Fig. 5: NHA2_ΔN ion-binding site and its electroneutral activity.

a, Cartoon representation of NHA2_ΔN in nanodiscs with the electrostatic surface representation through the ion-binding site of one monomer (colored blue to red, for positive to negative charge). The strictly conserved ion-binding residue Asp278 is labeled and shown in yellow sticks. b, Surface representation of NHA2_ΔN in nanodiscs, showing the 6-TM core domain (pink), the dimerization domain (green) and the N-terminal domain-swapped transmembrane helix TM −1 (blue). The crossover of half helices TM5a-b and TM12a-b (cartoon) are unique to the NhaA-fold and the half-helical dipoles that they create are highlighted. In NHA2_ΔN, a lysine residue (K459 in stick form) is well positioned to neutralize the negatively charged half-helical dipoles, but the positively charged dipoles lack a negatively charged residue that is conserved in all other Na⁺/H⁺ antiporter structures¹⁹. Instead, the polar residue (T461 in stick form) that is conserved in all NHA2 members is enough for protein stability (Extended Data Fig. 1). c, Electrostatic surface representation of the extracellular view of the monomers of outward-facing NHA2 (left), outward-facing NapA (middle; PDB 4BWZ) and outward-facing human NHE1 (right; PDB 7DSX). The circles show the positions of the respective outward-facing funnels. d, Left: the ion-binding site of NHA2 has the two aspartates seen in electrogenic Na⁺/H⁺ antiporters (D277 and D278, in stick form) and a NHA2-specific salt bridge between R431 and E214 that is connected to W456 (residues shown in stick form). Dashed lines represent hydrogen bonding. Right: ion-binding site comparison between NHA2 (pink sticks) and electrogenic NapA (gray sticks and residue number in italic), highlighting the differences in E214 (P98 in NapA) and T461 (E333 in NapA).