Periodic Reporting for period 3 - EXCHANGE (Dynamic Complexes and Allosteric Regulation of Small Molecule Transporters)
Berichtszeitraum: 2022-06-01 bis 2023-11-30
To tackle this problem, the EXCHANGE project aims to provide molecular models for allosteric regulation by focusing on a family of sodium/protein exchangers (NHEs) belonging to the SLC9 family. The outcome will not only enable a better understanding of how NHEs work, but will also provide a deeper understanding for how NHEs and other SLC transporters work in general and how they can be better targeted in drug development.
The overall objectives of EXCHANGE are: (i) The architecture and the transporter conformations of mammalian Na+/H+ exchangers (ii) To establish the coupling between ion-binding and structural transitions (iii) To dissect the allosteric regulation of mammalian Na+/H+ exchangers (iv) Determined how lipids, tool compounds and drug interact with NHEs.
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.
Taken together, we have established the overall architecture of both SLC9A and SLC9B 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. Ongoing is efforts to obtain an ion-bound structure of an NHE protein.
(objective 3): To dissect the allosteric regulation of mammalian Na+/H+ exchangers we would like to obtain structures of NHE complexes and understand how these complexes regulate NHE activity.
We were unable to build the C-terminal tail of NHE9 in the absence of complex interacting partners. Ongoing is approaches to make this possible. In parallel, taking advantage of AlphaFold, we have computer-generated models for NHE9 with different interacting proteins, as well complex pairing to all other NHEs. These NHE-protein complex models will be assessed by a number of different approaches including cross-linking, HDX-MS, cryo EM as well as SSM electrophysiology.
(objective 4): We have further developed an 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 are pursuing the hypothesis that controlling oligomerization by specific lipids is a regulatory-switch for NHE activation with interesting physiological ramifications.