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 Å 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 Ca2+.
INTRODUCTION
Osmoregulation is the ability of cells to detect and counterbalance osmotic concentration variations in their surroundings1. In their natural environment, unicellular eukaryotic organisms like yeast, which live on plants and animals, can experience rapid water efflux (hyperosmotic shock) leading to shrinkage, or water influx (hypoosmotic shock) causing them to swell1. To survive hyperosmotic shocks, yeasts have been shown to release Ca2+ from intracellular stores to initiate a defense response that counterbalances rapid changes of osmotic concentration in their environment2.
The vacuole acts as a Ca2+ buffering system that maintains low free cytosolic Ca2+ concentration in yeast3. It has been reported that Ca2+ concentration in the yeast vacuole is around 1.3 mM, while cytosolic Ca2+ is only 260 nM3–5. The Saccharomyces cerevisiae vacuolar transient receptor potential yeast 1 (TRPY1, also known as yeast vacuolar conductance 1 or YVC1), considered the closest extant representative of the ancestral state of eukaryotic TRP channels, has been shown to play a major role in Ca2+ release from the vacuole to the cytosol in response to hyperosmotic stress5. TRPY1 is a non-selective Ca2+ permeable and polymodal cation channel, like many other TRP channels6–9. It was originally characterized as a Ca2+-activated channel, but was later proposed to be also mechanosensitive10–12. Increased cytosolic Ca2+ concentration can enhance TRPY1 mechanosensitivity, consistent with its proposed in vivo function in osmotic regulation13,14. Recently, it was shown that a high concentration of Ca2+ in the vacuolar lumen inhibits TRPY1 through Ca2+-binding at the S5-S6 linker15. TRPY1 can be also activated by reducing agents and inhibited by phosphatidylinositol 3-phosphate (PI(3)P), suggesting that reactive oxygen species and PI(3)P lipid are endogenous modulators of the channel14.
A recent comprehensive study on TRP channel mechanosensitivity has shown that the majority of mammalian TRP channels are insensitive to membrane stretch16. Nevertheless, a Drosophila TRP channel known as NOMPC has been shown unequivocally to be mechanosensitive both in vitro and in vivo17. Electrophysiological experiments suggest that TRPY1 is responsive to membrane stretch, indicating that both channels are mechanosensitive, although their activation mechanisms might be different13,18. Recently, a NOMPC structure revealed how its large ankyrin-repeat domain organizes into a spring-like bundle that would interact with the cytoskeleton to control channel opening19. In contrast, TRPY1 does not have ankyrin-repeats, and its activation might be triggered by membrane stretch sensed by its transmembrane core or by Ca2+ binding to its cytosolic domain13,20.
To further understand the structural topology of this ancestral eukaryotic TRP channel and to shed light on mechanisms of TRPY1 channel modulation by Ca2+ and membrane lipids, we determined the structure of the full-length TRPY1 channel in the presence of Ca2+ by cryo-electron microscopy (cryo-EM). The transmembrane core of the channel revealed a “classic” TRP channel domain-swapped topology, but a different arrangement of N- and C-terminal domains contributed to a distinct overall TRPY1 channel architecture. This structure of the TRPY1 channel has also revealed key PI(3)P and Ca2+-binding sites. Insights from all-atom molecular dynamics (MD) simulations confirmed that PI(3)P and vacuolar Ca2+ play a central role in maintaining a closed state of the channel, while cytosolic Ca2+ is important for the structural integrity of the TRPY1 cytosolic domain. In addition, our findings provide structural insights that might guide the exploration of the molecular basis of TRPY1 channel modulation by hyperosmotic stress.
RESULTS
We overexpressed the full-length Saccharomyces cerevisiae TRPY1 channel in Saccharomyces cerevisiae and purified the channel using digitonin and glyco-diosgenin detergents. Since TRPY1 is activated by cytosolic Ca2+ between 10 μM to 1 mM and inhibited at 1 mM Ca2+ from the vacuolar luminal side15, we determined TRPY1 structures in an apo condition at 3.0 Å and in a 2 mM Ca2+-supplemented condition at 3.1 Å resolution by cryo-EM (Extended Data Figs. 1–2). Both of these cryo-EM density maps were of sufficient quality for de novo model building for the majority of the TRPY1 channel (Extended Data Figs. 1–4). These structures are nearly indistinguishable from each other (RMSD 0.307), including the bound Ca2+ ions and PI(3)P molecules (Extended Data Figs. 1–2). While Ca2+ was not added to the buffer solutions for the structure determined at the apo condition, there was still sufficient Ca2+ present that the channel was trapped in an apparently saturated Ca2+-bound state. This may have been due to residual Ca2+ leeching from the filter paper during grid preparation, a possibility that has been reported in a separate cryo-EM study of inositol triphosphate receptors21. As both TRPY1 structures appear to be in identical closed states, we will only discuss the 2 mM Ca2+-supplemented state in this manuscript (Fig. 1, Extended Data Fig. 1–4, Table 1).
Similar to previously determined TRP channel structures22–25, TRPY1 forms a domain-swapped homo-tetramer with each subunit having a cytosolic N-terminal linker helical domain (LHD) consisting of eight tightly packed α-helices, six transmembrane α-helices (S1-S6) with a pore helix (P helix), followed by a TRP helix and a C-terminal domain (CTD) mainly comprising two long α-helices (Fig. 1). Due to the absence of the ankyrin-repeat domains at the N-terminus (Extended Data Fig. 5), the entire cytosolic domain spanning LHDs and CTDs across the four TRPY1 subunits assumes the shape of a cytosolic “skirt” (Fig. 1b), discussed in detail along with computational models in later sections. Although we worked with the full-length TRPY1 protein construct (Met1-Glu675), parts of the N-terminal domain (residues Met1-Asn24, Asp55-Glu65), the pre-S1 elbow (residues Leu216-Phe225), the loop between S3 and S4 helices (residues Pro323-Lys327), the TRP-CTD linker (residues Ala487-Ser529), and some parts of the C-terminal domain (residues Leu572-Ser580, Asp608-Glu675) were not resolved in our structure likely due to flexibility (Fig. 1) and models were not built for them. We also did not build models for other ambiguous densities in the transmembrane region that are most probably annular lipids or detergent molecules. Overall, the TRPY1 structure has several “classical” TRP channel domains seen in other TRP channel structures (Fig. 1c, d), yet there are specific features relevant for its regulation by Ca2+ and the channel’s role in mechanosensation in the yeast vacuolar membrane (Fig. 1).
In our TRPY1 structure, the LHDs have distinct features in terms of the number and length of the helices, their relative arrangement, and the length of LH5-LH6 loop (Fig. 1c, Fig. 2a and Extended Data Fig. 6a). Structural comparison with counterparts from NOMPC (PDB: 5VKQ)19, TRPM4 (PDB: 5WP6)26 and TRPC6 (PDB: 5YX9)27 reveals good conservation for the five LHD helices LH4-LH8 that are located adjacent to the pre-S1 helix (Fig. 1c, d and Extended Data Fig. 6b). However, LH3 in TRPY1 is two helical turns longer than in NOMPC, TRPM4 and TRPC6 (Extended Data Fig. 6b). Toward the N-terminus, like TRPM4, TRPY1 LHD has two more helices, LH1 and LH2. This is in contrast with NOMPC and TRPC6 where three such helices are present at the N-terminus, two of which are very short while the other is similar in length to LH2 of TRPY1. The LH5-LH6 loop (Arg154-Asn169) in TRPY1 is comparable to TRPC6 but longer than in NOMPC and TRPM4 (Extended Data Fig. 6b). Strikingly, the LH5-LH6 loop in TRPY1 interacts with the CTD of the neighboring subunit (discussed later) (Fig. 2a and Extended Data Fig. 6a). This loop-mediated interaction is a distinct feature of TRPY1’s cytosolic skirt and is not observed in other TRP channels. It is remarkable that such a lengthy loop remained stable at this position and consequentially a high-quality electron density was captured to model side chains for the entirety of the loop. The reason for this stability can be partly attributed to several interactions within the LHD such as three salt bridges between Arg155 and Glu172, Lys102 and Glu160, and Arg33 and Asp162 (Fig. 2a). Coordination of a bridging cytosolic Ca2+ at the distal end of the LH5-LH6 loop provides additional stability (Fig. 2a). In the absence of the ankyrin-repeat domains in TRPY1, the cytosolic skirt, especially the LHDs may harbor the docking site for binding partners in the S. cerevisiae cytosol. Therefore, the structural differences in LHD helices along with the longer LH5-LH6 loop and its capability to coordinate a Ca2+ ion might have implications for TRPY1 channel function.
Another unique feature of the TRPY1 structure is the position of the CTD relative to the vacuolar membrane and the interaction of the CTD with the N-terminal LHD of a neighboring subunit (Fig. 1 and Extended Data Fig. 6a). The CTD is composed of a TRP-CTD linker that is not resolved in our cryo-EM structure, two long α-helices labeled CH1 and CH2, and a connecting CH1-CH2 loop (Fig. 1). The CH1 helix is partially embedded in the membrane (Fig. 1b) and connected to the CH2 helix by a loop that harbors the cytosolic Ca2+-binding site (Fig. 1b, c and Fig. 2a). This cytosolic Ca2+ is coordinated by backbone and sidechain oxygen atoms from Asp562, Thr563, Asp566 and Asp569 on the CH1-CH2 loop, as well as from Asn161 on the LH5-LH6 loop of the neighboring LHD (Fig. 2a). Previously, it was suggested that the acidic patch of the four tandem aspartates between Asp573-Asp576 comprises the cytosolic Ca2+ binding site in TRPY113 (Extended Data Fig. 6a and 7). Because we could not resolve the density for the seven amino acids between Asp574-Ser580 (Extended Data Fig. 6a and 7) due to flexibility, it is not conclusive from our structure whether this acidic patch indeed is capable of binding Ca2+. However, it is conceivable that, if a Ca2+ ion were to bind at this patch in our purified protein, then it would lead to a greater stabilization of the CH1-CH2 loop allowing us to elucidate the complete structure of the same loop. Moreover, a recent mutagenesis study has reported that this acidic patch is not essential for Ca2+ activation of TRPY114. Furthermore, the acidic patch is not conserved as revealed by the sequence alignment from multiple fungal genomes (Extended Data Fig. 7). Therefore, it is unlikely that this acidic patch binds Ca2+ in purified TRPY1 or in S. cerevisiae. Rather, TRPY1 activation by cytosolic Ca2+ is mediated by Ca2+ binding at the subunit interface as discussed above. Interestingly, Asn161 at the subunit interface is conserved among three (S. cerevisiae TRPY1, K. lactis TRPY2, C. albicans TRPY3) of the six sequences aligned across different fungal species (Extended Data Fig. 7). In the other three (N. crassa, A. niger, A. flavus), an acidic aspartate variation has replaced this asparagine and the CH1-CH2 loop (Arg553-Asp581) is shortened by 6-7 amino acids (Extended Data Fig. 7). The implications of this sequence variation and loop shortening in these three fungal genomes remain unknown, but our analyses suggest the presence of a similar cytosolic skirt structure and cytosolic Ca2+-induced activation mechanism for at least TRPY1, TRPY2 and TRPY3.
The transmembrane domain of the channel was resolved to around 2.8 Å resolution (Extended Data Fig. 1f), which allowed us to visualize a lipid density that we assigned to PI(3)P (Fig. 2b) and a small non-protein density that we assigned to a luminal Ca2+ (Fig. 2c). The PI(3)P lipid was co-purified with the channel from yeast, as lipids were not added during purification. Endogenous yeast PI(3)P is typically found on the intraluminal vesicles of endosomes and vacuoles28, and it has been shown to inhibit TRPY1 channel activity14. The PI(3)P binding site is located between the S1-S4 domain, the S4-S5 linker and the TRP helix of one subunit and S5 of an adjacent subunit (Fig. 2b). The inositol ring of PI(3)P is positioned underneath the S1-S4 domain and is wedged above the TRP helix. In this position, the inositol ring interacts with TRP helix residues Arg483 and Gln479 via its 3’ phosphate and 2’ hydroxyl groups, respectively (Fig. 2b). The PI(3)P position in this pocket is further stabilized by hydrogen bonding between Glu357 and the 5’ hydroxyl on the inositol ring as well as between the backbone amine of Lys370 and the carboxyl from the ester moiety on one of the acyl tails of the lipid (Fig. 2b). Apart from this, the 1’ phosphate of PI(3)P maintains hydrogen bonding with the backbone amines of Phe298 and Trp299 and thus locks the hydrophilic headgroup of PI(3)P in position. The two acyl chains of PI(3)P branch out in a ‘V’ shape. One tail stabilizes in a cleft formed by S4 and the S4-S5 linker of one subunit and S5 and S6 of an adjacent subunit, through hydrophobic interactions. The other tail resides near the S4-S5 linker and we did not capture most of its density probably due to flexibility. A similar position for an inhibitory phosphatidylinositol lipid was observed in the TRPV1 structure23, suggesting a conserved mechanism of lipid-mediated modulation among TRP channel homologues.
The luminal Ca2+-binding site is formed by the loop connecting the S5 and P helices. The bound Ca2+ ion is coordinated by the sidechains of residues Asp398, Asp401 and Asp405, as well as by the backbone carbonyls of Asp398 and Lys403 (Fig. 2c). Since we have captured a closed state of TRPY1, this observation agrees with recent results15 suggesting that two of these residues, Asp401 and Asp405, are involved in the direct binding of Ca2+ and are therefore critical for Ca2+-dependent inhibition of TRPY1. The functional importance of this Ca2+-binding site is further underscored by the fact that Gly402Ser mutation resulted in a constitutively active channel29. However, how the luminal Ca2+ leads to rearrangement of the S6 helix to stabilize the closed conformation of the channel remains to be elucidated. The removal of the luminal Ca2+ would render the P helix flexible, transmitting a gating signal to the pore in S6. We also observe some additional features in TRPY1 such as a π-hinge in the middle of S6, similar to some other TRP channels and two proline residues, Pro432 and Pro433, at the beginning of S6 (Extended Data Fig. 7 and 8). In the absence of luminal Ca2+, these features might have implications in channel gating by allowing more flexibility in S6.
It is interesting to note that the TRPY1 pore structure is almost identical to the NOMPC pore in the closed state, and similar to the pore structures from TRPA1, TRPM4 and TRPC6 (Fig. 3 and Extended Data Fig. 8). Like all other TRP channels, the TRPY1 pore is comprised of a selectivity filter and a lower gate (Fig. 3b). The selectivity filter is formed by the backbone carbonyl oxygen atoms of residues Leu419 and Gly420 (Fig. 3a). Just above the selectivity filter, two negatively charged residues, Asp425 and Glu428, could attract and interact with luminal cations entering the pore. Overall, the luminal side of the pore is strongly negatively charged (Extended Data Fig. 9) as the luminal Ca2+ binding site residues such as Asp398, Asp401 and Asp405 also line the surface (Fig. 2c and 3a). The diameter of the selectivity filter is around 5.6 Å at the Gly420 carbonyl and is large enough to accommodate partially hydrated cations. This is similar to other TRP channels solved in the open state22,30. Therefore, although the selectivity filter is open in TRPY1, the pore is still closed to ion permeation due to a hydrophobic seal at the lower gate formed by Ile455, where the pore diameter is constricted to 1.4 Å (Fig. 3b).
Another interesting aspect of the TRPY1 structure is the network of interactions within each of the subunits that we identify as crucial in stabilizing the closed state of the channel (Fig. 4). These interactions involve the TRP helix, the S4-S5 linker, the LHD, and the CH1-CH2 loop (Fig. 4). The S4-S5 linker forms multiple contacts with the TRP helix which includes a salt bridge interaction between the side chains of Arg360 and Asp470, multiple hydrogen bonds between the side chain hydroxyl of Tyr473 and backbone amide of Arg360, and backbone carbonyls of Glu357 and Ser358 (Fig. 4a). Evidently, Tyr473 is centrally positioned among these residues and the majority of its interacting partners are backbone atoms from the S4-S5 linker. Tyr473 is also conserved across the fungal genomes that we aligned (Extended Data Fig. 7). Together, these observations suggest that Tyr473 is a pivotal residue in the stabilization of the closed state of TRPY1. Earlier studies showed that mutations Tyr458His and Tyr473His increase the open probability of the channel29. These two mutants caused unstable open and closed states of the channel, suggesting their involvement in channel gating29. As deducible from our structure, the first mutant likely destabilizes the S6 helix with either a direct effect on the pore or an indirect effect on the stability of the Arg360-Asp470 salt bridge, or both. The second mutant directly disrupts the three hydrogen bonding interactions between the TRP helix and the backbone of the S4-S5 linker. Intriguingly, PI(3)P is positioned right next to the TRP helix and the S4-S5 linker and provides additional stability to this region, as discussed earlier (Fig. 2b). Therefore, it is possible that PI(3)P interaction and channel gating is coupled in TRPY1. Finally, two salt bridges that are worth mentioning are formed within the same subunit between Arg190 and Arg197 from the LH8 of LHD with Asp557 and Glu560 from the CH1-CH2 loop of the CTD, respectively (Fig. 4b). Together, they seem to stabilize the communication between LHD and CTD in each of the subunits. Overall, an extensive network of salt bridges and hydrogen bonding between different domains of TRPY1 play an important role to maintain the closed state of the channel.
To further elucidate the roles of PI(3)P and Ca2+ in the stabilization of the TRPY1 closed conformation, all-atom MD simulations were carried out on the TRPY1 structure. Five systems were built and simulated (Extended Data Table 1), each representing a different potential state that the channel may exist in vivo or in vitro. Four separate systems (Sim1 to Sim4 in Extended Data Table 1) had the TRP-CTD linker (Ala487-Ser529) in a “vertical” configuration (Extended Data Fig. 10). These include All-Bound (Sim1), in which all PI(3)P lipids and Ca2+ ions are present; None-Bound (Sim2), in which all have been removed; No-Inh (Sim3), in which only the cytosolic Ca2+ sites are occupied while luminal Ca2+ ions and PI(3)P lipids are removed; and the Y473H model (Sim4), in which this gain of function mutation31 (see “network of interactions within each of the subunits” above) was introduced to the No-Inh system in all four subunits. A fifth system featured the TRP-CTD linker in a “horizontal” configuration (Extended Data Fig. 10) with all PI(3)P lipids and Ca2+ ions present. Each system was prepared and simulated with an identical protocol that included 100 ns of production simulations.
The All-Bound horizontal and vertical configurations exhibited noticeable differences in water permeation across the lipid bilayer during simulation (Extended Data Fig. 11). When averaged over the entire simulation trajectory, some water density was observed within the lipid bilayer near the TRPY1 S1-S4 helices and the TRP-CTD linker in both systems, but to a far greater extent in the vertical configuration compared to the horizontal configuration (Extended Data Fig. 11a, c, d and f). There is precedence for the facilitation of ion permeation across the lipid bilayer by similar domains from potassium channels, as the isolated voltage-sensing domain (VSD) of the Shaker Kv channel has been shown to form a cation selective pore32. Voltage-dependent permeation of protons was also shown for the ion channel HV133,34, which has only four transmembrane helices in a VSD-like arrangement (S1-S4) with a predicted hydrated pathway33. The individual S1-S4 subunits of TRPY1 coupled to the TRP-CTD linker and the CH1 helix featuring several charged residues at its N-terminus (Lys514 to Asp518), may function in a similar manner, explaining the observed water permeation. Although both vertical and horizontal configurations are feasible, we focused all subsequent analyses and discussion on our simulations of the channel with the TRP-CTD linker in the vertical configuration.
Simulations revealed that removal of PI(3)P resulted in early indications of pore opening and an overall increase in the dynamics of the TRPY1 structure. In the None-Bound, No-Inh, and Y473H systems, the S4 helix exhibits a greater degree of displacement from its initial position compared to the All-Bound system (Extended Data Fig. 12a-c). A similar result is seen for the S4-S5 linker (Extended Data Fig. 12d-f). The removal of PI(3)P facilitates a greater displacement of key helices likely involved in gating and that may lead to opening of the pore at longer timescales (Extended Data Fig. 12a-f). Additionally, both helices (S4 and S4-S5 linker) in the Y473H system exhibited the greatest degree of deviation, indicating that this mutation influences the dynamics of the transmembrane helices. Removal of PI(3)P also had an apparent effect on the dynamics of the TRP helix, as the distance between opposing TRP helices between two subunits maintained a smaller separation in the All-Bound system compared to the other three simulated systems (Extended Data Fig. 13g-i). These events may facilitate opening of the pore when PI(3)P is removed. Finally, removal of PI(3)P disrupted networks of allosteric communication between the different domains within the same subunit of TRPY1, as revealed by a dynamical network analysis35 (Extended Data Fig. 14). In the All-Bound system, the optimal path of allosteric communication from the N-terminus to the end of the S4-S5 linker (Gly26-Ser376) goes directly from the TRP helix to the S4-S5 linker through Tyr473 (Extended Data Fig. 14a, b), while in every other simulated system the optimal path does not pass through Tyr473 (Extended Data Fig. 14e-f, i-j, and m-n), indicating that the presence of the PI(3)P lipid maintains a more compact network of communication between the TRP helix and the S4-S5 linker. Similarly, the optimal path from the end of the S4-S5 linker to Ile291 at the base of the S2 helix first passes through the S3 and the S2 helix in the All-Bound system (Extended Data Fig. 14c, d). In the other three systems, in which PI(3)P is not present, the optimal path does not pass through the S2-S3 helices (Extended Data Fig. 14g-h, k-l, and o-p). All our analyses suggest that PI(3)P stabilized the closed state.
Analyses of interactions required for the stabilization of the closed state revealed that the Arg197-Glu560 salt-bridge was disrupted to a greater degree in simulations of the None-Bound and No-Inh systems relative to the All-Bound system (Extended Data Fig. 13a-c). Other interactions required for the stabilization of the closed state, such as Arg360-Tyr473 and Lys193-Tyr473, were disrupted by mobile lipid molecules from the bilayer (Extended Data Fig. 13d-f). As these lipid molecules formed interactions with one of the residues involved, the native interaction was disrupted, demonstrating the importance of the surrounding lipid environment to the function of TRPY1.
Next, ion density was analyzed by averaging the location of all K+ ions over the 100-ns long trajectories for all systems. Significant differences between the All-Bound and No-Inh systems were recorded (Extended Data Fig. 15a-d). In contrast to the All-Bound system, K+ density was observed beyond the selectivity filter formed by Leu419 and Gly420 in the No-Inh system (~95-100 Å in the z-axis; Extended Data Fig. 15c, d). This indicates that the removal of bound luminal Ca2+ and more mobility of the associated loops, removal of PI(3)P and greater displacement of transmembrane helices, or a combination of both resulted in the positively charged species having an increased accessibility to the pore of TRPY1, in support of the experimental observations indicating that luminal Ca2+ inhibits opening of the channel15.
As with the luminal Ca2+-binding sites, Ca2+ binding to the cytosolic sites also affected the dynamics and permeation properties of the channel. The Val50-Ser68 loop forms the neck of the cytosolic skirt that may modulate cytosolic ion permeability. The dynamics of this domain depend on the presence of Ca2+ as revealed by simulations. In the None-Bound system, the distance between the center of mass of these loops in opposing subunits was smaller than for the other three systems, and the N-terminal domain exhibited greater deviation from its starting position, indicating that Ca2+ “locks” these domains in place (Extended Data Fig. 13j-l, 12g-i, respectively). This is also revealed in the analysis of average pore radius over the entire trajectory (Fig. 5). Closing of this cytosolic skirt constricts the pore radius within the skirt in the None-Bound system (Fig. 5f), and provides a mechanistic explanation for how the presence of cytosolic Ca2+ leads to a higher open probability observed in experiments14,15. In addition, the density of K+ varies in the cytosolic domain depending on the presence or absence of Ca2+. In all systems where cytosolic Ca2+ is bound, there is an apparent concentration of K+ density in the 40-60 Å range in the z-axis (Extended Data Fig. 15a, c, e, and g). In the None-Bound system, this region is mostly devoid of K+ density, indicating that the closing of the cytosolic skirt as described above precludes K+ entry in this region.
DISCUSSION
TRP channels are absent in bacteria, archaea and plants, but present in all other eukaryotes including algae and fungi36,37. There are 28 family members in mammals that are polymodal and activated by a variety of physical stimuli and compounds. As eukaryotes evolved from single-celled to more complex multicellular organisms, TRP channel proteins may have acquired the ability to sense and respond to stimuli in more sophisticated ways. Thus, TRP channels from single-celled organisms, such as yeast, can be considered as archetypical channels providing insights into the most basic and fundamental properties common to all family members.
The Saccharomyces cerevisiae TRP channel homolog TRPY1 has been extensively studied over the last twenty years using yeast genetic manipulations, functional assays, and electrophysiology, yet its structure has remained elusive14,15,31,38. Biophysical studies have shown that TRPY1 plays a critical role in yeast Ca2+ homeostasis3. It has also been proposed that changes in Ca2+ concentration and mechanical force gate TRPY113. Nevertheless, the molecular basis underlying this channel’s function has not been established. The high-resolution structure of TRPY1 presented here serves as a stepping-stone towards understanding TRP channels’ evolution and TRPY1 gating mechanisms modulated by Ca2+, lipids and membrane stretch in fungi.
Since 2003, structures for representative members from almost all mammalian TRP channel families have been determined24,39. These structures have revealed that mammalian TRPs share structural and functional features, which are also seen in our TRPY1 structure. The architecture of the S1-S4 domains, the pore domains comprising S5, S6 helices and the connecting pore helix, and the TRP box are all structurally conserved between TRPY1 and mammalian TRP channels, despite having low sequence similarity. This is expected since protein-folds exhibit fewer and slower changes compared to sequence during evolution40. Apart from the aforementioned domains, the structural similarity of TRPY1 extends further to NOMPC, TRPM4, and TRPC6 channels in terms of the overall structure of the N-terminal LHD. Like all other TRPs, TRPY1 is also a tetramer. Nevertheless, when compared to TRPM4 and TRPC6 (Extended Data Fig. 5), it is clear that TRPY1 also has some distinct structural features that might have been acquired throughout evolution or might be representative of ancestral features lost in the mammalian members. Interestingly, mammalian TRP channel structures are different compared to the TRP channel homolog in algae, TRP141. This channel adopts a 2-fold symmetrical rose-shape architecture with additional structural elements not seen in TRPY1 or any of the mammalian TRP channels, suggesting that TRPY1 is more evolutionarily related to the mammalian TRP channels than TRP141.
Several unique structural features of TRPY1 are likely to be relevant for channel gating. The TRP-CTD linker and the CH1 helix are tucked against the S1-S4 bundle and insert into the membrane significantly in our model (Fig. 1b, c). This arrangement provides a direct connection between the TRP helix, the periphery of the channel and the surrounding lipid bilayer, which could be relevant for mechanisms of mechanosensation involving force from lipids42. In addition, the CH1 helix wraps on the back of pS1 helix to connect to the cytosolic Ca2+-binding site and CH2, which in turn interacts with the periphery of the neighboring subunit’s LHD, a unique arrangement not seen in any other TRP channel. The stabilization of this architecture by the cytosolic Ca2+ that activates the channel further implicates the TRP helix in channel gating.
The presence of an endogenous PI(3)P lipid in our TRPY1 structure parallels the presence of a phosphatidylinositol lipid in a TRPV1 structure in a closed conformation, with the lipid occupying TRPV1’s vanilloid binding site23. The position of PI(3)P lipids in the TRPY1 structure is therefore suggestive of an active role played by lipids and compounds in channel modulation, also supporting the hypothesis of a basal and evolutionary conserved gating mechanism among TRP channels. It is conceivable that mechanical force generated by hyperosmotic shocks in yeast could induce the release of PI(3)P lipids from their binding site, thus facilitating opening of the channel and Ca2+ flow from the vacuole to the cytosol to start a hyperosmotic shock defense mechanism in yeast (Fig. 6).
The two Ca2+-binding sites revealed in our TRPY1 structure are distinct from those found in other TRP channels that are confined to a cavity between S2 and S3 helices, and the S2-S3 linker that resides in the membrane region30,43,44. Rather, in each TRPY1 subunit, one Ca2+ binds to the cytosolic side and another Ca2+ binds at the vacuolar lumen side (Fig 2a, c). It is fascinating that the two Ca2+-binding sites orchestrate opposite effects on the channel gating, the vacuolar lumen site being inhibiting and the cytosolic site being activating14,15. Simulations presented here offer insights into the possible mechanistic explanations for the experimental results15 concerning the role of the Ca2+ ions. Removal of cytosolic Ca2+ resulted in a domain constriction and a more occluded pore radius in this region (Fig. 5 and Extended Data Fig. 13). Removal of luminal Ca2+, however, led to an increase in the presence of K+ beyond the selectivity filter (Extended Data Fig. 15a, c), demonstrating how these unique Ca2+-binding sites can modulate channel activity on a molecular scale.
All sites were fully occupied by Ca2+ in our TRPY1 structure captured in a closed state, but it appears that only the vacuolar lumen site would remain occupied in the native yeast conditions that favor channel closure. Channel activation may require removal of Ca2+ from the vacuolar lumen site to create a Ca2+-flux directed from the vacuole to the cytosol. Yet, the concentration of Ca2+ in the vacuole is 3,000 times higher than in the cytosol under normal conditions3–5. It has been argued that vacuolar free-Ca2+ is scarce, as most Ca2+ ions are bound to vacuolar polyphosphate45, thus adding another layer of complexity to TRPY1 function in calcium-induced calcium release (CICR) feedback loops in vivo. Notably, TRPY1 is able to integrate various stress signals from the environment to produce a combined effect inside yeast cell in terms of Ca2+ signals. Two of the eukaryotic channels involved in CICR and studied extensively are the Inositol 1,4,5-trisphosphate and the Ryanodine receptors responsible for intracellular Camrelease in cardiac and skeletal muscles46. It is fascinating to note that such feedback loop to regulate an intracellular ion channel was preserved during evolution.
The TRPY1 structure reported here is in a closed state. Given that PI(3)P lipids are strongly bound to the channel, we suggest that an abundance of PI(3)P in the vacuole keeps TRPY1 in its closed conformation. The presence of PI(3)P in the observed binding site brings the TRP helix closer to the S4-S5 linker through interactions between Tyr473 and Arg360 residues. The destabilization of this interaction, which is probably the cause of gain-of-function31 in the TRPY1 Y473H mutant, might lead to opening of the channel upon application of different endogenous stimuli that are proposed to gate TRPY1. Our proposed model is in agreement with the notion that the S4-S5 linker is the “gear box” conveying gating force in TRP channels and suggests that TRP channels maintained a conserved gating mechanism throughout evolution. This is supported by simulations, in which the removal of PI(3)P had several effects on the dynamics and interactions implicated in channel activity. In particular, allosteric communication between the NTD and the S4-S5 linker was directly mediated by the Tyr473-Arg360 interaction only when PI(3)P was present (Extended Data Fig. 14).
The open state of TRPY1 has a conductance >300 pS (180 mM KCl), suggesting that gating requires conformational changes that can lead to a pore opening of as much as 10 Å in diameter estimated using Hille’s equation47,48. It is tempting to speculate that mechanical force from lipids is communicated through the TRP-CTD linker and the S1-S4 domains to the TRP and S4-S5 linker helices to gate the channel, and that this requires an intact skirt with bound Ca2+. However, how various parts of TRPY1 re-arrange to accommodate a larger pore that can sustain its conductance remains to be elucidated.
MATERIALS AND METHODS
Protein expression and purification
Wild type TRPY1 containing YepM plasmid49 was transfected into BJ5457 Saccharomyces cerevisiae (ATCC) for constitutive expression of the protein. The cells were grown at 30°C until OD600 reached mid log phase (1.2-1.4) and thereafter harvested to store at −80°C for future use. Steps hereafter were carried out at 4°C. Membranes containing expressed TRPY1 were prepared by thawing the cells on ice and breaking them using a M110Y microfluidizer (Microfluidics) in homogenization buffer containing 25 mM Tris-HCl, pH 8.0, 300 mM Sucrose and 5 mM EDTA and protease inhibitor cocktail (Sigma). Membranes were then pelleted by first discarding cellular debris with 3,000 x g and 14,000 x g centrifuge runs, then 100,000 x g ultra-centrifugation to pellet the membranes before storing them in 5 mM Tris-HCl, pH 8.0, 300 mM Sucrose and 1 mM PMSF. The membranes were solubilized in 20 mM HEPES pH 8.0, 150 mM NaCl, 10% glycerol, 1% digitonin, 2 mM TCEP, and 1 mM PMSF for 2 h. Insoluble material was removed via ultra-centrifugation at 100,000 x g and the solubilized TRPY1 was purified by binding to 1D4 antibody coupled to CnBr-activated Sepharose beads. The beads were used to make a column and extensively washed with Wash Buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl, 0.01% glyco-diosgenin and 2 mM TCEP. The protein was eluted from the column with Wash Buffer supplemented with 3 mg mL-1 1D4 peptide and concentrated to 4.3 mg mL-1 using a 100-kDa concentrator (Millipore) before further purifying by size exclusion chromatography using a Superose 6 increase 10/300 GL column (GE Healthcare). The eluted protein was concentrated to 2 mg mL-1 and used for vitrification.
Specimen preparation and cryo-EM data acquisition
Prior to preparing cryo-EM grids, purified protein was incubated with 2 mM CaCl2 for 10 min. This sample was blotted once (3.5 mL per blot) onto glow discharged 200 mesh Quantifoil 1.2/1.3 grids (Quantifoil Micro Tools) at 4°C and 100% humidity and plunge frozen in liquid ethane cooled to the temperature of liquid nitrogen (Thermo Fisher Vitrobot Mark IV). Cryo-EM images were collected using a 300 kV Thermo Fisher Krios microscope equipped with a Gatan K3 direct detector camera in super resolution mode. Forty frame movies were collected with a total dose of 45 e/ Å2 and a super resolution pixel size of 0.53 Å/pix. Defocus values of the images ranged from −0.5 to −2.5 μm.
Image processing
Relion 3.1 (version: beta-commit-b86482)50–52 was used for data processing of 11,099 super-resolution image stacks (Extended Data Fig. 3). Relion’s implementation of MotionCor250,53 was employed to compensate for beam-induced movement and to set the pixel size to 1.06 Å following 2×2 binning. CTFFIND-4.154 was used to estimate CTF parameters. Laplacian-of-Gaussian autopicking was performed on a small subset (100) of the motion-corrected images followed by two rounds of 2D classification to obtain representative templates for autopicking from the full dataset. Thus, 20,947 initial particles were autopicked, which after two rounds of 2D sorting yielded the best representative template for autopicking on the complete dataset. Autopicking on complete dataset yielded ~3.1 million particles. These particles were 4x-binned and subjected to two rounds of 2D sorting to discard false positives and bad particles. The remaining class averages containing 254,509 particles showed clear signs of being originated from a tetrameric TRP-like channel. These particles were 2x-binned and used to generate an initial model that was subsequently used as a reference to perform a 3D classification with six classes. One class among these six, containing 100,918 particles, aligned well and therefore was better resolved than the rest. Using the map from this class, a loose mask was generated surrounding the tetramer and detergent belt, and thereafter used for 3D refinement and 3D classification of 100,918 unbinned particles applying C4 symmetry and mask. One among the three 3D classes generated, containing 55,593 particles, was subsequently 3D refined to obtain a 3.93 Å map which was then subjected to CTF refinement with corrections for higher-order aberration, anisotropic magnification and per particle defocus, and Bayesian polishing. 3D refinement after Bayesian polishing yielded a 3.35 Å map. A second round of CTF refinement and Bayesian polishing, and a subsequent 3D refinement generated a 3.19 Å map which after postprocessing yielded a 3.08 Å map. This 3.08 Å map was used to build atomic models. The local resolution map was generated using Relion50.
Model building and refinement
An initial model for a single subunit of the full length TRPY1 was generated using the online server ITASSER55,56. This model was roughly partitioned into cytosolic, transmembrane and C-terminal domains and rigid body docked into their respective densities. Except for the transmembrane domain, which showed satisfactory fitting, the other two domains did not fit well and therefore were manually adjusted in COOT57 to fit the density. The quality of density was comparatively lower in the C-terminal domain and therefore extra care was taken to account for correct registers in the side chain assignments. To assign side chains in the C-terminal helix CH1 and the loop thereafter, spanning Lys517-Asp573, confidence was derived from the distinctive identity of the bulky side chains from Lys538, Arg541, Arg544, Arg545, Tyr548, Arg550, Tyr561, Trp565, Tyr571 and the kinks produced by Pro530 and Pro564. Due to poorer density of CH2, the original I-TASSER model that predicts a helix in this region was fitted. A model for the PI(3)P lipid was built in COOT and thereafter the eLBOW58 tool from the PHENIX59 software package was used to generate restraints for refinement. Models for Ca2+ ions were generated in COOT and were kept unlinked to the protein during refinement. The complete model was then iteratively refined using phenix.real_space_refine from the PHENIX59 software package with secondary structure and NCS restraints for the protein and eLBOW-generated restraints for PI(3)P. In the final model, side chains for Leu216, Phe225, Leu572, Asp573 and Lys517-Ser529 were pruned due to insufficient density. Model validation was carried out in Molprobity60 and EMRinger61. For cross-validation, each final model was randomized by 0.5 Å in PHENIX59 and refined against a single half map. These models were converted into volumes in Chimera62 and then EMAN2.163 was used to generate FSC curves between these models and each half map as well as between each final model and the final maps. HOLE64 was used to generate the pore radii. Electrostatic potential was calculated using APBS-PDB2PQR65. Figures were prepared using PyMOL66 and Chimera62 software.
TRPY1 molecular dynamics simulations and analyses
The cryo-EM structure of TRPY1 generated in the current study (PDB: 6WHG) was modified as described below and thereafter used for all MD simulations and their associated analyses. The cryo-EM structure has seven missing segments in each chain due to a lack of electron density, from residues Met1 to Asn24 (segment 1; N-terminus), Asp55 to Glu65 (segment 2; loop in cytosolic skirt), Leu216-Phe225 (segment 3; pre-S1 elbow), Pro323-Lys327 (segment 4; loop between S3 and S4 helices), Ala487-Ser529 (segment 5; TRP-CTD linker), Leu572-Ser580 (segment 6; part of C-terminus), and Asp608-Glu675 (segment 7; C-terminal tail). Segments 1 and 7 were not included in our models. Segments 3, 4, and 6 were built using the interface to the Modeler server in Chimera62. The top result for each was chosen and was minimized in vacuum for 10,000 steps in NAMD67,68 with constraints placed on all but the modeled segments (this and subsequent harmonic constraints used k = 1 kcal mol-1 Å-2). Real space refinement was performed on the resulting model to better fit the cryo-EM map. Segment 2 was built in COOT57, based on the cryo-EM map with C4 symmetry applied and no sharpening. This was then minimized in vacuum for 50,000 steps in NAMD with all but segment 2 constrained. Because segment 5 was predicted to contain a helix based on its sequence, this segment was submitted to the Phyre2 homology-modeling server69. The top model from Phyre2 contained an amphipathic helix and was inserted into the TRPY1 structure using COOT. This segment was then minimized in vacuum for 10,000 steps in NAMD with all but segment 5 constrained. This was done for a single chain. To insert the missing segments for the remaining three chains, the completed chain was aligned to the other three chains in COOT, and the coordinates of the missing segments were saved. These were then inserted into the remaining chains, and all completed chains were saved as a single model, which was then vacuum minimized for 50,000 steps in NAMD, with all but the missing segments constrained.
The TRPY1 structure (PDB: 6WHG) also contains four PI(3)P molecules bound as ligands with a partial missing tail. To complete these ligands, the internal coordinates from the SAPI13 molecule in the toppar_all36_lipid_inositol.str CHARMM parameter file were used with the autopsf plugin. The entire structure was then vacuum minimized in NAMD for 50,000 steps with all but the PI(3)P molecules constrained. The final coordinates were saved, and this entire structure was embedded in a 150 × 150 Å membrane patch that contained 50% POPC, 18% POPA, 16% POPS, and 16% POPI. The membrane patch was generated using the Membrane Builder function on the CHARMM-GUI server. The transmembrane region was placed based on hydrophobic regions of the core helices, proximity of tryptophan residues to lipid head groups, and previous simulations performed on TRP channels70,71. Then, the solvate plugin in VMD72 was used to add TIP3P water molecules to the system, and the autoionize plugin was used to neutralize the system and add KCl to a final concentration of 150 mM.
Simulations were ran using NAMD 2.1267,68 and the CHARMM36 force field73. The TIP3P74 explicit model of water was used. Long-range electrostatic forces were computed using the Particle Mesh Ewald method with a grid point density of >1 Å-3, and a cutoff of 12 Å was used for van der Waals interactions. The SHAKE algorithm was used with a timestep of 2 fs. The NpT ensemble was used at 1 atm with a hybrid Nosé-Hoover Langevin piston method and a 200 fs decay period, with a 50 fs damping time constant. The completed simulation system was minimized for 1,000 steps, followed by a lipid-melting step in which all but the lipid tails were constrained for 0.5 ns of equilibration. This was followed by 0.5 ns of equilibration in which the protein was constrained and the water, lipids, and ions were free. Next, constraints were released on the modeled segments of the protein for 10 ns, allowing them to equilibrate with their surroundings. Finally, all constraints were lifted and the system was ran for 20 ns using a Langevin damping coefficient of γ = 1, after which γ was set to 0.1 for 100 ns of free equilibration. All analyses were performed on the final 100 ns for every simulation.
A total of 5 simulations were prepared and ran as described above. The first four are variations of a “vertical” configuration, in which the CTD-linker was modeled with its helix segment vertically across the membrane (Extended Data Fig. 10). The first is the All-Bound system, in which all Ca2+ and PI(3)P sites are occupied. The second is the None-Bound system, in which all Ca2+ and PI(3)P are removed. For the third system, labeled No-Inh, only the non-inhibitory (cytosolic) Ca2+-binding sites remain occupied, while the PI(3)P and inhibitory (luminal) Ca2+ ions were removed. Finally, in the fourth system, a gain of function Y473H mutation was introduced to the non-inhibitory system using VMD’s Mutate Residue plugin.
An additional system was built based on the “horizontal” configuration, in which the amphipathic helix of segment 5 (TRP-CTD linker) was modeled as laying horizontally at the lipid-cytosolic interface (Extended Data Fig. 10). This system, besides segment 5, is identical to the All-Bound system of the vertical configuration.
The network analysis was performed following standard protocols35. First, a dynamic network is created from the MD trajectory in VMD using Carma75, a program that calculates correlation between atoms in an MD simulation. Optimal and suboptimal paths are calculated by first choosing a source and sink node, and the communication pathways between these nodes are calculated for the minimal number of nodes. Those nodes that occur in the most sub-optimal paths are critical nodes for allosteric communication between source and sink nodes. All visualization of molecular images and allosteric pathways was performed in VMD. Ion and water densities were calculated using the VolMap tool plugin in VMD. The average density was computed and combined for all frames with no weight after alignment. These data, given in number of atoms/Å3, were then converted to a contour map and reported in molarity.
For each frame of the given simulation, RMSD was calculated based on the alignment of a selection of Cα atoms to the same selection of atoms in the first frame of the trajectory. In this way, the RMSD analysis of every system was done based on the same starting structure.
The average pore radius (Fig. 5) was calculated using the program HOLE64. After alignment of the protein to the first frame of the trajectory, HOLE calculates the pore radius in every frame of the trajectory along the path of the pore in the z-dimension. Next, these values are averaged throughout the trajectory at each position in the pore, with error bars in Fig. 5 indicating the standard deviation.
Data availability
The cryo-EM density map and the atomic coordinate of PI(3)P and Ca2+-bound full-length TRPY1 in detergent are deposited into the Electron Microscopy Data Bank and Protein Data Bank under accession codes EMD-21672 and 6WHG. To avoid repetition, we have not submitted the apo condition map and model in the databases.
Acknowledgements
We thank Sabine Baxter for assistance with hybridoma and cell culture at the University of Pennsylvania Perelman School of Medicine Cell Center Services Facility. We acknowledge the use of instruments at the Electron Microscopy Resource Lab and at the Beckman Center for Cryo Electron Microscopy at the University of Pennsylvania Perelman School of Medicine. We also thank Darrah Johnson-McDaniel for assistance with Krios microscope operation. This work was supported by grants from the National Institute of Health (R01GM103899 and R01GM129357 to VYM-B). Use of TACC-Stampede2 and PSC-Bridges supercomputers was supported by the National Science Foundation through XSEDE (XRAC MCB140226 to MS). Use of resources at the Ohio Supercomputer Center was supported by grants PAS1037 and PAA0217 to MS. CN was supported by an OSU/NIH molecular biophysics training grant (T32GM118291).