Thermodynamics of Protein−Protein Interactions of cMyc, Max, and Mad:  Effect of Polyions on Protein Dimerization^†

The Myc−Max−Mad network of proteins activates or represses gene transcription depending on whether the dimerization partner of Max is c-Myc or Mad. To elucidate the physical properties of these protein−protein interactions, fluorescence anisotropy of TRITC-labeled Max was used. The binding affinities and thermodynamics of dimerization of the Max−Max homodimer and c-Myc−Max and Mad−Max heterodimers were determined. Our results indicate that c-Myc and Max form the most stable heterodimer. Previous work [Kohler, J. J., Metallo, S. J., Schneider, T. L., and Schepartz, A. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 11735−9] has shown that instead of dimerizing first and then binding to DNA, these proteins use a monomer pathway in which a monomer binds to DNA followed by dimerization on the surface of the DNA. The DNA E-box affects the dimerization, but nonspecific effects may also play a role. The influence of polyions, poly-l-lysine and poly-l-glutamic acid, were investigated to determine the effects of charged polymers other than DNA on homodimerization and heterodimerization. While the positively charged poly-l-lysine, PLL, did not show any significant effect, negatively charged poly-l-glutamic acid, PLG, stabilized both heterodimers and homodimers by 2−3 kJ/mol. These data suggest that in the cell nucleus the presence of negatively charged DNA or RNA could nonspecifically aid in association of these proteins. Calculations of ΔH° and ΔS° from the temperature dependence of K_d indicated that although the thermodynamic parameters for the dimer are different, the reactions for all three dimers are driven by negative (favorable) enthalpic and negative (unfavorable) entropic contributions. In the presence of PLG, entropy became more negative with the effect being largest for c-Myc−Max heterodimers. This suggests that van der Waals and H-bonding interactions are predominant in dimerization of these proteins.