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. 2009 Aug 11;106(32):13260-5.
doi: 10.1073/pnas.0906770106. Epub 2009 Jul 27.

Structural basis for subversion of cellular control mechanisms by the adenoviral E1A oncoprotein

Josephine C Ferreon  1 , Maria A Martinez-Yamout, H Jane Dyson, Peter E Wright
Affiliations

Structural basis for subversion of cellular control mechanisms by the adenoviral E1A oncoprotein

Josephine C Ferreon et al. Proc Natl Acad Sci U S A. .

Abstract

The adenovirus early region 1A (E1A) oncoprotein mediates cell transformation by deregulating host cellular processes and activating viral gene expression by recruitment of cellular proteins that include cyclic-AMP response element binding (CREB) binding protein (CBP)/p300 and the retinoblastoma protein (pRb). While E1A is capable of independent interaction with CBP/p300 or pRb, simultaneous binding of both proteins is required for maximal biological activity. To obtain insights into the mechanism by which E1A hijacks the cellular transcription machinery by competing with essential transcription factors for binding to CBP/p300, we have determined the structure of the complex between the transcriptional adaptor zinc finger-2 (TAZ2) domain of CBP and the conserved region-1 (CR1) domain of E1A. The E1A CR1 domain is unstructured in the free state and upon binding folds into a local helical structure mediated by an extensive network of intermolecular hydrophobic contacts. By NMR titrations, we show that E1A efficiently competes with the N-terminal transactivation domain of p53 for binding to TAZ2 and that pRb interacts with E1A at 2 independent sites located in CR1 and CR2. We show that pRb and the CBP TAZ2 domain can bind simultaneously to the CR1 site of E1A to form a ternary complex and propose a structural model for the pRb:E1A:CBP complex on the basis of published x-ray data for homologous binary complexes. These observations reveal the molecular basis by which E1A inhibits p53-mediated transcriptional activation and provide a rationale for the efficiency of cellular transformation by the adenoviral E1A oncoprotein.

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Conflict of interest statement

Conflict of interest: The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Domain organization in E1A and CBP. (A) Schematic diagram of E1A showing the 4 conserved regions (CR1–CR4). The sequences (residues 1–139) of representative E1A adenoviral serotypes are shown; conserved residues are indicated by colored shading. (B) Schematic diagram of CBP/p300 showing domain organization.
Fig. 2.
Fig. 2.
1H-15N HSQC spectra of the TAZ2:E1A complex. (A) HSQC spectra of 15N TAZ2 free (black) and bound to equimolar E1A(53–91) (red). (B) HSQC spectra of 15N E1A(53–91) free (black) and bound to equimolar TAZ2 (red). Corresponding cross peaks from the free and bound forms are connected. Asterisks denote cross peaks arising from E1A containing cis peptide bonds.
Fig. 3.
Fig. 3.
NMR structure of the TAZ2:E1A complex. (A) Ensemble of 20 lowest energy structures. The TAZ2 backbone is shown in blue, E1A is in coral, and the N and C termini of each protein are labeled. (B) Ribbon representation of the lowest-energy structure, colored as in A. Zinc atoms are shown as gray spheres. Helices α1–α4 of TAZ2 are labeled. (C) Electrostatic potential mapped to the surface of TAZ2 showing basic residues in blue and acidic residues in red. The orientation is the same as in B. The backbone of the bound E1A is represented as a coral ribbon. (D) Close-up view showing E1A hydrophobic side chains (green, yellow for M71) that form the intermolecular interface. The surface formed by helices α1, α2, and α3 of TAZ2 is shown, together with a tube representation of the E1A backbone. (E) Electrostatic interactions in the TAZ2-E1A interface. The backbone of the bound E1A is represented as a coral ribbon, superimposed on the TAZ2 surface. Asp and Glu side chains of E1A are colored red, and Asn, Gln, and Ser side chains are shown in beige. The cluster of hydrophobic side chains exposed on the E1A surface is shown in green. The nitrogen atoms of Arg and Lys side chains of TAZ2 are colored blue. (F) Superposition of the complexes of TAZ2 (represented as a surface) with E1A (coral ribbon) and the STAT1 TAD (green ribbon). This figure and Figs. 4B and 5C were drawn using MOLMOL (52).
Fig. 4.
Fig. 4.
E1A and the p53 transactivation domain compete for binding to TAZ2. (A) Region of the HSQC spectrum of 15N-labeled TAZ2 free (black); following addition of p53(13–61) at molar ratios of 1:0.25 (blue), 1:0.5 (orange), and 1:1 (red); in the presence of p53(13–61) and E1A(53–91) at 1:1:1 mol ratio (green); and in the 1:1 TAZ2:E1A binary complex (purple). The shifts caused by p53 binding are highlighted by red arrows, and the shifts caused by addition of E1A to the p53:TAZ2 complex are indicated by green arrows. The full HSQC spectra are shown in Fig. S3A. (B) Surface of TAZ2 colored according to the weighted average amide chemical shift difference (Δδ(N, H)av) between HSQC spectra of the free 15N-labeled TAZ2 domain and the 1:1 complex with p53(13–61). Residues with Δδ(N, H)av > 0.2 ppm are colored red, those with Δδ(N, H)av > 0.1 ppm are colored orange, and residues with Δδ(N, H)av < 0.1 ppm are white. The full histograms are shown in Fig. S3B. E1A is shown as a cyan ribbon.
Fig. 5.
Fig. 5.
Ternary complex formation by CBP, E1A, and pRb. (A) Region of the HSQC spectrum of 15N-labeled E1A(27–91) free (black), in the binary complex with TAZ2 (1:1.5 mol ratio, red), and in the ternary complex with both TAZ2 and pRb (1:1.5:3 mol ratio, green). (B) Regions of the same spectrum showing the cross peak of A53. As a reference, the spectrum of the binary E1A:pRb complex is superimposed in blue. Although the green and red cross peaks become broader due to formation of a high molecular weight complex, it is clear that pRb does not displace TAZ2 and vice versa. The full HSQC spectra are shown in Fig. S5. (C) Structural model of the ternary pRb-E1A-TAZ2 complex. The model was generated using the crystal structure of the complex of pRb with E1A (CR1, residues 37–49) (PDB entry 2R7G) (17), the NMR structure of the TAZ2-E1A(53–91) complex determined in this work, and the crystal structure of the HPV E7 peptide (DLYCYEQLN, homologous to residues 121–129 of E1A) (in CR2) containing the LXCXE motif that interacts with pRb (PDB entry 1GUX) (15). The flexible linker between residues 83 and 120 of E1A is indicated schematically as a dotted line. The backbone structures of pRb, E1A, and TAZ2 are represented as ribbons and are colored gray, coral, and blue, respectively.
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