Purification and biophysical characterization of a minimal functional domain and of an N-terminal Zn2+-binding fragment from the human papillomavirus type 16 E6 protein.
The E6 Zn(2+)-binding protein of high-risk human papillomaviruses (HPVs) is one of the major transforming proteins encoded by these tumor viruses. A bacterial system was used to express wild type and truncated forms of HPV-16 E6 linked to GST. The recombinant proteins were released from GST through cleavage of a factor Xa site. Functional analysis of these proteins demonstrated that amino acids 2--142 comprise the minimal domain of E6 required to promote the degradation of p53 in vitro in a rabbit reticulocyte lysate. This purified protein, E6(Delta 143--151), required a high salt concentration for maximum solubility, eluted as a monomer on gel filtration, and was shown to bind two Zn(2+) ions by atomic absorption analysis. An N-terminal subdomain of E6 (amino acids 2--77, E6-N) was similarly purified. Unlike E6(Delta 143--151), E6--N was very soluble in low-salt buffers and hence was highly amenable to biophysical characterization. E6-N was shown to bind one Zn(2+) ion by electrospray mass spectrometry and by atomic absorption analysis. UV--visible spectroscopic analysis of Co(2+)-substituted E6--N revealed that four cysteine residues coordinate the metal ion. Mutational studies of all the cysteine residues in E6--N substantiated a critical role for Cys 30, 33, 63, and 66 in Zn(2+) binding and in proper folding of the subdomain. Equilibrium sedimentation of E6-N demonstrated that it is a monomer, like E6(Delta 143--151), at low concentrations, but dimerization occurs at high concentrations (K(d) = 0.1 mM). Finally, circular dichroism studies revealed significant secondary structure for both E6(Delta 143--151) and E6--N. The results support a model of monomeric E6 possessing two functionally critical Zn(2+)-binding motifs.
Lipari
F
F
Department of Biological Sciences, Boehringer Ingelheim (Canada) Ltd., 2100 Cunard Street, Laval, Quebec, Canada H7S 2G5.
McGibbon
G A
GA
Wardrop
E
E
Cordingley
M G
MG
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D027383:Papillomaviridae / Q000737:chemistry* / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011485:Protein Binding / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012097:Repressor Proteins; D017384:Sequence Deletion; D012995:Solubility; D015032:Zinc / Q000737:chemistry*
D002352:Carrier Proteins; C068651:E6 protein, Human papillomavirus type 16; D008024:Ligands; D009856:Oncogene Proteins, Viral; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C017800:zinc-binding protein; D015032:Zinc; D003545:Cysteine