NMR Investigations of the Structure and Dynamics of New KcsA inhibitors

Peptide channel-inhibiting neurotoxins are commonly used for research and as therapeutic agents. Recently two toxins, Hui1 and HmK, were found to inhibit KcsA, a prototypical bacterial potassium channel, at nanomolar affinity. These toxins were isolated from a library of over one million toxins derived from ShK, a sea-anemone toxin which does not bind KcsA. While HmK promiscuously binds both KcsA voltage-gated channels, Hui1 is selective for KcsA. Having determined the Hui1 structure using standard 2D NMR spectra, we found that structurally Hui1 and the parent ShK toxin are quite similar, and that changes in the electrostatic surface potential of both toxins are the source for their different selectivity profiles [1]. In addition, dynamic measurements, obtained from NMR relaxation dispersion experiments, showed that as ShK [2], Hui1 exhibits millisecond motion in the toxin backbone which implies that it has a minor conformer which may have a role in channel binding. We are currently refining the structure of HmK, and the comparison of its structure with those of Hui1 and ShK will provide new insights into the recognition elements of the various toxins and channels.

Initial measurements of binding between Hui1 and KcsA were performed using nanodiscs as membrane mimic environment [3] for KcsA. Addition of nanodisc-embedded KcsA to Hui1 led to chemical shift perturbations indicating that a complex had been obtained. Labelling of specific amino acids in KcsA will enable us to obtain distance constraints between specific toxin and channel residues, and these will eventually be used for docking simulations for obtaining the Hui1-KcsA complex structure.

References

[1] Ruiming Zhao , et al. PNAS, 112, 7013-7021 (2015).

[2] Sher Inabal, et ak. ChemBioChem, 2402-2410 (2014).

[3] A. Nath, W. M. Atkins, and S. G. Sligar, Biochemistry, 46, 2059–2069 (2007).