Determination of the binding affinity of different human papillomavirus E7 proteins for the tumour suppressor pRb by a plate-binding assay.
A plate-binding assay was developed to quantify the affinity of the E7 oncoprotein from different human papillomavirus (HPV) types for the tumour suppressor pRb. The method is highly reproducible, sensitive and easy to handle. It could be easily adapted for the quantitative study of other interacting proteins and for screenings of inhibitors of protein/protein interactions. The pRb-binding affinity of six different E7 proteins has been quantified. The K(D) values vary from approximately 4.5x10(-9) M for HPV16 E7 to more than 1x10(-7) M for HPV10 and HPV48 E7. Point mutation C24G in the high affinity pRb-binding domain of HPV16 E7 results in a 3-fold affinity reduction. The data indicate that the high affinity pRb-binding domain of E7, LXCXE, is essential for the association between the viral and cellular proteins. However, other E7 domain(s), which appear(s) not to be present in all E7s, contribute to stabilize the E7-pRb association.
Dong
W L
WL
Angewandte Tumorvirologie, Abt. F0200, Deutsches Krebsforschungszentrum, INF 242, D-69120, Heidelberg, Germany.
Caldeira
S
S
Sehr
P
P
Pawlita
M
M
Tommasino
M
M
D006801:Humans; D007118:Immunoassay / Q000379:methods; D007700:Kinetics; D009856:Oncogene Proteins, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D027383:Papillomaviridae / Q000378:metabolism*; D050725:Papillomavirus E7 Proteins; D010455:Peptides / Q000138:chemical synthesis; D017354:Point Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D016160:Retinoblastoma Protein / Q000737:chemistry / Q000378:metabolism*
D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D010455:Peptides; D011994:Recombinant Proteins; D016160:Retinoblastoma Protein; C059731:oncogene protein E7, Human papillomavirus type 16