Metal thiolate coordination in the E7 proteins of human papilloma virus 16 and cottontail rabbit papilloma virus as expressed in Escherichia coli.
The oncogenic E7 proteins of human papilloma virus (HPV 16) and of cottontail rabbit papilloma virus (CRPV) have been purified from an expression system in Escherichia coli. The proteins as purified from E. coli contain one tightly bound Zn(II) ion per molecule. The metal site shows facile exchange with either Cd(II) or Cu(I). The HPV 16 E7 maximally bound one Cd(II) or two Cu(I) ions, while the CRPV E7 bound two Cd(II) or three Cu(I) ions. The Cd(II) and Cu(I) E7 molecules exhibited optical transitions in the ultraviolet suggestive of metal:thiolate coordination. E7 proteins from HPV 16 and CRPV contain 7 and 8 cysteines/molecule, respectively. Reaction of the E7 proteins with the sulfhydryl reagent, dithiodipyridine, revealed that all the cysteinyl sulfurs are present in the reduced thiol state. Cu(I)-E7 molecules are luminescent with maximal emission at 570 nm. The observed emission at room temperature is indicative of metal coordination within a compact protein environment shielded from solvent interactions. The emission maxima occurs at the same wavelength (570 nm) as Cu(I)-cysteinyl sulfur clusters in Cu(I)-metallothioneins. The single Zn(II) atom in each protein can be removed from E7 in the presence of EDTA. The resulting apoE7 molecules remain soluble and can be partially reconstituted with Cd(II) to regain the ultraviolet charge transfer transitions.
Roth
E J
EJ
Department of Obstetrics and Gynecology, University of Utah, Salt Lake City 84132.
Kurz
B
B
Liang
L
L
Hansen
C L
CL
Dameron
C T
CT
Winge
D R
DR
Smotkin
D
D
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism; D003001:Cloning, Molecular; D003300:Copper / Q000378:metabolism; D003545:Cysteine; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D027383:Papillomaviridae / Q000378:metabolism*; D050725:Papillomavirus E7 Proteins; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D015678:Viral Structural Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015032:Zinc / Q000032:analysis / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D011994:Recombinant Proteins; D015678:Viral Structural Proteins; C059731:oncogene protein E7, Human papillomavirus type 16; D002104:Cadmium; D003300:Copper; D015032:Zinc; D003545:Cysteine