Extended Data Fig. 4: Topology comparison of NHA2 to 13-TM and 12-TM Na⁺/H⁺ antiporter homologs and structural comparison to NapA.

a. top: NHA2 has 14 TMs with the N-and C-terminus located in the cytoplasm and is made up of two 7-TM structural-inverted repeats that form the 6-TM core domain (pink), dimerization domain (green) and N-terminal domain-swapped helix TM -1 (blue); note, the linker helix TM7 has been assigned with the dimer domain as is TM -1, even though technically both these helices could be assigned as independent from both core and dimer domains. middle: NapA, NHE and NhaP members with 13 TMs (N-terminus extracellular and C-terminus cytoplasm) is the most common topology seen in the Na⁺/H⁺ exchangers, which are made up of two 6-TM structural-inverted repeats that form the 6-TM core domain (pink), dimerization domain (green) and non-symmetry-related linker helix (gray). bottom: NhaA is the only Na⁺/H⁺ exchanger seen with 12-TMs and it is made up of two 5-TM structural-inverted repeats and forms the 6-TM core domain (pink), dimerization domain that contains a β-hairpin between TM1 and TM2 (green) and two non-symmetry related helices (gray). b. left: cartoon representation of the dimer domain of the NHA2 monomer (pink) showing the structural conservation compared to the dimer domain of the bacterial homolog NapA domains (gray) following superimposition. middle: as in left but for the transport domain. right: structural superimposition of the NHA2 (pink and green) and NapA (gray) monomers. The arrow highlights the structural differences in the core domain helices TM12b and TM6 between outward-facing NHA2 and NapA structures, with the strictly conserved Asp278 (Asp157 in NapA) shown in stick form (encircled).