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

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


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Binding Sites / genetics
  • Binding, Competitive
  • Biosensing Techniques / methods
  • DNA / chemistry*
  • DNA / genetics
  • DNA / pharmacokinetics*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / pharmacokinetics*
  • Distamycins / pharmacology
  • Mice
  • Models, Molecular
  • Protein Structure, Tertiary / genetics
  • Proto-Oncogene Proteins / antagonists & inhibitors
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / pharmacokinetics*
  • Static Electricity
  • Surface Plasmon Resonance / methods
  • Trans-Activators / antagonists & inhibitors
  • Trans-Activators / genetics
  • Trans-Activators / pharmacokinetics*


  • DNA-Binding Proteins
  • Distamycins
  • Proto-Oncogene Proteins
  • Trans-Activators
  • proto-oncogene protein Spi-1
  • stallimycin
  • DNA