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

Weißhart, Klaus; Friedl, Sabine; Taneja, Poonam; Nasheuer, Heinz-Peter; Schlott, Bernhard; Grosse, Frank; Fanning, Ellen

doi:10.1074/jbc.m406159200

Cited by 18 publications

(18 citation statements)

References 37 publications

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“…There is only one discrepancy in the assignment of the cleavage sites. The discrepancy occurs at residue 698 where cleavage between 698 and 699 was deduced by Weisshart et al (32) but not detected in this study. However, a closer examination of the cleavage pattern in our study reveals the possible presence of this cleavage site.…”

Section: Discussioncontrasting

confidence: 63%

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

Gai

Finkielstein

et al. 2004

Journal of Biological Chemistry

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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.

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Section: Discussioncontrasting

confidence: 63%

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

Gai

Finkielstein

et al. 2004

Journal of Biological Chemistry

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“…It is tempting to speculate that phosphorylation of the N-terminal domain could be involved in regulating the interaction of the helicase domain with DNA. This notion is supported by the findings that the N-terminal domain can be cross-linked to the helicase domain (63) and that mutant T124A T-ag molecules assemble into double hexamers that are less stable (64) and require longer sequences in the EP or AT region for their assembly on single assembly units (46). …”

Section: Discussionmentioning

confidence: 90%

Quantitative Analysis of the Binding of Simian Virus 40 Large T Antigen to DNA

Fradet-Turcotte

Vincent

Joubert

et al. 2007

J Virol

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SV40 large T antigen (T-ag)is a multifunctional protein that successively binds to 5-GAGGC-3 sequences in the viral origin of replication, melts the origin, unwinds DNA ahead of the replication fork, and interacts with host DNA replication factors to promote replication of the simian virus 40 genome. The transition of T-ag from a sequencespecific binding protein to a nonspecific helicase involves its assembly into a double hexamer whose formation is likely dictated by the propensity of T-ag to oligomerize and its relative affinities for the origin as well as for nonspecific double-and single-stranded DNA. In this study, we used a sensitive assay based on fluorescence anisotropy to measure the affinities of wild-type and mutant forms of the T-ag origin-binding domain (OBD), and of a larger fragment containing the N-terminal domain (N260), for different DNA substrates. We report that the N-terminal domain does not contribute to binding affinity but reduces the propensity of the OBD to self-associate. We found that the OBD binds with different affinities to its four sites in the origin and determined a consensus binding site by systematic mutagenesis of the 5-GAGGC-3 sequence and of the residue downstream of it, which also contributes to affinity. Interestingly, the OBD also binds to single-stranded DNA with an ϳ10-fold higher affinity than to nonspecific duplex DNA and in a mutually exclusive manner. Finally, we provide evidence that the sequence specificity of full-length T-ag is lower than that of the OBD. These results provide a quantitative basis onto which to anchor our understanding of the interaction of T-ag with the origin and its assembly into a double hexamer.

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“…The same conclusion was drawn from limited proteolysis experiments carried out with double hexamers. 28 We have found that the tiers from each hexamer have different quaternary structure. This is very clear for the OBD domains ( Figure 5(a) and (b)).…”

Section: Discussionmentioning

confidence: 90%

Structural Basis for the Cooperative Assembly of Large T Antigen on the Origin of Replication

Valle

Chen

Donate

et al. 2006

Journal of Molecular Biology

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

Cited by 18 publications

References 37 publications

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

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

Quantitative Analysis of the Binding of Simian Virus 40 Large T Antigen to DNA

Structural Basis for the Cooperative Assembly of Large T Antigen on the Origin of Replication

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