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 papillomavir us types 16 and 18 (HPV-16 and HPV-18), and possess unknown protein st ructures. E7 interacts with the cellular tumour-suppressor protein pRB and contains a zinc-binding site with two Cys-Xaa(2)-Cys motifs space d 29 or 30 residues apart. E6 interacts with another cellular tumour-s uppressor protein p53 and contains two zinc-binding sites, each with t wo Cys-Xaa(2)-Cys motifs at a similar spacing of 29 or 30 residues. By using the GOR I/III, Chou-Fasman, SAPIENS and PHD methods, the effect iveness of consensus secondary structure predictions on zinc-finger pr oteins was first tested with sequences for 160 transcription factors a nd 72 nuclear hormone receptors. These contain Cys(2)His(2) and Cys(2) Cys(2) zinc-binding regions respectively, and possess known atomic str uctures. 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 orde r beta beta alpha beta in the zinc-binding region of E7 at its C-termi nus. The prediction was tested by Fourier transform infrared spectrosc opy of recombinant HPV-16 E7 in H2O and (H2O)-H-2 buffers. Quantitativ e integration showed that E7 contained similar amounts of alpha-helix and beta-sheet structures, in good agreement with the averaged predict ion of alpha-helix and beta-sheet structures in E7 and also with previ ous circular dichroism studies. Protein fold recognition analyses pred icted 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, T his is consistent with the E7 structure predictions, despite the low s equence similarities with E7, This predicted motif is able to position four Cys residues in proximity to a zinc atom. A model for the zinc-b inding motif of E7 was constructed by combining the Protein G coordina tes with those for the zinc-binding site in transcription factor TFIIS . Similar analyses for the two zinc-binding motifs in E6 showed that t hey have different alpha/beta secondary structures from that in E7. Wh en compared with 12 other zinc-binding proteins, these results show th at E7 and E6 are predicted to possess novel types of zinc-binding stru cture.