Structure-Function Studies of DNA Binding Domain of Response Regulator KdpE Reveals Equal Affinity Interactions at DNA Half-Sites

<div><p>Expression of KdpFABC, a K⁺ pump that restores osmotic balance, is controlled by binding of the response regulator KdpE to a specific DNA sequence (<em>kdpFABC_BS</em>) via the winged helix-turn-helix type DNA binding domain (KdpE_DBD). Exploration of <em>E. coli</em> KdpE_DBD and <em>kdpFABC_BS</em> interaction resulted in the identification of two conserved, AT-rich 6 bp direct repeats that form half-sites. Despite binding to these half-sites, KdpE_DBD was incapable of promoting gene expression <em>in vivo</em>. Structure-function studies guided by our 2.5 Å X-ray structure of KdpE_DBD revealed the importance of residues R193 and R200 in the α-8 DNA recognition helix and T215 in the wing region for DNA binding. Mutation of these residues renders KdpE incapable of inducing expression of the <em>kdpFABC</em> operon. Detailed biophysical analysis of interactions using analytical ultracentrifugation revealed a 2∶1 stoichiometry of protein to DNA with dissociation constants of 200±100 and 350±100 nM at half-sites. Inactivation of one half-site does not influence binding at the other, indicating that KdpE_DBD binds independently to the half-sites with approximately equal affinity and no discernable cooperativity. To our knowledge, these data are the first to describe in quantitative terms the binding at half-sites under equilibrium conditions for a member of the ubiquitous OmpR/PhoB family of proteins.</p>