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.