title: DNA binding regulates the self-association of the ETS domain of PU.1 in a sequence-dependent manner.
abstract: The current paradigm of ETS transcription factors holds that their DNA-binding (ETS) domain binds to a single sequence-specific site with strict 1:1 stoichiometry. PU.1 (Spi-1) is a lineage-restricted member of the ETS family that is essential in normal hematopoietic development. Characterization of the binding properties of the ETS domain of PU.1 by isothermal titration calorimetry revealed that it binds a single sequence-specific binding site with 1:1 and 2:1 stoichiometry in a discrete, sequential, and negatively cooperative manner. While both high-affinity- and low-affinity-specific sites exhibit this behavior, the thermodynamics for each complex are highly differentiated. In the unbound state, the PU.1 ETS domain exists as a weak noncovalent homodimer that dissociates and unfolds cooperatively. Thus, the PU.1 ETS domain exists as a monomeric and dimeric species in both DNA-bound and free states. Structural characterization of the protein-DNA interface by quantitative DNA footprinting revealed new minor groove contacts and changes in the core consensus suggestive of increased DNA distortion in the 2:1 complex. Together, the structural and thermodynamic data support a model in which DNA binding dissociates a PU.1 ETS dimer to a 1:1 protein-DNA complex followed by, at higher concentrations, an asymmetric 2:1 complex. The implications of distinct monomeric and dimeric states on the known structural biology of ETS domains as well as potential ETS-protein interactions are discussed.
authors.affiliation: Department of Pharmaceutical Sciences, Washington State University, Pullman, WA 99164-6534, USA. gpoon@wsu.edu
authors.forename: Gregory M K
authors.initials: GM
authors.lastname: Poon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D011518:Proto-Oncogene Proteins; D013816:Thermodynamics; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1
chemical_list: D004247:DNA; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1; C423918:Spic protein, mouse