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Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells
Published on: November 12, 2015
Mapping the interactions of adenoviral E1A proteins with the p160 nuclear receptor coactivator binding domain of CBP
Peter Haberz1, Munehito Arai2, Maria A Martinez-Yamout1
1Department of Integrative Structural and Computational Biology and The Skaggs Institute of Chemical Biology, The Scripps Research Institute, La Jolla, California.
Abstract:
Many viruses deregulate the cell and force transcription of viral genes by competing with cellular proteins for binding to the transcriptional co-activators CREB-binding protein (CBP) and p300. Through its interactions with CBP/p300 and the retinoblastoma protein, the adenovirus (AdV) early region 1A (E1A) oncoprotein hijacks the cell cycle and, in rodents, transforms the cell; the mechanistic and structural basis for these effects remain unclear. In this study we compare the affinity of protein constructs from the E1A proteins from two adenovirus serotypes, non-oncogenic AdV5 and highly oncogenic AdV12, for binding to the nuclear receptor coactivator binding domain (NCBD) of CBP. NMR spectra show that the E1A constructs from both serotypes are intrinsically disordered in the free state and that each contains three homologous binding sites for the NCBD, one in the N-terminal region and two within conserved region 1 (CR1) of E1A. The binding sites in CR1 correspond to the motifs that bind the retinoblastoma protein and the TAZ2 domain of CBP/p300. The E1A and NCBD peptides fold synergistically upon complex formation. Binding affinities determined from NMR titrations show that, although the overall affinities for AdV5 and AdV12 E1A are comparable, there are significant differences between the two E1A serotypes in the relative strength with which their constituent interaction motifs bind to the NCBD. The individual E1A interaction motifs were unable to compete effectively with p53 for binding to the NCBD and both the N-terminal region and CR1 region of E1A are required for efficient competition with p53.
Insights
Adenovirus E1A proteins bind to CBP/p300 coactivators. Differences in binding affinities between AdV5 and AdV12 E1A serotypes were observed, with both N-terminal and CR1 regions of E1A required for effective p53 competition.
Area of Science:
- Molecular Biology
- Virology
- Structural Biology
Background:
- Viruses deregulate cellular transcription by competing for coactivators like CBP/p300.
- Adenovirus (AdV) early region 1A (E1A) oncoprotein hijacks the cell cycle and transforms cells, but the mechanism is unclear.
- Understanding E1A interactions with CBP/p300 is crucial for elucidating viral oncogenesis.
Purpose of the Study:
- To compare the binding affinity of E1A protein constructs from non-oncogenic AdV5 and highly oncogenic AdV12 to the NCBD of CBP.
- To investigate the structural basis of E1A-CBP interactions and their role in viral oncogenesis.
Main Methods:
- Nuclear Magnetic Resonance (NMR) spectroscopy was used to analyze protein structures and binding.
- NMR titrations were performed to determine binding affinities between E1A constructs and CBP NCBD.
- Comparative analysis of E1A serotypes (AdV5 and AdV12) was conducted.
Main Results:
- E1A constructs from both AdV5 and AdV12 are intrinsically disordered and contain three homologous binding sites for CBP NCBD.
- E1A and NCBD peptides exhibit synergistic folding upon complex formation.
- While overall affinities were comparable, significant differences in the relative binding strengths of individual motifs between AdV5 and AdV12 E1A were observed.
- Individual E1A motifs could not effectively compete with p53 for NCBD binding; both N-terminal and CR1 regions of E1A are necessary for efficient p53 competition.
Conclusions:
- Adenovirus E1A proteins interact with CBP/p300 through specific binding sites within intrinsically disordered regions.
- Differential binding affinities between E1A serotypes suggest a role in oncogenic potential.
- Cooperative binding involving multiple E1A regions is essential for effective competition with cellular proteins like p53, impacting cell cycle control.
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