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.

•Pseudomonas exotoxin A (PE3) kills cells by ADP-ribosylating elongation factor 2.•We dissected PE3 into two inactive fragments that reassemble to an active enzyme.•Complementing PE3 inhibits protein synthesis in vitro and in live cells.•Delivery of both PE3 fragments as transgenes conditionally triggers cell death.•Complementing PE3 may enable conditional bispecific targeting in a single cell.The catalytic moiety of Pseudomonas exotoxin A (domain III or PE3) inhibits protein synthesis by ADP-ribosylation of eukaryotic elongation factor 2. PE3 is widely used as a cytocidal payload in receptor-targeted protein toxin conjugates. We have designed and characterized catalytically inactive fragments of PE3 that are capable of structural complementation. We dissected PE3 at an extended loop and fused each fragment to one subunit of a heterospecific coiled coil. In vitro ADP-ribosylation and protein translation assays demonstrate that the resulting fusions—supplied exogenously as genetic elements or purified protein fragments—had no significant catalytic activity or effect on protein synthesis individually but, in combination, catalyzed the ADP-ribosylation of eukaryotic elongation factor 2 and inhibited protein synthesis. Although complementing PE3 fragments are catalytically less efficient than intact PE3 in cell-free systems, co-expression in live cells transfected with transgenes encoding the toxin fusions inhibits protein synthesis and causes cell death comparably as intact PE3. Complementation of split PE3 offers a direct extension of the immunotoxin approach to generate bispecific agents that may be useful to target complex phenotypes.Download high-res image (232KB)Download full-size image

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 (≤ 105 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 (> 107 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.Download high-res image (252KB)Download full-size imageHighlights► PU.1 ETS is a transcription factor that binds to specific DNA sequences and controls gene expression in cells. ► The DNA binding kinetics of PU.1 ETS are strongly salt dependent but mutually balanced to produce a relatively moderate dependence in the equilibrium constant. ► We propose therefore at least two mechanisms of sequence recognition for this family. ► We also demonstrate pharmacological inhibition of sequence-specific ETS binding, using distamycin as a model compound. ► We show that biosensor-SPR results agree quite well with solution methods for DNA–transcription factor complexes.

Designed ligands that self-assemble noncovalently via an independent oligomerization domain have demonstrated enhancement in affinity for a variety of chemical and biological targets. To better understand the thermodynamic linkage between enhanced receptor binding and self-assembly, we have developed linkage models for the three commonly encountered types of noncovalently oligomeric ligands: homofunctional oligomeric ligands, heterodimeric ligands that target a single receptor, and bispecific ligands that crosslink noninteracting receptors. Expressions and numerical approaches for exact analysis as a function of total ligand concentrations are provided. We apply the linkage models to the binding data for two published noncovalently oligomeric ligands: one targeting a small molecule (phosphocholine) and the other targeting a soluble protein (tumor necrosis factor α). The linkage models provide a quantitative measure of the potential and realized enhancement in affinity that could inform and guide design optimization efforts, and they reveal physical insight that would elude model-free analysis. Incorporation of the linkage models, therefore, is expected to be valuable in the rational engineering of noncovalently oligomeric ligands.