The nucleotide binding site detected by affinity labeling in the large T proteins of polyoma and SV40 viruses is distinct from their ATPase catalytic site

• Published in: The Journal of biological chemistry Vol. 259; no. 24; pp. 15196 - 15203
• Main Authors: Clertant, P, Gaudray, P, May, E, Cuzin, F
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 25.12.1984; American Society for Biochemistry and Molecular Biology
• Subjects: Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - metabolism; Affinity Labels - metabolism; Animals; Antibodies; Antigen-Antibody Complex; Antigens, Polyomavirus Transforming; Antigens, Viral, Tumor - metabolism; Binding Sites; Biological and medical sciences; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Chlorocebus aethiops; Fundamental and applied biological sciences. Psychology; Kidney; Kinetics; Mice; Microbiology; Polyomavirus - enzymology; Protein Binding; Protein Kinases - metabolism; Replicative cycle, interference, host-virus relations, pathogenicity, miscellaneous strains; Simian virus 40 - enzymology; Viral Proteins - metabolism; Virology; Molecular structure; Affinity labelling; ATPase; Polyomavirus; Identification; Binding site; Papovaviridae; Structure function relationship; Virus; Oncogenic virus; Nucleotide; Catalytic site; Mechanism of action; SV40 virus; Catalyst activity; T antigen
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)42534-8

Binding of nucleotides to a specific site of the large T proteins of polyoma and SV40 viruses was demonstrated by covalent affinity labeling with periodate-oxidized [alpha-32P]ATP (oxATP) (Clertant, P., and Cuzin, F. (1982) J. Biol. Chem. 257, 6300-6305). This site appears different from the catalytic site of these proteins for ATP hydrolysis: (i) nucleotide binding and ATPase activities exhibited different ionic requirements and kinetic parameters; (ii) different antibodies directed against the polyoma large T protein either completely inhibited ATPase activity and not affinity labeling, or vice versa; (iii) a truncated form of polyoma large T, with its carboxyl-terminal third deleted, does not bind oxATP but exhibits normal ATPase activity; (iv) conversely, a “super T” SV40 protein, resulting from a duplication within the coding region of large T, was efficiently labeled with oxATP, although it lacks detectable ATPase activity. Cyanogen bromide cleavage after affinity labeling mapped the nucleotide binding site of the polyoma and SV40 large T proteins within a carboxyl-terminal amino acid sequence highly homologous between the two polypeptides. A survey of the phenotypes of the known mutations in these multifunctional proteins suggests that their ATPase and nucleotide-binding activities, although distinct, might both be required to ensure crucial steps in the lytic cycle.