Characterization of Ad5 E3-14.7K, an adenoviral inhibitor of apoptosis: structure, oligomeric state, and metal binding.
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-alpha 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 beta-strands and one alpha-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.
Kim
Hee-Jung
HJ
Biophysics Program, The Ohio State University, 484 W 12th Avenue, Columbus, OH 43210, USA.
Foster
Mark P
MP
D000256:Adenoviridae / Q000378:metabolism*; D017503:Adenovirus E3 Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D017209:Apoptosis / Q000502:physiology*; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D004926:Escherichia coli; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D015032:Zinc / Q000378:metabolism*; D039221:mRNA Cleavage and Polyadenylation Factors
D017503:Adenovirus E3 Proteins; D004676:Myelin Basic Protein; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D039221:mRNA Cleavage and Polyadenylation Factors; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D015032:Zinc