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J. Biol. Chem., Vol. 279, Issue 37, 38952-38959, September 10, 2004

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

Dahai Gai, Dawei Li¶, Carla V. Finkielstein, Robert D. Ott||, Poonam Taneja||**, Ellen Fanning||, and Xiaojiang S. Chen

From the Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, School of Medicine, Denver, Colorado 80262 and ^||Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235

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 Mg²⁺. 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 C-terminal fragments cleaved by trypsin were associated with other parts of the molecule, suggesting the interdomain interactions for the fragments at both ends.

Received for publication, June 3, 2004

^* This work was supported in part by a fellowship (to D. G.) from Colorado Cancer League, a postdoctoral fellowship (to C. V. F.) from the American Heart Association (0225388Z), and a Scholar Research grant (to X. S. C.) from the American Cancer Society (GMC-106782). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Molecular and Computational Biology, University of Southern California, Los Angeles, CA 90089.

^¶ Present address: National Key Laboratory, China Agriculture University, Beijing 100094, China.

^** Present address: Arcturus Engineering, Inc., 400 Logue Ave., Mountain View, CA 94043.

To whom correspondence should be addressed: Molecular and Computational Biology, University of Southern California, Los Angeles, CA 90089-1340. Tel.: 213-740-5487; Fax: 213-740-8631; E-mail: Xiaojiac{at}usc.edu.

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