1.5 X-ray structural analysis of E2 protein from human papillomavirus

Authors: Fred Antson, Olga Moroz, David Scott, Igor Bronstein, Guy Dodson and Keith Wilson

Papillomaviruses cause wart lesions of normal epithelium cells; several types of human papillomavirus (HPV), including HPV types 16, 18, 31, 33, 45 and 56, could cause further transformation of the wart lesions producing tumours [ref. 1]. HPVs have evolved a sophisticated system of control, mediated by protein:DNA and protein:protein interactions, that involves both host cell and viral proteins. The papillomavirus E2 protein has two central roles in this control: it acts as the principal virally encoded transcription factor and, in association with the viral E1 protein, it creates the molecular complex at the origin of the viral DNA replication [ref. 2].  E2 has three distinct modules, Figure 1. The N-terminal module (E2NT) of about 200 amino acids is responsible for interactions with viral and host cell transcription factors. It is followed by a flexible, proline-rich, linker module and a C-terminal module (E2CT), each of about 100 amino acids. Both the primary transcription and replication activities of the E2 are governed by the E2NT module.  Recently we have determined the crystal structure for the E2NT module from high-risk type 16 HPV [ref. 3]. We found that the E2NT module forms a dimer with several conserved residues implicated in transactivation being buried in the dimer interface. This suggested a novel hypothesis, namely that these residues are important for dimer formation rather than for direct interaction with other protein factors and that the dimer formation could be crucial for HPV transactivation. To test if the crystallographically observed dimer forms in solution, we measured the dissociation constant by analytical ultracentrifugation. The Kd of 8x10-6 M indicated medium-strength association which is physiologically significant.  We further proposed that the viral transactivation is driven by formation of DNA loops, the process which brings distally bound transcription factor to the site of transcription initiation. These loops were previously observed by electron microscopy, when intact E2 molecules were added to DNA templates with multiple E2 binding sites [ref. 4]. It was also known that E2CT dimers interact strongly with their cognate DNA binding sites. In our model, E2NT modules from distantly bound E2CT dimers cross-interact with each other through formation of dimers observed in the crystal structure [ref.3]; this inter-dimer interaction could stabilise the DNA loops, Figure 2.  Funding: BBSRC and YCR Collaborators: Dr Julie Burns and Professor Norman Maitland, Biology Department, University of York References: 1. zur Hausen, H. Molecular pathogenesis of cancer of the cervix and its causation by specific human papillomavirus types. Curr. Top. Microbiol. Immunol. 186, 131-156 (1994).  2. McBride, A. & Myers, G. The E2 proteins. In Human Papillomaviruses 1997 (eds Myers, G., Sverdrup, F., Baker, C., McBride, A., Münger, K. &     Bernard, H.-U.) III-54-III-73 (Theoretical Biology and Biophysics, Los Alamos, 1997).  3. Antson AA, Burns JE, Moroz OV, Scott DJ, Sanders CM, Bronstein IB, Dodson GG, Wilson KS, Maitland NJ: Structure of the intact transactivation domain of the human papillomavirus E2 protein. (2000) Nature, 403: 805-809.  4. Knight, J. D., Li, R. & Botchan, M. The activation domain of the bovine papillomavirus E2 protein mediates association of DNA-bound dimers to form DNA loops. Proc. Natl. Acad. Sci. USA 88, 3204-3208 (1991).  5.Hegde, R. S., Grossman, S. R., Laimins, L. A. & Sigler, P. B. Crystal structure at 1.7 Å of the bovine papillomavirus-1 E2 DNA-binding domain bound to its DNA target. Nature 359, 505-512 (1992).

Click on an image to enlarge Figure 1.5.1 Figure 1.5.2