Conformation of the c-Fos / c-Jun complex in vivo: a combined FRET, FCCS and MD-modeling study
György Vámosi 1, Nina Baudendistel 2, Claus Wilhelm von der Lieth 3, Nikoletta Szalóki 1, Gábor Mocsár 1, Gabriele Müller 2, Péter Brázda 4, Waldemar Waldeck 2, Sándor Damjanovich 1, Jörg Langowski 2 and Katalin Tóth 2*
1 University of Debrecen, Cell Biology and Signaling Research Group, Debrecen, Hungary
2 German Cancer Research Center, Div. Biophysics of Macromolecules, Heidelberg, Germany
3 German Cancer Research Center, Spectroscopic Department, Molecular Modeling, Heidelberg, Germany
4 University of Debrecen, Department of Biochemistry and Molecular Biology, Debrecen, Hungary
* To whom correspondence should be addressed. E-mail: kt{at}dkfz.de.
Submitted on September 9, 2007
Revised on October 17, 2007
Accepted on 20 November 2007
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Abstract |
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The activator protein 1 (AP-1) transcription factor is a heterodimer containing one of each of the Fos and Jun subfamilies of basic-region leucine-zipper (bZIP) proteins. We have previously shown by fluorescence crosscorrelation spectroscopy (FCCS) that the fluorescent fusion proteins Fos-EGFP and Jun-mRFP1, co-transfected in HeLa cells, formed stable complexes in situ. Here we studied in addition the relative position of the C terminal domains via FRET measured by flow cytometry and confocal microscopy. To get a more detailed insight into the conformation of the C terminal domains of the complex we constructed C terminal labeled full length and truncated forms of Fos. We developed a novel iterative evaluation method to determine accurate FRET efficiencies regardless of relative protein expression levels, using a spectral or intensity based approach. The full length C terminal labeled Jun and Fos proteins displayed a FRET-measured average distance of 8±1 nm. Deletion of the last 164 AA at the C terminus of Fos resulted in a distance of 6.1±1 nm between the labels. FCCS shows that Jun-mRFP1 and the truncated Fos-EGFP also interact stably in the nucleus, albeit bind to nuclear components with lower affinity. Thus, the C terminal end of Fos may play a role in the stabilization of the interaction between AP-1 and DNA. Molecular dynamic simulations predict a dye-to-dye distance of 6.6±0.1 nm for the dimer between Jun-mRFP1 and the truncated Fos-EGFP, in good agreement with our FRET data. A wide variety of models could be developed for the full length dimer, with possible dye-to-dye distances varying largely between 6 and 20 nm. However, from our FRET results we can conclude that more than half of the occurring dye-to-dye distances are between 6 and 10 nm.
Key Words:
AP-1, EGFP, mRFP1, in vivo protein-protein interaction, transcription factor
