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

TTYH family members form tetrameric complexes at the cell membrane

“…While in situ cross-linking indicated that mTTYH proteins are composed of multiple subunits, we cannot exclude the possibility that heteromeric assembly formation of mTTYH dimers 14 , 15 with additional endogenous proteins underlies the observed migration patterns. Thus, to directly examine the number of mTTYH subunits within a complex in living cells, we resorted to the single-molecule subunit counting approach 22 .…”

“…This is in contrast to the predicted tetrameric organization inferred from electrophysiological studies 9 . Interestingly, in the absence of Ca 2+ in the solution, mTTYH2 demonstrated a different organization, consisting of a head-to-head trans -dimeric interaction between the ECDs of subunits from juxtaposing membranes 15 .…”

“…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.…”

“…The structures revealed a novel subunit topology common to all TTYH members, consisting of five transmembrane helices and a large α-helical extracellular domain (ECD). hTTYH paralogs and mTTYH3 were shown to exhibit a side-by-side cis -dimeric organization, involving interaction interfaces within the ECD and transmembrane regions 14 , 15 . This is in contrast to the predicted tetrameric organization inferred from electrophysiological studies 9 .…”

“…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 toward their functional and cellular roles. Indeed, the extraction of membrane proteins from their native environment can result in dramatic changes to their ternary and quaternary organizations 21 , hampering the elucidation of TTYH physiological roles.…”