Changing the spacing between metal-binding motifs decreases stability and transforming activity of the human papillomavirus type 18 E7 oncoprotein.
Spacing composed of 29 amino acids (AAs) between a pair of metal-binding motifs, Cys-X-X-Cys, is common to human papillomavirus (HPV) zinc-binding oncoproteins E6 and E7 from various HPV types. From the HPV 18 E7 gene encoding a 105-AA protein with the motifs at AAs 65-68 and 98-101, we constructed expression plasmids for two mutants, 18del73 and 18ins84, with varied spacing between the motifs. Mutant proteins 18del73 and 18ins84 had a deletion from AAs 74 to 88 and an insertion of three AAs substituting for AA 85, respectively. The mutations lowered the efficiency of E7-mediated focal transformation of rat 3Y1 cells, approximately parallel to the reduced level of steady-state E7 expressed in COS-1 cells. It is likely that, besides the presence of the motifs, the conserved spacing between the motifs is important for a stable structure of E7, but is not essential for the E7-transforming activity.
Watanabe
S
S
Division of Molecular Genetics, National Institute of Health, Tokyo, Japan.
Sato
H
H
Furuno
A
A
Yoshiike
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D002472:Cell Transformation, Viral / Q000502:physiology*; D004268:DNA-Binding Proteins; D006801:Humans; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D009856:Oncogene Proteins, Viral / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D027383:Papillomaviridae / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D051381:Rats
D004268:DNA-Binding Proteins; C052633:E7 protein, Human papillomavirus type 18; D008670:Metals; D009856:Oncogene Proteins, Viral; D011505:Protein-Tyrosine Kinases