The structure of the Caenorhabditis elegans TMC-2 complex suggests roles of lipid-mediated subunit contacts in mechanosensory transduction

Significance One mechanism by which organisms sense their environment is through the perception of mechanical stimuli such as sound, touch, and vibration. Transmembrane channel-like (TMC) proteins are ion channels whose function has been linked to a variety of mechanosensitive processes, including hearing and balance in vertebrates and touch sensation in worms. The molecular mechanisms by which TMCs respond to mechanical stimuli are unknown. Here, we present the structure of the TMC-2 complex isolated from worms. Comparison of the TMC-2 complex to the recently solved structure of the worm TMC-1 complex highlights common structural features that are likely important for sensing mechanical stimuli yet also illuminates key differences that may explain the distinct functional roles of TMC-1 and TMC-2 in the worm.


Figures S1 to S6
Representative FSEC profile of the TMC-2 complex, detected via mVenus fluorescence.Inset shows a silver stained SDS-PAGE gel of the purified TMC-2 complex.Red asterisk indicates TMC-2.c, Mass spectrometry analysis of the purified TMC-2 complex, showing the five proteins with the highest peptide spectral counts (pSC).Components of the TMC-2 complex that were also identified in the TMC-2 single particle cryo-EM structure are highlighted in red.

Fig. S1 :
Fig. S1: Isolation of TMC-2 from C. elegans.a, Spectral confocal images of mVenus fluorescence and transmitted light in a tmc2::mVenus worm showing fluorescence in the body wall and vulval muscles.b,Representative FSEC profile of the TMC-2 complex, detected via mVenus fluorescence.Inset shows a silver stained SDS-PAGE gel of the purified TMC-2 complex.Red asterisk indicates TMC-2.c, Mass spectrometry analysis of the purified TMC-2 complex, showing the five proteins with the highest peptide spectral counts (pSC).Components of the TMC-2 complex that were also identified in the TMC-2 single particle cryo-EM structure are highlighted in red.

Fig. S2 :
Fig. S2: Cryo-EM data analysis of the TMC-2 complex.a, Flow chart of cryo-EM data analysis of the TMC-2 complex.b, Representative 2D classes of the dimer TMC-2 complex (top) as well as the 'transdimer' and monomeric classes, the latter two of which were discarded during heterogeneous refinement.

Fig. S3 :
Fig. S3: Cryo-EM classes, statistics, angular distributions and selected sections of density maps.a, A representative cryo-EM micrograph of the TMC-2 particles.Scale bar = 200 Å.b, Selected 2D classes of the TMC-2 complex.c, Angular distributions of the final reconstruction.d, Density maps colored by local resolution values.e, Fourier shell correlations (FSEC) curves for each map and model.f, Fragments of cryo-EM density map and atomic models of the auxiliary subunits, CALM-1 and TMIE.Cryo-EM maps are shown as yellow and red mesh.g, Fragments of cryo-EM density map and atomic models of TMC-2.Cryo-EM maps are shown as purple mesh.

Fig. S4 :
Fig. S4: Density feature differences between the TMC-1 and TMC-2 maps.The TMC-2 map and model are shown in purple and the TMC-1 map and model are shown in cyan.A sequence alignment for each fragment is shown at the bottom of the panel with the same coloring as in panel (d) of Figure 2. Underlined residues are non-conservative substitutions that are clearly distinguished in the density maps.The sequence of TMC-2 is on top and the sequence of TMC-1 is on bottom.

Fig. S5 :
Fig. S5: Sequence alignment of TMC proteins from C. elegans and H. sapiens.Amino acid sequences from C. elegans TMC-1 and TMC-2 are aligned with sequences from human TMC1-8 using the program Promals3D (1).The secondary structure, based on the cryo-EM structure of C. elegans TMC-2, is indicated above the sequence as cylinders ( -helices), black lines (loop regions), or dashed lines (regions not visible in the structure).Conserved residues (conservation score of 9) are highlighted in orange and residues with a conservative substitution (conservation score of 5-7) are highlighted in blue.Red arrows indicate glycosylation sites in C. elegans TMC-2.

Fig. S6 :
Fig. S6: TMC-1 and TMC-2 exhibit differences in their conformations and sequences at the dimer interface.a, View of the dimer interfaces, shown from three perspectives, following superposition using backbone atoms.TMC-1 is colored cyan and TMC-2 is colored purple.Basic amino acids that are proximal to the inner membrane leaflet are labeled and highlighted in blue.b, Multiple sequence alignment of TM10 from TMC-1 and TMC-2 from C. elegans, humans, and mice.Residues shown in black are not conserved, those in red are conservatively substituted, and those in bold red are fully conserved.Positively charged residues proximal to the membrane are highlighted in blue.Non-conserved residues in C. elegans TMC-2 are marked with black arrows.A schematic representation of TM10, showing the region that is associated with the membrane, is shown above the sequence alignment.

Table S1 .
Statistics for 3D reconstruction and model building