TTYH family members form tetrameric complexes at the cell membrane

Abstract

The conserved Tweety homolog (TTYH) family consists of three paralogs in vertebrates, displaying a ubiquitous expression pattern. Although considered as ion channels for almost two decades, recent structural and functional analyses refuted this role. Intriguingly, while all paralogs shared a dimeric stoichiometry following detergent solubilization, their structures revealed divergence in their relative subunit orientation. Here, we determined the stoichiometry of intact mouse TTYH (mTTYH) complexes in cells. Using cross-linking and single-molecule fluorescence microscopy, we demonstrate that mTTYH1 and mTTYH3 form tetramers at the plasma membrane, stabilized by interactions between their extracellular domains. Using blue-native PAGE, fluorescence-detection size-exclusion chromatography, and hydrogen/deuterium exchange mass spectrometry (HDX-MS), we reveal that detergent solubilization results in tetramers destabilization, leading to their dissolution into dimers. Moreover, HDX-MS demonstrates that the extracellular domains are stabilized in the context of the tetrameric mTTYH complex. Together, our results expose the innate tetrameric organization of TTYH complexes at the cell membrane. Future structural analyses of these assemblies in native membranes are required to illuminate their long-sought cellular function.

Fig. 1. In situ cross-linking analyses of mTTYH family members.

a SDS-PAGE western blot analysis of HEK 293 cells transiently transfected with the indicated FLAG-tagged mTTYH paralogs. Arrowheads indicate the bands corresponding to mTTYH1 and mTTYH3. Note that there is a nonspecific band in mTTYH2, which can also be found in non-transfected cells (mock). b, c SDS-PAGE western blot of mTTYH1 (b) and mTTYH3 (c) cross-linking with increasing concentrations of BS³ (concentrations used were 0, 10, 50, 100, 200, and 2000 μM from left to right). Molecular weights and oligomeric states are shown on the left and right, respectively.

Fig. 2. Single-molecule subunit counting of mTTYH family members.

Analyses of EGFP-fused mTTYH1 (a–c), mTTYH3 (d–f), and KCNH1 (g–i). a, d, g Representative images sampled from 2 min movies, 1-day post-injection of 1 ng cRNA, using TIRFM. Spots of interest are highlighted by white squares. Scale bar, 2 μm. b, e, h Representative fluorescence intensity traces obtained from single spots showing four steps of photobleaching, as indicated by arrows. c, f, i Probability distributions of bleaching steps (black bars), with error bars indicating SEM. Binomial distribution (p = 0.7) for tetrameric complexes is presented as a dashed green line. n indicates the number of spots analyzed.

Fig. 3. Analyses of detergent-solubilized mTTYH paralogs.

a BN-PAGE western blot analysis of HEK 293 cells transiently transfected with mTTYH1 (left) or mTTYH3 (right). b FSEC chromatograms depicting the elution profiles of DDM solubilized HEK 293 cells expressing the indicated EGFP-fused mTTYH paralogs. BSA and EGFP profiles are provided for reference. c Purified mTTYH1-GFP SEC-MALS analysis. The normalized refractive index is shown as a black line, while the calculated masses of the protein, detergent, and protein–detergent complex are shown as blue, yellow, and green lines, respectively. d, e FSEC chromatograms of mTTYH1 (d) and mTTYH3 (e) preincubated for 10 min at the indicated temperatures. f, g FSEC chromatograms of mTTYH1 (f) and mTTYH3 (g), solubilized using different detergents, as indicated. Orange circles represent the oligomeric states of the complex. Column void volume (v) is indicated by arrow where applicable.

Fig. 4. HDX-MS analysis of mTTYH1.

a Cartoon representations of mTTYH1 homology model. A single chain is colored as a heat map, representing the HDX level following 10 sec (s) incubation in D₂O for the dimeric (left) and tetrameric (right) populations. The highest and lowest exchange levels are indicated in red and blue, respectively. b The HDX difference between the tetrameric and dimeric populations, following 10 s incubation in D₂O, is mapped onto the mTTYH1 homology model. c Deuteration levels at the indicated time points for the dimer (upper panel) and tetramer (middle panel). The difference between the tetrameric and dimeric populations is shown (bottom panel). The secondary structure elements are indicated above the heat maps.

Fig. 5. mTTYH disulfides contribute to tetramerization.

a Cryo-EM structures of hTTYH1 (upper panel; PDB: 7P5J) and hTTYH3 (lower panel; PDB: 7P5C) in cartoon representation. The dashed rectangles frame zoom perspectives of the disulfide bridges within the ECDs. Cysteine residues are colored green and shown as spheres. b FSEC chromatograms depicting the elution profiles of DDM solubilized HEK 293 cells expressing the indicated EGFP-fused mTTYH1 (upper panel) and mTTYH3 (lower panel) before (gray curve) and following incubation with 20 mM 2-ME (colored curve). c Tetramer/dimer ratio of mTTYH1 (upper panel) and mTTYH3 (lower panel) before and following incubation with 2-ME. d Protein sequence alignment of the ECD region from mTTYH1 and mTTYH3, highlighting conserved positions and cysteine residues in green and yellow, respectively. e FSEC chromatograms depicting the elution profiles of mTTYH3 ECD cysteine mutants. f Tetramer/dimer ratio of the mTTYH3 ECD cysteine mutants. (n = 3, t-test, ns non-significant, ** p < 0.01).

Fig. 6. mTTYH3 ECDs form tetramers in solution.

a A model of ECD1-T4L-ECD2 was generated using AlphaFold 2. b Western blot analyses of mTTYH3 ECD1-T4L-ECD2 expression using anti-FLAG. Note the single band observed in the secreted fraction (Medium), compared with the multiple intracellular forms (Lysate) (left panel), and the effect PNGase F treatment (500 units) (right panel). c Cross-linking using increasing concentrations of DSS (concentrations used were 0, 10, 50, 100, 200, and 2000 μM from left to right) resulted in the exposure of higher oligomeric assemblies, culminating in a mass corresponding to that expected for a tetramer.