Hydrational Control of ETS-Family Transcription Factors: A Possible Resolution of the “Specificity Conundrum”

Abstract

The ETS family of transcription factors is widely distributed among the metazoan phyla and regulates the expression of a wide range of genes. Despite their functional diversity, all ETS proteins share a structurally conserved DNA-binding (or ETS) domain. Given the highly overlapping sequence preferences among ETS members, it is as yet unclear how ETS proteins achieve functional specificity, a problem known as the “specificity conundrum.” Compounding this problem is a current lack of understanding of the biophysical mechanism of sequence selectivity among ETS binding sites. We hypothesize that the structural conservation among ETS domains disguises physicochemical heterogeneity in their mechanisms of sequence recognition. We have previously demonstrated that the ETS-family member PU.1 (Spi-1) recruits a cooperative network of water-mediated contacts along the protein-DNA interface for high-affinity binding. We have now compared the thermodynamics and kinetics of sequence recognition between the ETS domains of PU.1 and ETS1 which represent extremes of sequence divergence (∼30% homology) in the ETS family. We found that the thermodynamics and kinetics between the two structurally conserved ETS domains are highly differentiated under physiological conditions. More precisely, whereas high-affinity PU.1 ETS-DNA binding is enthalpically driven against an entropic penalty, ETS1 ETS-DNA binding is entropically driven. Kinetically, whereas ETS1 ETS associates rapidly with a high-affinity cognate site (ka > 107 M−1 s−1), PU.1 ETS is strikingly slow (ka ∼104 M−1 s−1). This profound difference in association rate constants means that the high-affinity PU.1 ETS-DNA complex, despite being somewhat thermodynamically less stable than the corresponding complex with ETS1, is significantly longer-lived. If these differences that underlie the intrinsic heterogeneity in site recognition by ETS proteins extend to protein-protein and domain-domain interactions, they offer one potential biophysical resolution to the specificity conundrum.