Conformational analysis of toxins affinity and selectivity to K channels – NMR/MD study

Potassium (K+) channels are common ion channels among membrane proteins that form selective pores controlling a wide variety of cell functions. Toxins of the sea-anemone K+ channel (SAK-I) family, including ShK, AeTX-K, HmK, and Hui1, inhibit K+ channels by interacting with their pore regions in a specific manner. Here we combine experimental and computational methods to probe our hypothesis that bound conformations of toxins deviate from their free state structures, and that minor conformers contribute to channel specificity and affinity.

Using relaxation dispersion (RD) experiments, sensitive to µs-ms motions typical of conformational equilibria, we establish the presence of a lowly-populated conformer in ShK and Hui1, which for HmK is hardly observed. This difference may explain the insertion of Arg24 or Lys22 into the channel pore by Hui1 and HmK, respectively. We also conducted experiments under hydrostatic pressure (prNMR), in which Hui1 was most sensitive to pressure, with conformational changes appearing at the N-terminal turn and the two α-helices. This is an agreement with the change in binding mode observed for the D14Q mutant of HmK.

In a complementary approach, we used metadynamics-metainference simulations to generate a probability-weighted trajectory of ShK including sampling of low-probability structures. Focusing on disulfide isomerization, we found that the (–) χ3 conformer of 12-28 rota-isomers is preferred (by ~2.4 kT). Extension of this treatment to other toxins is underway and will allow us to compare their conformational behaviors. We expect our synergistic NMR/MD approach to afford valuable information on the structural basis of toxin-channel interactions.