CRYSTALLISATION OF HPV-16 E2 DNA-BINDING DOMAIN APO- AND CO-CRYSTALS. Stephanie Roberts, Hilary Muirhead, Tony Clarke. Department of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK

The DNA binding domain of the viral transcription factor E2 has been crystallised in the presence and absence of its cognate DNA. It is also hoped to co-crystallise with non-cognate DNA molecules. HPV-16 E2 requires four AT/TA base parts at the centre of its 12 bp binding site. These are uncontacted but provide flexibility required for binding. Thus four central AT/TA base pairs in an otherwise unrelated oligonucleotide may provide a preferred non-cognate binding site, generating homogenous complexes for crystallisation.

Conditions for co-crystals have not been optimised due to problems with DNA supply, but a dataset for the apocrystals has been obtained to 2.1 angstroms. Data were obtained from a single crystal, frozen to low temperature. The space group is P3₁2 or P3₂1, with unit cell dimensions 44.19 x 44.19 x 76.86 and angles 90 90 120. There are 6 monomers in the unit cell, assuming a 38.4% solvent content. They are presumed dimeric, with a natural two fold symmetry. Initial analysis suggests the structure is too distinct from the published DNA-bound structure of the bovine papillomavirus E2 domain to solve by molecular replacement. Apocrystals were obtained form 3 - 8 mg/ml protein precipitated with 1.50-1.65M ammonium sulphate in 8 ul drops, at an approximate final pH of 8.2. Final concentrations of other solutes in drops was 32.5mM TRIS, 100mM sodium chloride, 0.6mM calcium chloride, 1.6mM potassium chloride. Co-crystals were obtained from 3mg/ml complex in the same salts, precipitated with 28% PEG 3500 at an approximate final pH of 7.5. The current co-crystals are too small for analysis.