Researchers resolved the atomic binding mechanism between the scorpion neurotoxin charybdotoxin and the MthK potassium channel using cryogenic electron microscopy, nuclear magnetic resonance spectroscopy, and molecular dynamics simulations, according to a study published in Nature Communications.
Structural architecture
Cryo-EM analysis of MthK reconstituted into POPE:POPG nanodiscs initially produced a closed-state channel structure at 3.2 Å resolution with C4 symmetry imposed. To resolve the asymmetric ~4.3 kDa toxin bound to the ~250 kDa homotetrameric channel, the authors applied particle subtraction, symmetry expansion, and focused 3D classification in Relion-5, yielding an asymmetric reconstruction at 4.1 Å overall resolution with 3.8 to 3.9 Å local resolution at the binding interface.
The atomic model revealed that charybdotoxin binds the extracellular entrance of the channel, anchoring its lysine 27 side chain into the S0 ion-binding site. The backbone carbonyl oxygens of MthK residue Y62 coordinate this lysine. The channel's selectivity filter preserved a canonical conductive conformation with an RMSD of 0.188 Å relative to the published open MthK structure, rather than adopting a collapsed state.
Spectroscopic measurements
Solid-state NMR on proteoliposome-reconstituted complexes confirmed that charybdotoxin becomes immobilized upon binding. Cross-polarization experiments detected rigid toxin regions, whereas polarization transfer experiments sensitive to fast dynamic motions yielded no signals. Resonances corresponding to residues W14, G26, K27, C28, M29, and R34 showed pronounced chemical shift perturbations or signal loss.
Isotope-labeled MthK pore domain spectra demonstrated chemical shift perturbations concentrated around selectivity filter residues Y62 through D64, which shifted by approximately 2 ppm in the nitrogen dimension. Amide proton signals for MthK residue G61 remained undetectable when toxin was present during reconstitution, consistent with restricted hydrogen-deuterium exchange caused by stable toxin binding.
