Structural insight into the allosteric inhibition of human sodium-calcium exchanger NCX1 by XIP and SEA0400

Sodium-calcium exchanger proteins influence calcium homeostasis in many cell types and participate in a wide range of physiological and pathological processes. Here, we elucidate the cryo-EM structure of the human Na + /Ca 2+ exchanger NCX1.3 in the presence of a specific inhibitor, SEA0400. Conserved ion-coordinating residues are exposed on the cytoplasmic face of NCX1.3, indicating that the observed structure is stabilized in an inward-facing conformation. We show how regulatory calcium-binding domains (CBDs) assemble with the ion-translocation transmembrane domain (TMD). The exchanger-inhibitory peptide (XIP) is trapped within a groove between the TMD and CBD2 and predicted to clash with gating helices TMs 1/6 at the outward-facing state, thus hindering conformational transition and promoting inactivation of the transporter. A bound SEA0400 molecule stiffens helix TM2ab and affects conformational rearrangements of TM2ab that are associated with the ion-exchange reaction, thus allosterically attenuating Ca 2+ -uptake activity of NCX1.3. Synopsis The exchange of sodium and calcium ions across membranes plays important (patho)physiological roles. Here, cryo-EM is used to study the inhibitor-bound human sodium-calcium exchanger NCX1.3, shedding light on the molecular mechanisms underlying SEA0400- and XIP-mediated inactivation. The XIP region is located within a groove between TMD and CBD2, mediating inactivation by obstructing the conformational transition of gating helices TMs 1/6 . An SEA0400 molecule is situated within a negatively charged cavity in the transmembrane region, exerting allosteric inhibition by inhibiting the conformational changes of TM2ab. The XIP and SEA0400, though not directly interacting, mutually stabilize each other’s binding pockets in the inward-facing conformation through loopTM1-2, thereby cooperatively inhibiting the Ca 2 + -uptake activity of NCX. Allosteric inhibition by SEA0400 is selectively effective in the antiporter’s reverse mode. Cryo-EM data show how two key endogenous and exogenous sodium/calcium ion-exchange inhibitors block conformational rearrangements associated with ion exchange by NCX1.3.