objective 1): We determined the first structure of a mammalian NHE (SLC9A9) and could demonstrate that they undergo similar elevator conformations as seen in the bacterial homologues. NHE9 is important for regulating endosomal pH and mutations of the protein are associated with autism and is also highly expressed in brain cancer, i.e. glioblastoma. Thus, work opens up structural-based selective inhibition of NHE9. We followed this by further structures of NHE9 in complex with PI(3,5)P2 lipids, showing a novel mechanism for transporter regulation. Following this theme we have determined structures of human and mouse NHE6 to show how oligomerization is determined by early endosome lipids PI(3)P (Ms, in preparation).
We further determined the first structure of a mammalian NHA2 protein that belongs to the SLC9B clade. NHA2 activities are linked to hypertension in humans and insulin resistance. NHA2 has one additional helix than NHE9 and this alters how they the NHEs oligomerizes and we find that its activity in linked to volume regulation. Lastly, we determined the structure of the sperm-specific transporter SLC9C1, which is essential for fertilization.
Taken together, we have established the overall architecture of both SLC9A, SLC9B and SLC9C members, their conformational changes, and established how oligomerization is different between exchangers.
(objective 2): To establish the coupling between ion binding and elevator transitions we carried out a structural bioinformatic approach to show that hydrophobic residues gate entry to the ion-binding site in NHE9. Sodium binding to NHE9 and NHA2 was further probed using molecular dynamic simulations. To understand the impact of mutations for ion-binding we have employed a yeast sensitive salt-strain for NHA2 and also implemented solid-state membrane electrophysiology. These functional measurements have provided a better understanding for ion-recognition and how binding might trigger larger conformational changes. More recently we published the first structure of a K+/H+ exchanger bound to K+ that has helped to determine how ion specificity is achieved. Lastly, we have been able to obtain the first NHE structure (mouse NHE6) in complex with Na+ bound (Ms. in preparation).
(objective 3): To dissect the allosteric regulation of mammalian Na+/H+ exchangers we have been able to determine structures of NHE6 and NHE9 in absence of binding partners to show how their C-terminal tail acts in an auto-inhibitory manner. The same auto-inhibition mechanism (by restricting the movement of the linker helix TM7) is also apparent in structures of KefC and SLC9C1. The difference between the ion-exchangers is the external stimuli that removes the interaction to the linker helix to activate the protein.
(objective 4): We have further developed n thermal shift assay (GFP-TS) to be able to rapidly assess lipid- and drug- interactions to SLC transporters. We have been able to combine the GFP-TS assay with native MS and the cryo EM structures to show how phosphatidylinositol lipids can regulate oligomerization of NHE transporters. We have been able to show that controlling oligomerization by specific lipids is a regulatory-switch that turns on NHE activation under specific cues, which we are beginning to understand the importance of physiological processes.
