Characterization of the knob domain of the adenovirus type 5 fiber protein expressed in Escherichia coli.
The adenovirus fiber protein is used for attachment of the virus to a specific receptor on the cell surface. Structurally, the protein consists of a long, thin shaft that protrudes from the vertex of the virus capsid and terminates in a globular domain termed the knob. To verify that the knob is the domain which interacts with the cellular receptor, we have cloned and expressed the knob from adenovirus type 5 together with a single repeat of the shaft in Escherichia coli. The protein was purified by conventional chromatography and functionally characterized for its interaction with the adenovirus receptor. The recombinant knob domain bound about 4,700 sites per HeLa cell with an affinity of 3 x 10(9) M-1 and blocked adenovirus infection of human cells. Antibodies raised against the knob also blocked virus infection. By gel filtration and X-ray diffraction analysis of protein crystals, the knob was shown to consist of a homotrimer of 21-kDa subunits. The results confirm that the trimeric knob is the ligand for attachment to the adenovirus receptor.
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235-8573.
L J
LJ
Henry
D
D
Xia
M E
ME
Wilke
J
J
Deisenhofer
R D
RD
Gerard
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism*; D000914:Antibodies, Viral / Q000276:immunology; D001483:Base Sequence; D002213:Capsid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D036022:Capsid Proteins; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003460:Crystallization; D004279:DNA, Viral; D004926:Escherichia coli; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins; D012091:Repetitive Sequences, Nucleic Acid; D014961:X-Ray Diffraction
D000914:Antibodies, Viral; D036022:Capsid Proteins; D004279:DNA, Viral; D011994:Recombinant Proteins; C018315:hexon capsid protein, Adenovirus