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Characterization of Ad5 E3–14.7K, an adenoviral inhibitor of apoptosis: Structure, oligomeric state, and metal binding
Author(s) -
Kim HeeJung,
Foster Mark P.
Publication year - 2002
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.4180102
Subject(s) - biology , protein g , fusion protein , apoptosis , microbiology and biotechnology , biochemistry , chemistry , biophysics , recombinant dna , antibody , immunology , gene
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‐α 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 β‐strands and one α‐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.