Kinetic analysis of the interaction of b/HLH/Z transcription factors Myc, Max, and Mad with cognate DNA

Abstract

Myc, Mad, and Max proteins belong to the basic helix-loop-helix leucine zipper family of transcription factors. They bind to a specific hexanucleotide element of DNA, the E-box (CACGTG). To be biologically active, Myc and Mad require dimerization with Max. For the route of complex assembly of these dimers, there are two proposed pathways. In the monomer pathway, two monomers bind DNA sequentially and assemble their dimerization interface while bound to DNA. In the dimer pathway, two monomers form a dimer first prior to association with DNA. The monomer pathway is kinetically favored. In this report, stopped-flow polarization was utilized to determine the rates and temperature dependence of all of the individual steps for both assembly pathways. Myc.Max dimerization had a rate constant approximately 5- and approximately 2-fold higher than those of Max.Max and Mad.Max dimerization, respectively. The protein dimerization rates as well as the dimer-DNA rates were found to be independent of concentration, suggesting conformational changes were rate-limiting. The Arrhenius activation energies for the dimerization of Myc, Mad, and Max with Max were 20.4 +/- 0.8, 29 +/- 0.6, and 40 +/- 0.2 kJ/mol, respectively. Further, rate constants for Max.Max homodimer DNA binding are significantly higher than for Myc.Max and Mad.Max heterodimers binding to DNA. Monomer-DNA binding showed a faster rate than dimer-DNA binding. These studies show the rate-limiting step for the dimer pathway is the formation of protein dimers, and this reaction is slower than formation of protein dimers on the DNA interface, kinetically favoring the monomer pathway.

Figure 1

Stopped-flow kinetic binding measurements of Max, Myc and Mad to FITC labeled Max protein. Representative kinetic data show the time dependent increase in anisotropy after mixing 50 nM ^FITCMax (final concentration) with 0.2 µM unlabeled (final concentration) of (A) Myc, (B) Mad and (C) Max at 21°C. Residuals for the fits are shown in the lower panels. The experimental conditions are described under “Experimental Procedures”.

Figure 2

Kinetic plots of 1/kobs versus 1/[C] for the interaction of 50 nM ^FITCMax with varying concentrations of Max (—○—), Myc (—●—) and Mad (—△—) protein. The rate constant k₂ was obtained as the reciprocal of the y-intercept.

Figure 3

Arrhenius plots for the interaction of Max, Myc and Mad with ^FITCMax. The rate constant values for Max (—○—), Myc (—●—) and Mad (—△—) protein with ^FITCMax protein at different temperatures were used to construct an Arrhenius plot according to equation 5. The activation energy was calculated from the slope of the fitted linear plot of ln k versus T⁻¹ (Kelvin).

Figure 4

Stopped-flow kinetic binding measurements of Max·Max, Myc·Max and Mad·Max protein dimer to FITC labeled 16-mer DNA E-Box. Representative kinetic data show the time dependent increase in anisotropy after rapid mixing of 50 nM ^FITCDNA E-Box (final concentration) with 0.2 µM unlabeled (final concentration) of (A) Max·Max (B) Max·Myc and (C) Max·Mad at 21°C. Residuals for the fits are shown in the lower panels. The experimental conditions are described under “Experimental Procedures”.

Figure 5

Kinetic plots of 1/kobs versus 1/[C] for the interaction of 50 nM ^FITCDNA E-Box with varying concentrations of Max·Max (—○—), Max·Myc (—●—) and Max·Mad (—△—) dimmer protein. The rate constant k₂ was obtained as the reciprocal of the y-intercept.

Figure 6

Arrhenius plots for the interaction of ^FITCDNA E-Box with Max·Max, Max·Myc and Max·Mad dimer protein. The rate constant values for Max·Max (—○—), Max·Myc (—●—) and Max·Mad (—△—) protein at different temperatures were used to construct an Arrhenius plot according to equation 5. The activation energy was calculated from the slope of the fitted linear plot of ln k versus T ⁻¹ (Kelvin).

Figure 7

Kinetic measurements for the binding of monomer protein to DNA E-box. Data shows the time dependent increase in anisotropy after rapid mixing (A) 50 nM ^FITCMax (final concentration) with 1 µM DNA E-box (final concentration), (B) Max-DNA complex with 1 µM Max, (C) Max-DNA complex with 1 µM Myc, and (D) Max-DNA complex with 1 µM Mad, respectively, at 21°C.

Figure 8

Schematic diagram of the Myc, Max, and DNA E-box interactions. The model shows the two pathways k1, k2 and k3, k4 for the formation of Myc•Max•DNA complex. The k values at different temperatures are shown in Table 1 and 2.