Predicted alpha-helix/beta-sheet secondary structures for the zinc-binding motifs of human papillomavirus E7 and E6 proteins by consensus prediction averaging and spectroscopic studies of E7.
The E7 and E6 proteins are the main oncoproteins of human papillomavirus types 16 and 18 (HPV-16 and HPV-18), and possess unknown protein structures. E7 interacts with the cellular tumour-suppressor protein pRB and contains a zinc-binding site with two Cys-Xaa2-Cys motifs spaced 29 or 30 residues apart. E6 interacts with another cellular tumour-suppressor protein p53 and contains two zinc-binding sites, each with two Cys-Xaa2-Cys motifs at a similar spacing of 29 or 30 residues. By using the GOR I/III, Chou-Fasman, SAPIENS and PHD methods, the effectiveness of consensus secondary structure predictions on zinc-finger proteins was first tested with sequences for 160 transcription factors and 72 nuclear hormone receptors. These contain Cys2His2 and Cys2Cys2 zinc-binding regions respectively, and possess known atomic structures. Despite the zinc- and DNA-binding properties of these protein folds, the major alpha-helix structures in both zinc-binding regions were correctly identified. Thus validated, the use of these prediction methods with 47 E7 sequences indicated four well-defined alpha-helix (alpha) and beta-sheet (beta) secondary structure elements in the order beta beta alpha beta in the zinc-binding region of E7 at its C-terminus. The prediction was tested by Fourier transform infrared spectroscopy of recombinant HPV-16 E7 in H2O and 2H2O buffers. Quantitative integration showed that E7 contained similar amounts of alpha-helix and beta-sheet structures, in good agreement with the averaged prediction of alpha-helix and beta-sheet structures in E7 and also with previous circular dichroism studies. Protein fold recognition analyses predicted that the structure of the zinc-binding region in E7 was similar to a beta beta alpha beta motif found in the structure of Protein G. This is consistent with the E7 structure predictions, despite the low sequence similarities with E7. This predicted motif is able to position four Cys residues in proximity to a zinc atom. A model for the zinc-binding motif of E7 was constructed by combining the Protein G coordinates with those for the zinc-binding site in transcription factor TFIIS. Similar analyses for the two zinc-binding motifs in E6 showed that they have different alpha/beta secondary structures from that in E7. When compared with 12 other zinc-binding proteins, these results show that E7 and E6 are predicted to possess novel types of zinc-binding structure.
Ullman
C G
CG
Department of Biochemistry and Molecular Biology, Royal Free Hospital School of Medicine, London, U.K.
Haris
P I
PI
Galloway
D A
DA
Emery
V C
VC
Perkins
S J
SJ
D000595:Amino Acid Sequence; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000737:chemistry* / Q000378:metabolism; D027383:Papillomaviridae / Q000737:chemistry*; D050725:Papillomavirus E7 Proteins; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D012097:Repressor Proteins; D016415:Sequence Alignment; D017550:Spectroscopy, Fourier Transform Infrared; D014157:Transcription Factors / Q000378:metabolism; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; C068651:E6 protein, Human papillomavirus type 16; C052603:E6 protein, Human papillomavirus type 18; C052633:E7 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D012097:Repressor Proteins; D014157:Transcription Factors; C059731:oncogene protein E7, Human papillomavirus type 16; D011505:Protein-Tyrosine Kinases