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Albany 2001

category image Biomolecular
Stereodynamics
SUNY at Albany
June 19-23, 2001

Structure, Dynamics and Thermodynamics of the Mbp1-DNA Interaction

The yeast transcription factor Mbp1 forms a complex with Swi 6 that regulates the cell cycle by activating genes involved in DNA biosynthesis. The 2.1Å resolution X-ray structure of the DNA-binding domain revealed a winged helix-turn-helix motif, but the C-terminal 21 residues essential for tight binding were invisible (1). The solution structure of Mbp1 is very similar to that determined by X-ray, except for the presence of a folded tail that is disorded in the crystal. We have used NMR relaxation methods for probing the internal dynamics of Mbp1 and the Mbp1-DNA complex. 15N and 13C NMR relaxation studies revealed complex dynamics on the ps to ms timescales. In particular the dynamics identified a C-terminal region of the protein invisible in the electron density map that is folded back onto the core of the protein in solution, and another region adjacent to the DNA recognition helix that shows fast intermediate exchange behaviour, with a submillisecond lifetime (2). The DNA ligand seems to select one of these two conformations.

The DNA binding site on Mbp1 has been mapped in solution using chemical shift perturbation and cross-saturation experiments. The experimentally-determined binding surface agrees largely with expectations from structure homology and the calculated electrostatic potential, but NOEs show it to be somewhat more extended. This is consistent with mutation analysis and measured electrostatic contributions to binding (3). The combined use of shift perturbations, cross-relaxation and paramagnetic perturbation to probing the details of DNA binding sites will be illustrated with simulations and data acquired on the Mbp1-DNA complex.

References and Footnotes

  1. Taylor, I.A., Treiber, M.K., Olivi, L. and Smerdon, S.J. (1997) J. Mol. Biol., 272, 1-8.
  2. McIntosh, P.B., Taylor, I.A., Frenkiel, T.A., Smerdon, S.J. & Lane, A.N. (2000) J. Biomolec. NMR 16. 183-196.
  3. Taylor, I.A., McIntosh, P.B, Pala, P., Treiber, M.K., Howell, S., Lane, A.N. & Smerdon, S.J. (2000) Biochemistry 39, 3943-3954.

Andrew N. Lane (1), Pauline B. McIntosh (1,2), Thomas A. Frenkiel (1), Ian Taylor (1,3), Teresa W-M. Fan (4) and Stephen J. Smerdon (1)

(1)Nat. Inst,/Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK (2)CUNY Mt Sinai Sch Med, New York, NY 10029 USA (3)Dep't./Biophysics, Univ./Oxford, (4)Univ./California, One Shields Avenue, Davis, CA 95616