Chelating Agents Stabilize the Monomeric State of the Zinc Binding Human Papillomavirus 16 E6 Oncoprotein

The E6 protein of human papillomavirus 16 is known to be difficult and, when overexpressed, insoluble and agglomerated. It has two putative zinc ion binding sites crucial for its function. No metallochaperone has yet been found to deliver zinc ions to the E6 protein. Here, we report that a specific...

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Bibliographic Details
Published in:Biochemistry (Easton) 2003-04, Vol.42 (13), p.3868-3873
Main Authors: Degenkolbe, Roland, Gilligan, Patrick, Gupta, Sanjay, Bernard, Hans U
Format: Article
Language:English
Subjects:
Binding Sites
Carrier Proteins - metabolism
Chelating Agents - pharmacology
Dimerization
Egtazic Acid - pharmacology
Escherichia coli - genetics
Humans
Ligands
Oncogene Proteins, Viral - isolation & purification
Oncogene Proteins, Viral - metabolism
Papillomaviridae - physiology
Penicillamine - pharmacology
Protein Binding - drug effects
Recombinant Fusion Proteins
Repressor Proteins
Solubility
Zinc - metabolism
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Summary:The E6 protein of human papillomavirus 16 is known to be difficult and, when overexpressed, insoluble and agglomerated. It has two putative zinc ion binding sites crucial for its function. No metallochaperone has yet been found to deliver zinc ions to the E6 protein. Here, we report that a specific chelating agent, which we think functionally mimics a metallochaperone, stabilized the soluble monomeric form of E6 and inhibited multimerization in vitro. This effect seemed to depend on the chelating strength of the agent. While strong chelating agents precipitated the E6 protein and weak chelating agents did not favor the monomeric form of E6, chelating agents of intermediate strength [l-penicillamine and ethylene glycol bis(β-aminoethyl)-N,N,N‘,N‘-tetraacetic acid (EGTA)] effectively support the formation of a monomer. We did not observe formation of a dimer or defined oligomers. Degradation assays imply that the monomer is the biologically active form of the protein. Since EGTA favors the formation of monomeric over agglomerated E6 protein, we propose that chelating agents of appropriate strength could assist zinc delivery to recombinant metalloproteins in vitro and may even destabilize existing agglomerates.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi027390h