Fig. 3: Lipid preferences of NHA2ΔN.
From: Structure, mechanism and lipid-mediated remodeling of the mammalian Na+/H+ exchanger NHA2
a, The native high-resolution mass spectrum of purified NHA2ΔN reveals a homodimer with multiple lipid adducts, as well as a small amount of lipid-free monomer (left inset). The masses of the first lipid adduct shown for the 19+ monomer are consistent with retention of a PI (829 ± 14.4 Da) or a PIP2 (1,036 ± 14.5 Da) molecule (right inset). Peaks shown as inserts are highlighted by grey bars in the full spectrum. b, Thermal stabilization of DDM-purified dimeric NHA2ΔN-GFP (blue bars) and NHA2ΔTM−1 (green bars) by lipids. Normalized mean fluorescence (r.f.u., relative fluorescence units) is shown after heating (TM + 5 °C, for the respective forms) and centrifugation in the presence of either the detergent DDM or DDM-solubilized lipids. Error bars represent the mean ± s.e.m. of n = 3 independent experiments (Methods). c, Thermal shift stabilization of purified dimeric NHA2ΔN-GFP in the presence of DDM addition (black) compared to PIP2 in DDM (blue), POPC in DDM (cyan) and PI in DDM (red). The data are normalized fluorescence mean ± s.e.m. of n = 5 independent experiments for DDM, n = 3 independent experiments for PI and PC and n = 2 independent experiments for PIP2. The apparent melting temperature TM was calculated with a sigmoidal four-parameter logistic regression function (Methods). d, Representative FSEC traces of DDM/CHS-purified NHA2ΔN after heating at 40 °C for 10 min in the presence of DDM addition (black) compared to PIP2 in DDM (blue) or PI in DDM (red). Inset: as in the main panel, but prior to heating. e, Cryo-EM density map of NHA2ΔN in nanodiscs with the 6-TM core ion-transport domains (colored in pink), the dimer domain (colored in green), cholesterol (gray) and the N-terminal domain-swapped helix TM −1 (blue).
