Activation of a nucleotide-dependent RCK domain requires binding of a cation cofactor to a conserved site.

Abstract

RCK domains regulate the activity of K⁺ channels and transporters in eukaryotic and prokaryotic organisms by responding to ions or nucleotides. The mechanisms of RCK activation by Ca²⁺ in the eukaryotic BK and bacterial MthK K⁺ channels are well understood. However, the molecular details of activation in nucleotide-dependent RCK domains are not clear. Through a functional and structural analysis of the mechanism of ATP activation in KtrA, a RCK domain from the <i>B. subtilis</i> KtrAB cation channel, we have found that activation by nucleotide requires binding of cations to an intra-dimer interface site in the RCK dimer. In particular, divalent cations are coordinated by the γ-phosphates of bound-ATP, tethering the two subunits and stabilizing the active state conformation. Strikingly, the binding site residues are highly conserved in many different nucleotide-dependent RCK domains, indicating that divalent cations are a general cofactor in the regulatory mechanism of many nucleotide-dependent RCK domains.