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. 2013 May 27;425(10):1655-69.
doi: 10.1016/j.jmb.2013.02.010. Epub 2013 Feb 14.

Probing the Electrostatics and Pharmacological Modulation of Sequence-Specific Binding by the DNA-binding Domain of the ETS Family Transcription Factor PU.1: A Binding Affinity and Kinetics Investigation

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Free PMC article

Probing the Electrostatics and Pharmacological Modulation of Sequence-Specific Binding by the DNA-binding Domain of the ETS Family Transcription Factor PU.1: A Binding Affinity and Kinetics Investigation

Manoj Munde et al. J Mol Biol. .
Free PMC article

Abstract

Members of the ETS family of transcription factors regulate a functionally diverse array of genes. All ETS proteins share a structurally conserved but sequence-divergent DNA-binding domain, known as the ETS domain. Although the structure and thermodynamics of the ETS-DNA complexes are well known, little is known about the kinetics of sequence recognition, a facet that offers potential insight into its molecular mechanism. We have characterized DNA binding by the ETS domain of PU.1 by biosensor-surface plasmon resonance (SPR). SPR analysis revealed a striking kinetic profile for DNA binding by the PU.1 ETS domain. At low salt concentrations, it binds high-affinity cognate DNA with a very slow association rate constant (≤10(5)M(-)(1)s(-)(1)), compensated by a correspondingly small dissociation rate constant. The kinetics are strongly salt dependent but mutually balance to produce a relatively weak dependence in the equilibrium constant. This profile contrasts sharply with reported data for other ETS domains (e.g., Ets-1, TEL) for which high-affinity binding is driven by rapid association (>10(7)M(-)(1)s(-)(1)). We interpret this difference in terms of the hydration properties of ETS-DNA binding and propose that at least two mechanisms of sequence recognition are employed by this family of DNA-binding domain. Additionally, we use SPR to demonstrate the potential for pharmacological inhibition of sequence-specific ETS-DNA binding, using the minor groove-binding distamycin as a model compound. Our work establishes SPR as a valuable technique for extending our understanding of the molecular mechanisms of ETS-DNA interactions as well as developing potential small-molecule agents for biotechnological and therapeutic purposes.

Figures

Fig. 1
Fig. 1
DNA sequences used in this study. The cognate sites are derived from the λB site of the Igλ2–4 enhancer (GGAA-1). The duplex sequences are formed as hairpin (CTCT- loop), biotinylated at the 5' end, and immobilized via streptavidin for SPR analysis. Mutations in the flanking sequences are shown in red. The non-cognate site is identical to λB except the core consensus is mutated to 5'-AGAG-3'.
Fig. 2
Fig. 2
Kinetic and steady-state analysis of PU.1 ETS binding to the cognate λB site and non-cognate DNA by SPR. Sensorgrams for the λB site GGAA-1 in (a) 400Na (2, 4, 8, 15, 25, 50 and 75 nM PU.1 ETS from bottom to top), (b) 500Na (10, 25, 50, 100, 150, 200, 250 nM PU.1 ETS), (c) 600Na (10, 25, 50, 100, 150, 200, 250 nM PU.1 ETS) and (d) Sensorgrams for non-cognate DNA, AGAG at 400Na (10, 25, 50 and 100 nM PU.1 ETS from bottom to top). All the data were acquired at 25°C and pH 7.4 (e) Comparative binding isotherm from steady state analysis for (●) GGAA-1 and (∎) AGAG.
Fig. 3
Fig. 3
Salt dependence of PU.1 ETS-λB binding as observed by SPR. The KD values obtained from SPR kinetic data for PU.1 ETS-GGAA-1 complex formation under different experimental conditions are compared in this plot. (●) SA and (■) CM4 chip without nonspecific DNA, and (▲) CM4 and (◆) filter binding data with nonspecific DNA.
Fig. 4
Fig. 4
Effect of nonspecific DNA on apparent ETS-λB kinetics as observed by SPR. Sensorgrams for PU.1-GGAA-1 binding were acquired at 400 mM NaCl in the presence of (a) 100, (b) 185 and (c) 300 μM bp salmon sperm DNA as nonspecific DNA. The concentration of protein was 6, 8, 15, 25, 50, 100, 200 nM from bottom to top in each panel. (d) Apparent KD as a function of nonspecific DNA concentration.
Fig. 5
Fig. 5
Salt dependent kinetics of PU.1 ETS-λB binding. Senorgrams were acquired in the presence of 300 μM bp salmon sperm DNA in buffer containing NaCl as follows: (a) 250 mM NaCl (10, 25, 50, 75, 100 and 150 nM PU.1 ETS from bottom to top), (b) 300 mM NaCl (25, 50, 100, 200 and 400 nM PU.1 ETS), (c) 350 mM NaCl (25, 50 and 100 nM), and (d) 400 mM NaCl (10, 25, 50, 100, 200 and 400 nM PU.1 ETS). For comparison, the sensorgram for (e) PU.1-AGAG binding in 250 mM NaCl (50, 100, 200, 400 and 1000 nM PU.1 ETS) is also shown. (f) Salt dependence of the association and dissociation rate constants.
Fig. 6
Fig. 6
Competitive binding by PU.1 ETS to λB mutant sequences. PU.1-DNA competition experiments for (a) GGAA-1 (b) GGAA-2, (c) GGAA-3 and (d) GGAA-4. In each experiment 100 nM protein (P) was injected mixed with DNA site as indicated (D). Nonspecific DNA was present at 100 μM bp in the samples and running buffer (400 mM NaCl). (e) Plot of normalized protein signal vs DNA concentrations. The plot is truncated at 800nM DNA so that the strong competitors are better resolved. The actual experiment went to 15 μM DNA in Figure 6e.
Fig. 6
Fig. 6
Competitive binding by PU.1 ETS to λB mutant sequences. PU.1-DNA competition experiments for (a) GGAA-1 (b) GGAA-2, (c) GGAA-3 and (d) GGAA-4. In each experiment 100 nM protein (P) was injected mixed with DNA site as indicated (D). Nonspecific DNA was present at 100 μM bp in the samples and running buffer (400 mM NaCl). (e) Plot of normalized protein signal vs DNA concentrations. The plot is truncated at 800nM DNA so that the strong competitors are better resolved. The actual experiment went to 15 μM DNA in Figure 6e.
Fig. 7
Fig. 7
Displacement of PU.1 ETS from the λB site by distamycin. (a) A constant concentration (100 nM) of protein (P) was titrated with up to 2.0 μM distamycin (Dist) from top to bottom (400 mM NaCl). The bottom-most sensogram is for free distamycin (2 μM). (b) Inhibition of protein binding vs distamycin concentration.
Fig. 8
Fig. 8
Model for allosteric inhibition of major groove protein by minor groove binder distamycin.

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