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,Xiaojiang S Chen

doi:10.1074/jbc.m406160200

Dahai Gai, Dawei Li + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m406160200

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

Highlights

SV40 large T (LT)1 antigen is a multi-functional protein that transforms cells and is important for viral DNA replication
The yields of LT polypeptides from E. coli varied depending on the constructs
The helicase assay showed that all of the LT constructs with the exception of LT303– 627 had helicase activity (Fig. 4B, lanes 3–7)

Summary

Introduction

SV40 large T (LT)1 antigen is a multi-functional protein that transforms cells and is important for viral DNA replication (reviewed in Refs. 1 and 2). Using the gel-filtration chromatography in buffers without any cross-linking reagents, a truncated LT fragment containing residues 251– 627 (LT251– 627) was shown to form hexamers even in the absence of ATPϩMg2ϩ [11].

Results

Conclusion