The E7 oncoprotein from human Papillomavirus (HPV) mediates cell transformation in part by binding to the human pRb tumor suppressor protein and E2F transcription factors, resulting in the dissociation of pRb from E2F transcription factors and the premature cell progression into the S-phase of the cell cycle. This activity is mediated by the LXCXE motif and the CR3 zinc binding domain of the E7 protein. In this study we report the x-ray crystal structure of the CR3 region of HPV E7 and a

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... re-based mutational analysis to investigate its mode of pRb and E2F binding and E2F displacement from pRb. The structure reveals a novel zinc-bound E7-CR3 obligate homodimer that contains two surface patches of sequence conservation. Mutation of residues within these patches reveals that one patch is required for pRb binding, whereas the other is required for E2F binding. We also show that both E7-mediated interactions are required to disrupt pRb⅐E2F complexes. Based on these studies we present a mechanistic model for how E7 displaces E2F from pRb. Because the CR3 region of HPV E7 has no detectable homology to other human proteins, the structure-function studies presented here provide an avenue for developing small molecule compounds that inhibit HPV-E7-mediated cell transformation. Human papillomaviruses (HPVs) 2 are the causative agents for more than 90% of cervical cancers (1). More than 200 HPV genotypes have been identified based on genomic differences (2), and these genotypes fall into low risk (for example types 1A, 6B, and 11) and high risk (for example, types 5, 8, 16, 18, 31, and 33) forms that are correlated with benign and malignant lesions, respectively (3) (supplemental Fig. 1 ). Host cell transformation by HPV is mediated by two viral oncogenes, E6 and E7 (4 -6). E6 proteins contain four CXXC motifs that presumably form two zinc binding regions (3, 7-9), and E6 proteins of high risk type 16 and 18 cooperate with the host cellular factor E6-AP (E6-associated . 2 The abbreviations used are: HPV, human Papillomavirus; CBP, cAMP-response elementbinding protein (CREB)-binding protein; Ches, 2-(cyclohexylamino)ethanesulfonic acid; GST, glutathione S-transferase; MAD, multiple anomalous diffraction; P/CAF, p300/CBP-associated factor.