Lipoprotein nanodiscs provide a new nmr-based view of toxin-channel recognition

Venom-derived toxins are of high pharmaceutical relevance, as these small peptides have provided useful molecular probes for investigating the structure and function of potassium channels. Sea anemone K⁺-channel (SAK) toxins, known inhibitors of various voltage dependent potassium (Kv) channels, contain three disulfide bonds that are linked in a conserved pairing pattern and share a common structural fold. Our goal is a structure-based model of how different K⁺-channels bind their respective inhibitors, and how modifications of this binding interface can modulate affinity and selectivity. To accomplish this, we focus on studying the behavior of free toxins as well as their complexes with channels.

NMR analysis of backbone motions reveals a different dynamics profile for each of the toxins. Increased flexibility in some of the toxins suggests the presence of a minor conformer that may contribute to toxin-channel affinity by a conformational selectivity mechanism. To explore this further, it is necessary to study the toxins in complex with the channel, requiring a membrane mimetic environment that is conducive to biophysical studies while maintaining native structure and function. We established a lipid protein nanodiscs (LPNs) system to stabilize the potassium channel-toxin complex at close-to-native conditions. Using KcsA-embedded LPNs we were able to obtain a spectrum of the bound toxin, revealing residues that are in proximity to the binding site. Identifying the multitude of molecular factors that contribute to the overall behavior of channels will lead us to a comprehensive mapping of the channel-toxin ‘interactome’ with implications on research and drug-design efforts.