title: Chelating agents stabilize the monomeric state of the zinc binding human papillomavirus 16 E6 oncoprotein.
abstract: 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(beta-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.
authors.affiliation: Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore. mcbrd@imcb.nus.edu.sg
authors.forename: Roland
authors.initials: R
authors.lastname: Degenkolbe
authors.forename: Patrick
authors.initials: P
authors.lastname: Gilligan
authors.forename: Sanjay
authors.initials: S
authors.lastname: Gupta
authors.forename: Hans U
authors.initials: HU
authors.lastname: Bernard
mesh_terms: D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D019281:Dimerization; D004533:Egtazic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008024:Ligands; D009856:Oncogene Proteins, Viral / Q000302:isolation & purification / Q000378:metabolism*; D027383:Papillomaviridae / Q000502:physiology*; D010396:Penicillamine / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D012995:Solubility; D015032:Zinc / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D002614:Chelating Agents; C068651:E6 protein, Human papillomavirus type 16; D008024:Ligands; D009856:Oncogene Proteins, Viral; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C017800:zinc-binding protein; D004533:Egtazic Acid; D010396:Penicillamine; D015032:Zinc