Structural characterization of the SARS-coronavirus spike S fusion protein core.
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 alpha-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 alpha-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.
Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
Brian
B
Tripet
Megan W
MW
Howard
Michael
M
Jobling
Randall K
RK
Holmes
Kathryn V
KV
Holmes
Robert S
RS
Hodges
D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D006389:Hemagglutinins, Viral / Q000737:chemistry / Q000302:isolation & purification; D008562:Membrane Glycoproteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011487:Protein Conformation; D015183:Restriction Mapping; D045473:Severe acute respiratory syndrome-related coronavirus / Q000737:chemistry*; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification
D017931:DNA Primers; D006389:Hemagglutinins, Viral; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus