Determination of the binding affinity of different human papillomavirus E7proteins for the tumour suppressor pRb by a plate-binding assay

A plate-binding assay was developed to quantify the affinity of the E7 onco protein from different human papillomavirus (HPV) types for the tumour supp ressor pRb. The method is highly reproducible. sensitive and easy to handle . It could be easily adapted for the quantitative study of other interactin g proteins and for screenings of inhibitors of protein/protein interactions . The pRb-binding affinity of six different E7 proteins has been quantified . The KD values vary from approximate to 4.5 x 10(-9) M for HPV 16 E7 to mo re than 1 x 10(-7) M for HPV10 and HPV48 E7. Point mutation C24G in the hig h affinity pRb-binding domain of HPV16 E7 results in a 3-fold affinity redu ction. The data indicate that the high affinity pRb-binding domain of E7, L XCXE, is essential for the association between the viral and cellular prote ins. However, other E7 domain(s), which appear(s) not to be present in all E7s, contribute to stabilize the E7-pRb association. (C) 2001 Elsevier Scie nce B.V. All rights reserved.