Inner-ear sensory hair cells convert mechanical stimuli from sound and head movements into electrical signals during mechanotransduction. Identification of all molecular components of the inner-ear mechanotransduction apparatus is ongoing; however, there is strong evidence that TMC1 and TMC2 are pore-forming subunits of the complex. We present molecular dynamics simulations that probe ion conduction of TMC1 models built based on two different structures of related TMEM16 proteins. Unlike most channels, the TMC1 models do not show a central pore. Instead, simulations of these models in a membrane environment at various voltages reveal a peripheral permeation pathway that is exposed to lipids and that shows cation permeation at rates comparable to those measured in hair cells. Furthermore, our analyses suggest that TMC1 gating mechanisms involve protein conformational changes and tension-induced lipid-mediated pore widening. These results provide insights into ion conduction and activation mechanisms of hair-cell mechanotransduction channels essential for hearing and balance.

Highlights d Cryo-EM reveals a closed-pore structure of tetrameric TRPY1 d TRPY1 displays conserved TM domain but distinct folds in NTD and CTD d Two occupied Ca 2+ -binding sites per subunit, one activating and the other inhibiting d One inhibitory PI(3)P lipid per subunit gets co-purified with the channel

Nonenzymatic template-directed RNA primer extension with 2-aminoimidazole activated nucleotides begins with the generation of an imidazolium-bridged dinucleotide intermediate that binds to the template adjacent to the primer. Crystal structures have illuminated the overall conformation of the primer/template/bridged-dinucleotide complex, but critical aspects of the structure remain unseen or unresolved. Most significantly the catalytic metal ion has not been visualized, and its coordination to functional groups in the reaction center remains unknown. In addition, the orientation of the imidazolium moiety of the bridged dinucleotide is unresolved, and static crystal structures do not provide insight into the dynamic aspects of catalysis. To address these questions, we performed atomistic molecular dynamics simulations of primer/template/bridged-dinucleotide/helper-oligonucleotide complexes. Our simulations suggest that one particular orientation of the imidazolium moiety of the bridged dinucleotide is likely to be more favorable for the reaction. Additionally, Mg2+ions are known to play an important role in the catalysis of RNA copying chemistry. We have examined potential Mg2+contacts with multiple oxygen atoms in the reaction center. Our simulations suggest a preferred coordination of the catalytic Mg2+between O3′ of the terminal primer nucleotide and one of the non-bridging oxygens of the reactive phosphate of the imidazolium-bridged dinucleotide. We also observe significant stabilization of the geometry of the reaction center in this preferred coordination of the catalytic Mg2+. Our simulations suggest that the catalytic metal ion plays an important role in overcoming electrostatic repulsion between a deprotonated O3′ and the incoming phosphate of the bridged dinucleotide.

No abstract

Transient Receptor Potential (TRP) channels have evolved in eukaryotes to control various cellular functions in response to a wide variety of chemical and physical stimuli. This large and diverse family of channels emerged in fungi as mechanosensitive osmoregulators. The Saccharomyces cerevisiae vacuolar TRP yeast 1 (TRPY1) is the most studied TRP channel from fungi, but the molecular details of channel modulation remain elusive so far. Here, we describe the full-length cryo-electron microscopy structure of TRPY1 at 3.1 A resolution. The structure reveals a distinctive architecture for TRPY1 among all eukaryotic TRP channels with an evolutionarily conserved and archetypical transmembrane domain, but distinct structural folds for the cytosolic N- and C-termini. We identified the inhibitory phosphatidylinositol 3‐phosphate (PI(3)P) lipid binding site, which sheds light into the lipid modulation of TRPY1 in the vacuolar membrane. The structure also exhibited two Ca2+-binding sites: one in the cytosolic side, implicated in channel activation, and the other in the vacuolar lumen side, involved in channel inhibition. These findings, together with data from molecular dynamics simulations, provide structural insights into the basis of TRPY1 channel modulation by lipids and calcium.