Structures of Tweety Homolog Proteins TTYH2 and TTYH3 reveal a Ca²⁺-dependent switch from intra- to inter-membrane dimerization

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 novel 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.

TTYH family members form tetrameric complexes within the cell membrane

“…Specifically, focusing on mTTYH3, we introduced a T4 lysozyme (T4L) sequence between ECD1 (positions 107-209) and ECD2 (positions 260-386) to increase protein stability and solubility 32 . Due to the presence of disulfide bridges and glycosyl moieties 14,15,33 , we expressed this FLAG-tagged construct as a secreted protein in insect cells. Indeed, western blot analysis of the cell lysate revealed a complex migration pattern consistent with different stages in the protein biogenesis pathway.…”

“…The structures revealed a novel subunit topology common to all TTYH members, consisting of five transmembrane helices and a large a-helical extracellular domain (ECD). However, while the hTTYH paralogs and mTTYH3 were shown to exhibit a side-by-side dimeric organization involving interaction interfaces within the ECD and transmembrane regions, such an organization was shared by mTTYH2 only in the presence of calcium 14,15 . Conversely, it was suggested that in the absence of calcium, the dimers form by a head-to-head interaction between the ECDs of subunits from juxtaposing membranes 14 .…”

“…However, while the hTTYH paralogs and mTTYH3 were shown to exhibit a side-by-side dimeric organization involving interaction interfaces within the ECD and transmembrane regions, such an organization was shared by mTTYH2 only in the presence of calcium 14,15 . Conversely, it was suggested that in the absence of calcium, the dimers form by a head-to-head interaction between the ECDs of subunits from juxtaposing membranes 14 . Another surprising finding concerned the dimeric interface organization among the hTTYH paralogs, resulting from rigid body rotation along the two-fold axis of the individual monomers 15 .…”

“…Moreover, TTYH-mediated Clcurrents were recorded using mammalian heterologous expression systems 6,13 . Importantly, two recent studies elucidated the long-sought structure of TTYH family members 14,15 . However, strikingly, anion conductive pathways were not identified in any of the human (hTTYH) or mouse (mTTYH) members.…”

“…As the structures of TTYH members, determined following detergent-mediated membrane solubilization, revealed distinct spatial organizations 14,15 , elucidation of their stoichiometry in the native membrane environment may hint towards the functional and cellular roles of TTYH members. This is of special importance given the involvement of TTYH members in human pathologies.…”