Basis of Specificity in Ets-1 DNA Binding Domain to Variable DNA Sequences

Abstract

Transcription factor Ets-1 is a member of the ETS family, characterized by a conserved winged helix-turn-helix DNA binding domain that specifically recognizes 5’-GGA(A/T)-3’. Nonspecific sites are dictated by the lack of this consensus, while high/low affinity binding arise from discrepancies in sequences flanking the consensus. Affinities for consensus bearing sequences are dispersed, but nonetheless possess biologically relevant binding. Structures of Ets-1/high affinity complexes are available, but corresponding structures for low affinity are lacking. To understand the molecular basis of how Ets-1 discriminates between DNA sequences, we performed molecular dynamics simulations of Ets-1 unbound, and bound to nonspecific, low, or high affinity DNA. Ets-1 binding to DNA results in Ets-1 becoming less dynamic relative to unbound in a rank order correspondent with affinity, and induces bending in the DNA in a trend inverse to affinity. Ets-1/nonspecific complexes exhibit a generalized reduction in DNA contacts, behaving similar to unbound Ets-1. Contrarily, Ets-1/ nonspecific complexes display the largest degree bending, suggesting a prelude to localized searching. High and low affinity bound Ets-1 form similar complexes, showing significant deviation at three residues- Q336, E343, and R378. Q336 contacts the DNA backbone in high and low affinity; E343 and R378 form a salt bridge aiding in the hydrophobic packing of α-helix H1. These interactions are maintained in Ets-1/high affinity DNA; in low affinity, Q336/DNA weakens significantly, and the E343-R378 salt bridge is severed. Loss of the salt bridge enables R378 to contact DNA by distorting the terminus of α-helix H2, thereby exposing the hydrophobic core of helix H1 to water. Mutations (Q336L and E343L) in high and low affinity perturbed binding affinity; this suggests a mechanism distal to the DNA interface that differentiates Ets-1 binding to high/low affinity DNA sequences.