation

Authors

  • Ajoy Basak Prof.,

    1. Hormone, Growth, and Development Program, Regional Protein Chemistry Center, Ottawa Health Research Institute, University of Ottawa, 725 Parkdale Ave., Ottawa, ON K1Y 4E9, Canada, Fax: (+1) 613-761-4355
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  • Abhijit Mitra Prof.,

    1. Department of Chemistry and Biochemistry, Manhattan College/College of Mount Saint Vincent, Riverdale, NY 10471, USA
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    • These authors contributed equally to this work.

  • Sarmistha Basak Dr.,

    1. Hormone, Growth, and Development Program, Regional Protein Chemistry Center, Ottawa Health Research Institute, University of Ottawa, 725 Parkdale Ave., Ottawa, ON K1Y 4E9, Canada, Fax: (+1) 613-761-4355
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  • Carolyn Pasko,

    1. Hormone, Growth, and Development Program, Regional Protein Chemistry Center, Ottawa Health Research Institute, University of Ottawa, 725 Parkdale Ave., Ottawa, ON K1Y 4E9, Canada, Fax: (+1) 613-761-4355
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  • Michel Chrétien Dr.,

    1. Hormone, Growth, and Development Program, Regional Protein Chemistry Center, Ottawa Health Research Institute, University of Ottawa, 725 Parkdale Ave., Ottawa, ON K1Y 4E9, Canada, Fax: (+1) 613-761-4355
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  • Pamela Seaton Prof.

    1. Department of Chemistry and Biochemistry, University of North Carolina, Wilmington, NC 28403, USA
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    • These authors contributed equally to this work.


Abstract

Human severe acute respiratory syndrome coronavirus (hSARS-CoV) is the causative agent for SARS infection. Its surface glycoprotein (spike protein) is considered to be one of the prime targets for SARS therapeutics and intervention because its proteolytic maturation by a host protease is crucial for host–virus fusion. Using intramolecularly quenched fluorogenic (IQF) peptides based on hSARS-CoV spike protein (Abz-755Glu-Gln-Asp-Arg-Asn-Thr-Arg-Glu-Val-Phe-Ala-Gln766-Tyx-NH2) and in vitro studies, we show that besides furin, other PCs, like PC5 and PC7, might also be involved in this cleavage event. Through kinetic measurements with recombinant PCs, we observed that the peptide was cleaved efficiently by both furin and PC5, but very poorly by PC7. The cleavage could be blocked by a PC-inhibitor, α1-PDX, in a dose-dependent manner. Circular dichroism spectra indicated that this peptide possesses a high degree of sheet structure. Following cleavage by furin, the sheet content increased, possibly at the expense of turn and random structures. 1H NMR spectra from 2D COSY and ROESY experiments under physiological buffer and pH conditions indicated that this peptide possesses a structure with a turn at its C-terminal segment, close to the cleavage site. The data suggest that the cleavable peptide bond is located within the most exposed domain; this is supported by the nearby turn structure. Several strong to weak NMR ROESY correlations were detected, and a 3D structure of the spike IQF peptide that contains the crucial cleavage site R761↓E has been proposed.

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