Insights into the Oligomeric States, Conformational Changes, and Helicase Activities of SV40 Large Tumor Antigen

Abstract

The large T (LT) antigen encoded by SV40 virus is a multi-domain, multi-functional protein that can not only transform cells but can also function as an efficient molecular machine to unwind duplex DNA for DNA replication. Here we report our findings on the oligomeric forms, domain interactions, and ATPase and helicase activities of various LT constructs. For the LT constructs that hexamerize, only two oligomeric forms, hexameric and monomeric, were detected in the absence of ATP/ADP. However, the presence of ATP/ADP stabilizes LT in the hexameric form. The LT constructs lacking the N-and C-terminal domains, but still retaining hexamerization ability, have ATPase as well as helicase activities at a level comparable to the full-length LT, suggesting the importance of hexamerization for these activities. The domain structures and the possible interactions between different LT fragments were probed with limited protease (trypsin) digestion. Such protease digestion generated a distinct pattern in the presence and absence of ATP/ADP and Mg 2؉ . The most C-terminal fragment (residues 628 -708, containing the host-range domain), which was thought to be completely unstructured, was somewhat trypsin-resistant despite the presence of multiple Arg and Lys, possibly due to a rather structured C terminus. Furthermore, the N-and Cterminal fragments cleaved by trypsin were associated with other parts of the molecule, suggesting the interdomain interactions for the fragments at both ends.

Partial Proteolysis of Simian Virus 40 T Antigen Reveals Intramolecular Contacts between Domains and Conformation Changes upon Hexamer Assembly

“…Because antibodies specific for domain III were not used, we cannot assess whether domain I may also interact with domain III. This interpretation is consistent with the independent observation that tryptic fragments of Tag from domains I, II, and III form complexes that remain stable through gel filtration (31). This intramolecular interaction between the N terminus and the central region of Tag also provides a plausible explanation for the previously observed requirement for domain IIb of Tag to permit casein kinase I to phosphorylate its targets in domain I (Ser-120 and Ser-123) (35).…”

“…1-3), the stable fragment 513-698 (domain III in Fig. 7A), detected here and independently by Chen and colleagues (31), is novel. The sequenced N termini reported here are completely consistent with those determined by Chen and colleagues (31).…”

“…7A), detected here and independently by Chen and colleagues (31), is novel. The sequenced N termini reported here are completely consistent with those determined by Chen and colleagues (31). Our use of monoclonal antibodies whose epitopes are small and well mapped affords somewhat greater resolution of the multiple cleavage sites in the C terminus of domain IIb and the extreme C terminus of Tag (Fig.…”

“…2). Minor differences in deduced cleavage sites based on the apparent molecular mass of peptides are probably due to technical differences between the two studies (31).…”

“…Domain IIa is multifunctional, being sufficient for origin DNA binding activity, binding to host RPA, topoisomerase I, transcription factors, and perhaps other proteins (1, 2, 3). Domain IIb-III contains the helicase activity of Tag (20,31) as well as binding sites for the tumor suppressor protein p53, DNA polymerase ␣-primase, and other host proteins (1-3, 33). In addition, a role for domain III in contacting origin DNA in the double hexamer was recently reported (34).…”