Mechanistic Diversity in DNA Site Discrimination by Structurally Homologous ETS-Family Transcription Factors

Abstract

Eukaryotic transcription factors are characterized by large families that share structurally homologous DNA-binding domains and similar DNA site preferences. The molecular mechanism by which these functionally non-interchangeable homologs achieve specificity remains poorly known. The ETS-family of transcription factors represent an excellent model system that embodies this “specificity conundrum.” The 28 human ETS paralogs share a structurally conserved DNA-binding domain (known as the ETS domain) and recognize sites harboring a central 5'-GGAA/T-3' consensus. Although ETS domains are highly conserved structurally, they share low amino acid homology. We hypothesize that this apparent sequence space encodes divergent physicochemical properties that confers specificity to site discrimination at the protein-DNA level. We compared the solution properties of site discrimination by the ETS domains of PU.1 and Ets-1, two highly structurally conserved by sequence-divergent proteins. The proteins exhibit profound differences in interfacial hydration: whereas Ets-1 is weakly sensitive to perturbation in water activity, PU.1 is strongly destabilized by osmotic stress. The contrasting thermodynamics and kinetics of site recognition by PU.1 and Ets-1 indicate significant differences in their mechanisms of selectivity. Specifically, PU.1 extensively integrates structural water into high-affinity binding, a feature that renders site specificity by PU.1 highly sensitive to the solution osmotic status. PU.1 is expressed only in immune and closely-related cell lineages, all of which develop and function in osmotically variable environments. PU.1 and Ets-1 direct distinct responses in cells in which they are co-expressed. The strong sensitivity to osmotic stress in sequence discrimination by which PU.1 may represent a mechanism by which it functions distinctly from other ETS members such as Ets-1 in cells that must adapt to physiological osmotic stress.