Supplementary Material
The three-dimensional structure of the
intact transactivation domain of the human
papillomavirus E2 protein
†
†‡
Alfred A. Antson* , Julie E. Burns , Olga V. Moroz*, David J. Scott¶*, Cyril M.
‡§
Sanders , Igor B. Bronstein*, G. Guy Dodson*^, Keith S. Wilson* and Norman J.
Maitland
*
‡
York Structural Biology Laboratory, Department of Chemistry, University of York,
York YO10 5DD, UK
‡
YCR Cancer Research Unit, Department of Biology, University of York, York YO10
5DD, UK
¶
Department of Biology, University of York, York YO10 5DD, UK
^ NIMR, London NW7 1AA, UK
§
Current address: Cold Spring Harbor Laboratory, New York, USA.
†
These authors contributed equally to this work
Supplementary Material
Figure 1
Sequence alignment of the E2NT modules from a subset of HPV types (HPV16, HPV18,
HPV11 and HPV2a) and one BPV type. Shaded blocks above the alignment indicate the
experimentally determined secondary structure. Shaded blocks below the sequences indicate
the minimal peptide sequences involved in protein:protein interactions, suggested by mutation
studies2,6,7. Residues with more than 90% identity among 86 PV types are coloured: internal
structural residues - red, residues within the fulcrum region - green, surface residues - blue.
Figure 2
Experiments were carried out in an Optima XL-A ultracentrifuge (Beckman-Coultier, CA,
USA) using scanning UV optics. Three concentrations of recombinant E2NT were loaded into
cells containing 12 mm pathlength six channel Epon centrepieces with quartz windows. The
solvent was 10 mM TrisHCl pH 8.0, 5 mM DTT, 0.2 mM EDTA, 300 mM NaCl. The solvent
density and partial specific volume of the protein were calculated as 1.01128 and 0.7284,
respectively, using the program SEDNTERP, according to the method of Laue et al.1. Data
were obtained at rotor speeds of 12,000 and 16,000 rpm, and the time to equilibrium was
typically 10-12 hours. All runs were carried out at 293 K, and all radial scans were at a
wavelength of 280 nm. The dissociation constant was obtained by nonlinear regression using
the Beckman ultracentrifuge software, and the variance of the fit was found to be 1.98 with
988 degrees of freedom.
Sedimentation equilibrium trace of E2 dimers at 22 000 rpm (black circles). The trace has
2
been deconvoluted into monomer (grey squares) and dimer (grey triangles) fractions using the
 analysis method2. The optical dissociation constant across the cell was calculated from the
individual absorbance profiles using the expression:
Kd=(Atotal-Adimer)2/Adimer.
The true dissociation constant was calculated using a molar extinction coefficient of 45 840
M-1 and a cell pathlength of 1.2cm. As can be seen the dissociation constant (Black diamonds)
is single valued across the extent of the cell, and lies within the value determined by global
analysis of all the equilibrium traces.
Reference
1.
Laue, T.M., Shah, B.D., Ridgeway, T.M. & Pelletier, S.L. Computer-aided interpretation
of analytical sedimentation data for proteins. In Analytical ultracentrifugation in
Biochemistry and Polymer Science (Harding, S.E., Rowe, A.J. & Horton, J.C., eds)
Royal Society of Chemistry, UK. (1992) pp 90-125.
2.
Winzor, D.J., Jacobsen, M.P. & Wills, P.R. Direct analysis of sedimentation equilibrium
distributions reflecting complex formation between dissimilar reactants. Biochem. 37,
2226-2233 (1998).
3