Investigating Affinity and Selectivity of an Artificial Toxin to an Orphan K⁺ Channel

Peptide channel-inhibiting neurotoxins are commonly used for research and as therapeutic agents. The goal of my research is to resolve the solution structure of a toxin-channel complex at atomic resolution. Hui1, a de-novo toxin, was isolated from a library of over one million toxins (based on the ShK toxin scaffold) as exhibiting high selectivity and nanomolar affinity to our target K⁺ channel for which inhibitors have yet to be identified. The ensemble of the 25 lowest energy structures of Hui1 was obtained using standard 2D NMR methods.

Alignment of Hui1 structure with ShK did not show any significant structural changes. However, comparison of the electrostatic surface potentials of Hui1 and ShK indicates that the electrostatic differences are the reason for the affinity and specificity of Hui1 towards the K⁺ channel. Additionally and surprisingly, the voltage-sensitivity of Hui1 off-rate is mediated by Arg23, thus unlike other K⁺ channel toxins (including ShK), Lys21 most likely does not penetrate the pore. In conjunction with additional biophysical data it becomes clear that selectivity is conferred by an accumulated combination of small effects.

In accordance with our ultimate goal we aim to establish conditions at which the channel-toxin complex can be studied. We have optimized a protocol that enables the toxin to bind the K⁺ channel in a DPC detergent solution needed to stabilize the channel. By NMR we have shown that the toxin binds to the channel`s turret region, leading to a structural change. This lays the groundwork for complex structure determination and understanding of the molecular basis of affinity and selectivity.