Crystallization, enzymatic cleavage, and the polarity of the adenovirus type 2 fiber.
Crystals of the fiber protein of adenovirus type 2 have been grown. Analysis of these crystals (type I crystals) showed that they were composed of fiber polypeptide with a lower apparent molecular weight (60 kDa) than that of the soluble or virion-incorporated fiber (62 kDa). N-terminal sequencing revealed that the fiber polypeptide chain of 60 kDa was cleaved at tyrosine17 from the N-end. The C-terminus remained intact. Assays with protease inhibitors suggested that the spontaneous cleavage of the fiber occurring upon its crystallization was due to a cellular, calcium-dependent, chymotrypsin-like protease co-purifying with the fiber and activated during hydroxyapatite chromatography. Crystallization of fiber purified in the presence of chymostatin provided crystals of a different structure under the electron microscope (crystals of type II), composed of 62-kDa fiber polypeptide units. The 62-kDa fiber from the type II crystals, as well as the 62-kDa fiber isolated from infected cell extracts, were able to associate with the penton base in vitro to form a penton capsomer. The 60-kDa fiber has lost this capacity. The accessibility of the N- and C-termini of the fiber inside the penton structure was probed by anti-peptide sera after limited proteolysis. The results are consistent with a polarity of the fiber in which its N-terminus is oriented toward the penton base, the C-terminal domain corresponding to the distal knob.
Devaux
C
C
European Molecular Biology Laboratory, Grenoble Outstation, France.
Caillet-Boudin
M L
ML
Jacrot
B
B
Boulanger
P
P
D000260:Adenoviruses, Human / Q000032:analysis* / Q000201:enzymology; D002213:Capsid / Q000032:analysis* / Q000378:metabolism; D036022:Capsid Proteins; D002268:Carboxypeptidases / Q000378:metabolism; D002918:Chymotrypsin / Q000378:metabolism; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007118:Immunoassay; D007700:Kinetics; D008854:Microscopy, Electron; D010447:Peptide Hydrolases / Q000378:metabolism*; D011480:Protease Inhibitors / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014764:Viral Proteins / Q000032:analysis / Q000378:metabolism
D036022:Capsid Proteins; D011480:Protease Inhibitors; D014764:Viral Proteins; C018315:hexon capsid protein, Adenovirus; D002268:Carboxypeptidases; D010447:Peptide Hydrolases; D002918:Chymotrypsin; D014357:Trypsin