2.1 A crystal structure and functional analysis of the human papillomavirus type 18 E2 activation domain

Integration of the normally extra-chromosomal genome of the high-risk variants of the human papillomavirus, including type 18, is associated with cervical cancer and other intraepithelial neoplasias, particularly when these events disrupt the viral E1 or E2 genes. The E2 protein regulates transcription, replication, and viral genome segregation through specific interactions with other cellular and viral proteins. E2's ∼200 amino acid activation domain, which represents a protein class whose structural themes have remained poorly understood, contains key residues that mediate these important contacts. I describe a series of experiments that lead from identification and functional characterization of protease-resistant cores of the activation domain, through preliminary structural analysis, and ultimately to the solution of the 2.1 A crystal structure of amino acids 66 to 215 of the human papillomavirus, type 18 E2 activation domain. I observe a novel fold creating a cashew-shaped form with a 17 residue amphipathic alpha-helix packed against a beta-sheet framework. Structural and functional analyses of the protein surface show extensive overlap of the replication and transcription determinants. Further mutagenesis extends our understanding of the importance of exposed residues on the initial helix's outer face in activation of transcription. Bolstered by these biological data, the structure broadens the concept of activators to include proteins with potentially malleable but certainly ordered structures. The protein's architecture also provides a unique tool for exploring E2's intermolecular interactions with such proteins as the viral replicative helicase E1, how to impact the virus' clinical manifestations, and for gaining mechanistic insight into the fundamental processes of transcription and replication.