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Characterization of Ad5 E3–14.7K, an adenoviral inhibitor of apoptosis: Structure, oligomeric state, and metal binding

¹ Biophysics Program, The Ohio State University, Columbus, Ohio 43210, USA
² Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, USA
³ Protein Research Group, The Ohio State University, Columbus, Ohio 43210, USA
⁴ Department of Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA

Reprint requests to: Mark P. Foster, Department of Biochemistry, The Ohio State University, 484 W. 12th Ave., Columbus, OH 43210, USA; e-mail: foster.281{at}osu.edu; fax: (614) 292-6773.

The adenovirus E3–14.7K protein, expressed early in the life cycle of human adenoviruses to protect the virus from the antiviral response of host cells, inhibits cell death mediated by TNF- and FasL receptors. To better understand its role in cell death inhibition, we have sought to characterize the biophysical properties of the protein from adenovirus serotype 5 (Ad5 E3–14.7K, or simply 14.7K) through a variety of approaches. To obtain sufficient quantities of recombinantly expressed protein for biophysical characterization, we explored the use of various expression constructs and chaperones; fusion to MBP was by far the most effective at generating soluble protein. Using limited proteolysis, mass spectrometry, and protein-protein interaction assays, we demonstrate that the C-terminal two-thirds of the protein, predicted to be composed of five ß-strands and one -helix, is highly structured and binds its putative cellular receptors. Furthermore, using atomic absorption and ultraviolet/visible spectroscopies, we have studied the metal binding properties of the protein, providing insight into the observation that cysteine/serine mutants of 14.7K lack in vivo antiapoptotic activity. Lastly, results from size exclusion chromatography, dynamic light scattering, sucrose gradient sedimentation, chemical crosslinking, and electron microscopy experiments revealed that 14.7K exists in a stable high-order oligomeric state (nonamer) in solution.

Keywords: Ad5 E3; 14.7K; adenovirus; apoptosis; oligomerization; metal binding; proteolysis

Abbreviations: 14.7K, Ad5 E3-14.7K, a protein encoded in the E3 early region of the genome of adenovirus serotype 5 (gi|58510) • FIPs (1-3), 14.7K-interacting proteins • FLICE/caspase 8, a cysteine protease in the interleukin-1ß • converting enzyme family • DED, death-effector domain • MBP, maltose-binding protein • GST, glutathione-S-transferase • TNF, tumor necrosis factor • ESI-MS, electrospray mass spectrometry • CD, circular dichroism • LB, Luria-Bertani • DTT, dithiothreitol • DMS, dimethyl suberimidate • AEBSF, 4 (2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride

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