Fig. 2: TM2-TM3 β-loop domain structure of NHE9* CC with negatively-charged PI(3,5)P₂ lipids bound at the dimer interface.

a left: Structure of the NHE9*CC structure determined by cryo-EM and guided by the refined NHE9* model for the β-hairpin TM2-TM3 loop domain. Domain-swapped βH, βH’ and ECH1, ECH1’ in green and brown respectively, and NHE9 core and dimer domains. An additional C-terminal helix in the CTD could be modelled (salmon, cartoon), but there was no density to support the 20 residue AF2 loop model located between the end of the core domain and the beginning of the interfacial helix right: The cryo-EM maps superimposed on the NHE9* structure including the positively charged residues in the loop domain (sticks). b left: In the NHE9* CC structure additional map density supported the modelling of two PIP₂ lipids (yellow sticks) located in the middle at the dimerization interface between the two protomers and these could interact with the positively charged β-hairpin TM2-TM3 loop domain residues (green and brown sticks). right: The fit of PI(3,5)P₂ vs PI(4,5)P₂ lipids (sticks) into the cryo-EM maps is compared. c In addition to the β-hairpin TM2-TM3 loop domain lysine residues, PI(3,5)P2 is coordinated at the dimerization interface by the aromatic and polar residues Trp321, Gln17, Asn136 (sticks, with interactions illustrated by dashed grey lines). TM segments TM1 and TM8 are not shown for better visualization of the bound lipids (yellow sticks, map as grey mesh). d Electrostatic surface potential map (colored blue: positive to red: negative) cross-section with two negatively-charged PI(3,5)P₂ lipids modelled in the hydrophobic and positively-charged dimerization interface. The interface between the loop domains and core domains (indicated by the black-line) are negatively-charged and provide an electrostatic pathway for cations in the outward-facing state. Protein shown as cartoon (colored as in a), PI(3,5)P₂ as sticks (yellow) and the ion-binding site is highlighted (dotted-circle).