Crystal Structure and Asymmetric Conformation of A K+ Channel Rck Domain

Regulator of conductance of K+ (RCK) domains govern K+ channel and transporter activity through binding of cytosolic nucleotides or ions, although the locations and ligand-selectivities of activation sites within these domains can vary. To gain further insight toward structures of activation sites and ligand-dependent conformational changes in RCK domains, we solved the crystal structure of the cytosolic RCK domain from a Ca2+-gated K+ channel cloned from Thermoplasma volcanium. Crystals were grown in the presence of Mg2+, an activator of the Thermoplasma K+ channel, and the structure was solved to 2.95 Å resolution. The structure was found to contain two RCK domains per asymmetric unit, forming a homodimer. Interestingly, this homodimer was captured in an asymmetric state, and structural alignment of the chains with one another revealed a “hinge” that immediately followed the domain's N-terminal Rossmann fold region. This asymmetry suggested that the RCK dimer might either be in an intermediate, partially-liganded state, or (alternatively) that the asymmetric conformation represents the fully-liganded state. To determine the locations of divalent cation binding sites, the domain was co-crystallized with Ba2+. The Ba2+-bound structure yielded the same asymmetric conformation observed in the Mg2+ co-crystal, and the anomalous difference Fourier map revealed only one Ba2+ binding site, located at the RCK domain dimer interface and thus forming a metal bridge between adjacent subunits. Crystallization of the domain in a four-fold symmetrical “gating ring” form and small-angle X-ray scattering profiles obtained over a range of [Ca2+] are consistent with the idea that formation of the intersubunit metal bridge may drive a conformational change in the gating ring, which in turn may modulate channel gating.