title: 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.
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.
authors: Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA.
authors: Manoj
authors: M
authors: Munde
authors: Gregory M K
authors: GM
authors: Poon
authors: W David
authors: WD
authors: Wilson
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D015374:Biosensing Techniques / Q000379:methods; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000493:pharmacokinetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000493:pharmacokinetics*; D004214:Distamycins / Q000494:pharmacology; D051379:Mice; D008958:Models, Molecular; D017434:Protein Structure, Tertiary / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000493:pharmacokinetics*; D055672:Static Electricity; D020349:Surface Plasmon Resonance / Q000379:methods; D015534:Trans-Activators / Q000037:antagonists & inhibitors / Q000235:genetics / Q000493:pharmacokinetics*; D000099232:Proto-Oncogene Protein Spi-1
chemical_list: D004247:DNA; D004268:DNA-Binding Proteins; D004214:Distamycins; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1; C030000:stallimycin