A fluorogenic peptide containing the processing site of human SARS corona virus S-protein: kinetic evaluation and NMR structure elucidation.
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-(755)Glu-Gln-Asp-Arg-Asn-Thr-Arg-Glu-Val-Phe-Ala-Gln(766)-Tyx-NH(2)) 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, alpha1-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. (1)H 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 R(761) E has been proposed.
Basak
Ajoy
A
Hormone, Growth, and Development Program, Regional Protein Chemistry Center, Ottawa Health Research Institute, University of Ottawa, 725 Parkdale Ave., Ottawa, ON K1Y 4E9, Canada.
Mitra
Abhijit
A
Basak
Sarmistha
S
Pasko
Carolyn
C
Chrétien
Michel
M
Seaton
Pamela
P
D000465:Algorithms; D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D005456:Fluorescent Dyes; D005663:Furans / Q000737:chemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D021281:Quantitative Structure-Activity Relationship; D045473:Severe acute respiratory syndrome-related coronavirus / Q000737:chemistry*; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000737:chemistry*
D000596:Amino Acids; D005456:Fluorescent Dyes; D005663:Furans; D008562:Membrane Glycoproteins; D010455:Peptides; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus