Extended Data Fig. 5: Lipid-induced structural changes at the dimeric interface.

a. Electrostatic surface of the SLC9C1 homodimer in detergent (left) and in nanodiscs (right) as viewed from the extracellular side, and bound fatty acid tails (grey-sticks) and PA lipids (green-sticks) are labelled b. Left: cartoon representation of the SLC9C1 structure of the dimerization interface in detergent (light brown) and in nanodiscs made with crude yeast polar lipids (grey). The N-terminal tail of TM1 extends across to the opposite-facing protomer and forms interactions with the extracellular end of TM3’. In the detergent structure (light-brown) H73B forms a salt-bridge with D134 as well as a backbone hydrogen bond to H71B, as shown in the zoomed in view below. In the nanodisc structure (grey), cryo-EM map density supporting two well-resolved PA lipids were able to be modelled (green sticks). To coordinate the PA lipid, the phosphate head-group hydrogen bonds to D129 in ECH1, most probably its uncharged state, as well as to H132 via a water-molecule, likely in the −1 charged state. The carbonyl oxygen from the fatty acid ester further hydrogen bonds to H73 in the N-terminal tail form the opposite-facing protomer. The intricate coordination of the negatively-charged PA lipids in the nanodisc SLC9C1 structure has stabilized a more compacted homodimer than in detergent. Other Na⁺/H⁺ exchangers have proposed to bind specific lipids at the oligomerization interface and are thought to regulate oligomerization and functional activity^{17,18,33,52,60}. Notably, the phosphomonoester headgroup of PA is the only lipid headgroup to alter its charged state around physiological pH values, and is thus also been referred to a pH-sensing lipid⁶¹. Right: as in the left panel showing the nanodisc structure and corresponding cryo EM map density (grey mesh) c. Quantification of the normalized homodimer FSEC peak height of SLC9C1-GFP after incubation of DDM/CHS purified fusion with either DDM or various DDM-solubilized lipids and heating (see Methods). Representative FSEC traces are shown in Supplementary Fig. 6c. Error bars are the mean value ± n = 5 independent experiments for SLC9C1 at 4 °C, at 45 °C, at 4 °C plus DOPA, at 45 °C plus DOPC, at 45 °C plus DOPC and mean value ± n = 3 independent experiments for 45 °C plus DOPE, 45 °C plus DOPS.

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