Abstract
The Tweety homologs (TTYHs) are members of a conserved family of eukaryotic membrane proteins that are abundant in the brain. The three human paralogs were assigned to function as anion channels that are either activated by Ca2+ or cell swelling. To uncover their unknown architecture and its relationship to function, we have determined the structures of human TTYH1–3 by cryo-electron microscopy. All structures display equivalent features of a dimeric membrane protein that contains five transmembrane segments and an extended extracellular domain. As none of the proteins shows attributes reminiscent of an anion channel, we revisited functional experiments and did not find any indication of ion conduction. Instead, we find density in an extended hydrophobic pocket contained in the extracellular domain that emerges from the lipid bilayer, which suggests a role of TTYH proteins in the interaction with lipid-like compounds residing in the membrane.
Highlights
The Tweety homologs (TTYHs) are members of a conserved family of eukaryotic membrane proteins that are abundant in the brain
To obtain a comprehensive overview of TTYH conformations, we have investigated the structural properties of all three paralogs in distinct environments (Supplementary Figs. 2–6 and Table 1): The structures of TTYH2 and 1 were determined in GDN under Ca2+-free conditions at 3.3 and 4.0 Å, respectively (Supplementary Figs. 2 and 4)
We investigated the impact of a lipid bilayer on the protein and reconstituted TTYH2 into lipid nanodiscs and determined its structure at 3.9 Å in a conformation that is indistinguishable from the structure of the same protein in detergent (Supplementary Fig. 3)
Summary
The Tweety homologs (TTYHs) are members of a conserved family of eukaryotic membrane proteins that are abundant in the brain. A subsequent study has assigned an equivalent function to TTYH1 and 2 in cancer cells[20] Despite their proposed function as chloride channels, little is known about the structural properties and the oligomeric organization of TTYHs, which are unrelated to any other characterized family of membrane proteins. To overcome this knowledge gap, we have here determined the structures of the three human paralogs by cryo-electron microscopy (cryo-EM). All structures provide equivalent views of a dimeric membrane protein that does not contain characteristic features of an anion channel After revisiting their proposed functional properties by patch-clamp electrophysiology, we did not observe any evidence of ion conduction mediated by either protein. The structures show features that hint at a potential involvement of TTYH proteins in the transport, binding, or metabolism of lipids or other membrane-associated compounds
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