Figure 5. Identification of the VSTx1-binding surface of VSD_K.

(a) Overlay of 2D ¹H-¹⁵N HSQC spectra of ²H/¹³C/¹⁵N-labeled VSD_K (100 μM) acquired in the absence (red) or presence (green) of 300 μM VSTx1. (b) Extent of peak broadening (%, right ordinate axis–orange/grey bars) and chemical shift perturbations (Δδ, left ordinate axis–brown bars) observed for VSD_K residues upon VSTx1 binding. Peak broadening was observed for residues in the S1-S2 and S3-S4 loops, indicating that both regions are involved in VSTx1 binding. (c) Molecular surface of VSD_K (PDB code 2KYH), viewed from the extracellular surface, with residues important for VSTx1 binding based on NMR-CSM and mutagenesis data highlighted in orange and red, respectively. The gating charges on S4 (R1, R2 and R3) are highlighted in blue.