title: Partial proteolysis of simian virus 40 T antigen reveals intramolecular contacts between domains and conformation changes upon hexamer assembly.
abstract: Simian virus 40 large tumor antigen (Tag) is a multi-functional viral protein that binds specifically to SV40 origin DNA, serves as the replicative DNA helicase, and orchestrates the assembly and operation of the viral replisome. Tag associated with Mg-ATP forms hexamers and, in the presence of SV40 origin DNA, double hexamers. Limited tryptic digestion of monomeric Tag revealed three major stable structural domains. The N-terminal domain spans amino acids 1-130, the central domain comprises amino acids 131-476, and the C-terminal domain extends from amino acid 513 to amino acid 698. Co-immunoprecipitation of digestion products of monomeric Tag suggests that the N-terminal domain associates stably with sequences located in the central region of the same Tag molecule. Hexamer formation protected the tryptic cleavage sites in the exposed region between the central and C-terminal domains. Upon hexamerization, this exposed region also became less accessible to a monoclonal antibody whose epitope maps in that region. The tryptic digestion products of the soluble hexamer and the DNA-bound double hexamer were indistinguishable. A low-resolution model of the intramolecular and intermolecular interactions among Tag domains in the double hexamer is proposed.
authors.affiliation: Institute for Molecular Biotechnology, Beutenbergstrasse 11, 07745 Jena, Federal Republic of Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Weisshart
authors.forename: Sabine
authors.initials: S
authors.lastname: Friedl
authors.forename: Poonam
authors.initials: P
authors.lastname: Taneja
authors.forename: Heinz-Peter
authors.initials: HP
authors.lastname: Nasheuer
authors.forename: Bernhard
authors.initials: B
authors.lastname: Schlott
authors.forename: Frank
authors.initials: F
authors.lastname: Grosse
authors.forename: Ellen
authors.initials: E
authors.lastname: Fanning
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D004265:DNA Helicases / Q000737:chemistry; D000939:Epitopes / Q000737:chemistry; D007313:Insecta; D008954:Models, Biological; D010455:Peptides / Q000737:chemistry; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D000952:Antigens, Polyomavirus Transforming; D000939:Epitopes; D010455:Peptides; D014357:Trypsin; D004265:DNA Helicases