Structures of tweety homolog proteins TTYH2 and TTYH3 reveal a Ca2+-dependent switch from intra- to intermembrane dimerization

Baobin Li,Christopher M Hoel,Stephen G Brohawn

doi:10.1038/s41467-021-27283-8

Baobin Li, Christopher M Hoel + Show 1 more

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https://doi.org/10.1038/s41467-021-27283-8

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Journal: Nature Communications	Publication Date: Nov 25, 2021
Citations: 12	License type: open-access

Affiliation: University of California, Berkeley, QB3

Abstract

Tweety homologs (TTYHs) comprise a conserved family of transmembrane proteins found in eukaryotes with three members (TTYH1-3) in vertebrates. They are widely expressed in mammals including at high levels in the nervous system and have been implicated in cancers and other diseases including epilepsy, chronic pain, and viral infections. TTYHs have been reported to form Ca2+- and cell volume-regulated anion channels structurally distinct from any characterized protein family with potential roles in cell adhesion, migration, and developmental signaling. To provide insight into TTYH family structure and function, we determined cryo-EM structures of Mus musculus TTYH2 and TTYH3 in lipid nanodiscs. TTYH2 and TTYH3 adopt a previously unobserved fold which includes an extended extracellular domain with a partially solvent exposed pocket that may be an interaction site for hydrophobic molecules. In the presence of Ca2+, TTYH2 and TTYH3 form homomeric cis-dimers bridged by extracellularly coordinated Ca2+. Strikingly, in the absence of Ca2+, TTYH2 forms trans-dimers that span opposing membranes across a ~130 Å intermembrane space as well as a monomeric state. All TTYH structures lack ion conducting pathways and we do not observe TTYH2-dependent channel activity in cells. We conclude TTYHs are not pore forming subunits of anion channels and their function may involve Ca2+-dependent changes in quaternary structure, interactions with hydrophobic molecules near the extracellular membrane surface, and/or association with additional protein partners.

Highlights

Tweety homologs (TTYHs) comprise a conserved family of transmembrane proteins found in eukaryotes with three members (TTYH1-3) in vertebrates
Dysregulation of TTYHs expression has been implicated in some cancers: TTYH2 is upregulated in renal cell carcinoma[2], colon carcinomas[11], and osteosarcomas[12]; TTYH1 is expressed in gliomas where it contributes to brain colonization[13] and a TTYH1-C19MC microRNA
Full-length Mus musculus TTYH2 was expressed in HEK293T cells with a cleavable C-terminal fusion to EGFP, extracted and purified in detergent, and reconstituted into lipid nanodiscs formed by the scaffold protein MSP1E3D1 and lipids in the presence of 1 mM Ca2+ (Supplementary Fig. 1)

Summary

Introduction

Tweety homologs (TTYHs) comprise a conserved family of transmembrane proteins found in eukaryotes with three members (TTYH1-3) in vertebrates. A fifth study reported TTYH1-3 form AQP4-dependent, Ca2+-insensitive volume-regulated anion channels activated by cell swelling in astrocytes or in cultured cells upon cotransfection[22]. Among these studies, two reports identified the small molecule DIDS as a channel blocker[3,20] and three reported point mutations with modest effects on ion selectivity (TTHY1R371Q, TTYH3H370D, and TTYH3R367Q)[3,20], DIDS block (TTHY1F394S)[20], or current magnitude (TTYH1R165A/R165C)22(numbering refers to human sequences)

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