Phosphorylation and active ATP hydrolysis are not required for SV40 T antigen hexamer formation.
ATP induces structural alterations in SV40 large T antigen and promotes changes in its interaction with the viral replication origin. We have analyzed nucleotide-induced changes in T antigen structure in the absence of origin DNA. Most preparations of immunopurified T antigen contain several discrete species ranging in size from monomers through oligomers larger than hexamers. The predominant species consist of monomers and dimers. Incubation of T antigen with ATP or dATP leads to a dramatic and rapid increase in the appearance of T antigen hexamers. Weakly and nonhydrolyzable analogs of ATP are effective as well, indicating that hexamer formation does not require active ATP hydrolysis. After incubation of T antigen with [gamma-35S]ATP, stable association of the labeled nucleotide with all detectable forms occurs. Removal of greater than 80% of the T antigen phosphate residues does not significantly affect the formation of T antigen hexamers, although changes in the distribution and mobility of the other species of T antigen are apparent. Furthermore, T antigen synthesized in and purified from Escherichia coli and, therefore, presumably un- or underphosphorylated, is capable of forming hexamers. Nucleotide-induced T antigen hexamer formation thus appears to require neither protein phosphorylation nor active ATP hydrolysis.
Reynisdóttir
I
I
Department of Biological Sciences, Columbia University, New York, New York 10027.
Lorimer
H E
HE
Friedman
P N
PN
Wang
E H
EH
Prives
C
C
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006868:Hydrolysis; D046911:Macromolecular Substances; D015636:Magnesium Chloride / Q000378:metabolism; D009036:Moths; D010766:Phosphorylation; D011994:Recombinant Proteins
D000952:Antigens, Polyomavirus Transforming; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D015636:Magnesium Chloride; D000255:Adenosine Triphosphate