Molecular dynamics characterization of the mechanism of inhibition of ClC-7 activity by PI(3)P

ClC-7 is a Cl-/H+ antiporter expressed in the lysosome and has been identified as a player in the control of lysosomal pH and ion homeostasis. Known pathologies associated with CLC-7 mutations include osteopetrosis and lysosomal storage diseases. Both pH and current measurements have shown that the signaling lipid PI(3,5)P2 is critical in tuning ClC-7 activity. However, the mechanism by which PI(3,5)P2 achieves this is still unknown. We hypothesized that PI(3,5)P2 stabilizes interactions at the interface between ClC-7's transmembrane domain (TMD) and the cytoplasmic cystathionine beta synthase (CBS) domain, resulting in a TMD-CBS docked conformation that inhibits ClC-7 currents. To test this hypothesis, we have used atomistic molecular dynamics simulations of the transporter in the presence and absence of PI(3)P, a phosphoinositide bound in a recent Cryo-EM structure of CLC-7. Our simulations show that coordination of residues on the CBS with PI(3)P, as well as interactions between residues on the CBS with residues on the TMD play a role in stabilizing the TMD-CBS docked conformation. furthermore, the absence of PI(3)P destabilizes the TMD-CBS docked conformation, increasing the dynamics of the CBS domain. Interestingly, a known disease-causing gain-of-function mutation has similar effects at the TMD-CBS interface. Together our results hint at a mechanism of lipid modulation of ClC-7 activity and pave a way for the development of targeted therapeutics for ClC-7 related pathologies.