The transient receptor potential (TRP) ion channel superfamily is involved in a broad range of 36 physiological processes and their dysregulation results in various diseases [1] [2] [3] . TRP 37 channels, formed from homo-or hetero-tetramers, contain a central pore that functions as a 38 cation channel. Mammalian TRP family proteins have been classified into six subfamilies 39 based on sequence similarity [2] . The vanilloid TRP subfamily (TRPV1-6) has been further 40 classified into two subgroups; the thermosensitive, Ca 2+ non-selective TRPV1-4 channels 41 (known as thermo-TRPs) and TRPV5-6, which are both insensitive to temperature and Ca 2+ 42 selective [4] [5] [6] [7] [8] . 43 44 TRPV4, first described as an osmotically activated channel [9, 10] , is a thermo TRP ion 45 channel that has been shown to play a prominent role in a multitude of biological processes 46 and dysregulation of its activity has been associated with several human diseases [11] [12] [13] . 47 -8 - of GSK1016790A after RNA injection for full-length TRPV4 (black) and truncated TRPV4 148 (gray) constructs. In these measurements the cells were clamped at a holding potential of -60 149 -9 -mV. The full-length TRPV4 channel reached its strongest signal after 48h and was stable for 150 at least 72h. Comparable currents were observed for the truncated construct after 72h. Based 151 on these observations dose-response curves shown in Fig. 1 were determined 48h and 72h 152 after RNA injection for full-length and truncated TRPV4, respectively. 153 (b) Representative oocyte two-electrode voltage clamp (TEVC) responses for hTRPV4 154 variants stimulated with GSK1016790A and 4α-PDD. Representative traces showing the 155 time-dependent current responses for oocytes expressing either truncated TRPV4 treated with 156 30µM GSK1016790A (blue) or full-length TRPV4 treated with 30µM 4α-PDD (gray). In the 157 measurements the cells were clamped at a holding potential of -60 mV. The truncated 158 hTRPV4 channel exhibited a fast response to GSK1016790A that reached a steady-state 159 current with the first 60s. In contrast, the full-length hTRPV4 construct exhibited a slower 160 response to 4α-PDD. In all 9 trials a continuously slow increase in the current was observed 161 throughout the course of the experiment (9 minutes). The truncated TRPV4 construct did not 162 respond at the concentrations of 4α-PDD tested. Based on the reduced GSK1016790A 163 efficacy in activating the truncated channel ( Fig. 1) it is likely that the concentrations of 4α-164 PDD required to activate the truncated channel are not technically achievable in this assay (upper trace). The full-length channel responded to both 0.3µm (shown) and 1µM (not shown) 175 GSK1016790A with currents of 2065 ± 220 nA (n=8 of 11 tested cells). A significant 176 truncated hTRPV4 response was also observed upon stimulation with 30µM GSK1016790A 177 n=2 of 3 tested cells). 178 -10 -We then used cryo-electron microscopy (cryo-EM) to determine the 3D structure of hTRPV4 180 in the detergent-solubilized state. The baculovirus-expressed hTRPV4 channel was stably 181 reconstituted in buffer containing the detergent glyco-diosgenin (GDN) and purified to 182 homogeneity. In order to obtain information about the 4α-PDD molecular mode-of-action, 183 hTRPV4 was incubated with an approximate 10-fold molar excess of 4α-PDD and the 184 structure of the resulting sample was solved using cryo-EM. Due to the limited aqueous 185 solubility of 4α-PDD and the need to minimize DMSO effects during grid freezing, higher 186 working concentrations of 4α-PDD were not achievable. 187
After optimization of the cryo-EM grid freezing conditions, hTRPV4 particles nicely entered 189 thin ice and a good particle distribution in recorded images was obtained (Extended Data 190 Fig. 2a) . Two-dimensional class averages or extracted particle images showed that the sample 191 adopted diverse orientations on the grid and secondary structural features were easily 192 discernable (Extended Data Fig. 2b ). Analysis and 3D sub-classification of the hTRPV4 193 particles indicated the presence of several conformational states (Extended Data Fig. 2d) . In 194 this contribution we present our analysis of the major conformational state which displayed 195 features associated with a single tetrameric assembly. A four-fold rotational C4 symmetry was 196 applied during the final stages of the cryo-EM structure reconstruction and the structure was 197 refined at a global resolution of 4.1Å (Extended Data Fig. 2e , Extended Data Table 1) . 198 Despite significant differences in the local resolution throughout the channel (Extended Data 199 Fig. 2g ), map quality allowed unambiguous placement of all secondary structural elements 200 and large bulky side-chains confirmed the correct registry assignment throughout the protein 201 sequence. The resulting cryo-EM 3D reconstruction represents the first high-resolution 202 hTRPV4 protein structural data (Fig. 2a) . transmembrane domain flanked by extra-and intra-cellular domains (Fig. 2a) . The 236 membrane-spanning TMD domain comprises six alpha-helices (S1-S6) and a TRP domain 237 together with several adjacent helices in the elbow and pore region (Fig. 2b) . The S1-S6 238 helices are ordered into two sub-domains whereby helices S1-S4 form an alpha-helical VSLD 239 bundle that is flanked by the pore-lining S5-S6 helices in a domain-swapped arrangement. 240
The adjacent intracellular domain comprises the ankyrin-repeat domain (ARD) and a helix- We hypothesized that an additional, strong non-protein feature in the cryo-EM map at the 255 interface between the S1, S2 and TRP helices within the VSLD site represented the binding of 256 4α-PDD to hTRPV4 (Fig. 2c) . The limited local resolution in this region of the map 257 (Extended Data Fig. 2g) , together with the inherently high degree of 4α-PDD structural -15 -flexibility, did not allow unambiguous modelling of the 4α-PDD binding mode. In order to 259 validate this binding site, we therefore designed and characterized a series of hTRPV4 point 260 mutants targeting both this and other known activity-modulating binding sites (Table 1) Fig. 3) . Furthermore, the N474A mutant also 286 displayed an increased EC 50 after treatment with 4α-PDD, albeit less strong than the N474Q 287 mutant. Both N474 and R594 are located directly adjacent to the 4α-PDD density, supporting -17 -the identification of the 4α-PDD binding site (Fig. 2c) . In contrast, W586 is located at the 289 inter-subunit interface approximately 15 Å from the proposed 4α-PDD binding site, where it 290 closely interacts with the S5 helix from an adjacent hTRPV4 protomer within the tetrameric 291 assembly (Extended Data Fig. 4) . Since W586 is surface exposed in the closed TRPV4 292 conformation, we hypothesize that the effects of the W586A mutation result from disruption 293 to the formation of the open hTRPV4 conformation and not via direct disruption of 4α-PDD 294 binding. It is, however, important to note that such conclusions cannot be fully supported by 295 calcium flux measurements alone. More detailed electrophysiological characterization 296 experiments will be required to fully characterize the disrupting mechanism of this mutation. 297
The fourth mutant with strong disruptive effects, S548V, was discovered serendipitously. It is 298 located within the S2/S3 loop adjacent to the putative 4α-PDD binding site. Whilst the loop is 299 close to the proposed binding site, a detailed molecular rationale of its effect has not been 300 possible, since this loop is not clearly resolved in the maps. A high degree of flexibility within 301 the S2-S3 loop has also been observed in other TRP channel structures (Extended Data Fig.  302   5) . 303
The other tested mutants displayed, comparatively, only weak to modest effects on 4α-PDD-305 mediated channel activation. The Q550A mutation is located in the vicinity of the 4α-PDD 306 binding site, and whilst the main-chain residues could be clearly modelled, the side-chain 307 could not be unambiguously built. However, considering its location and likely side-chain 308 conformations, it is conceivable that the Q550 mutation disrupts 4α-PDD activity. The Y556A 309 mutation, located at the back of the 4α-PDD binding site directly at the interface between the 310 S1 and S2 helices, resulted in an approximate 13-fold increase in the EC 50 . Two additional 311 mutants were characterized to probe the impact of mutating the vanilloid binding or EET site 312 on 4α-PDD activity. These mutants, F549A and K535A, resulted in either weak, or not -18 -significant changes to the 4α-PDD mediated activation and therefore suggest that 4α-PDD 314 activity is not mediated via direct interactions with these alternative activity modulating sites. 315
Collectively, these data support the identification of the 4α-PDD binding site at the interface 316 between S1/S2/TMD helices within the VSLD-binding site. 317 -19 - mutation has also been observed to differ between other TRPV4 stimuli. For example, whilst 357 W586A also disrupts the channel sensitivity to bisandrographolide A and heat, the mutation 358 has no effect on the channel response to cell swelling, arachidonic acid and 5,6-EET [35] . As 359 for 4α-PDD, vanilloid-binding sites mutants displayed only weak effects on GSK1016790A 360 potency, changing the potency by less than an order of magnitude. Taken together, these 361 shared trends suggest an overlap between the 4α-PDD and GSK1016790A binding sites. 362
Future in-depth electrophysiological characterizations, which are outside the scope of this 363 project, will be required to disentangle the molecular mechanisms. 364 
To investigate the molecular basis of 4α-PDD induced activation we next built an apo, closed 376 conformation hTRPV4 homology model using a closed conformation xTRPV4 structure as a 377 template [25] . Comparison of the pore structure of this model with our 4α-PDD-complexed 378 hTRPV4 structure revealed that whilst the upper gate architecture is retained between the two 379 states, the lower gate region changes significantly. Transition to the open-state is accompanied 380 by an approximate 90º counter-clockwise rotation of the S6 helix (as viewed from the 381 extracellular side), resulting in I715 replacing M718 as the residue defining the constriction 382 point ( Fig. 3a/ translates into an increased van der Waals radius (from 0.8 Å to 1.8Å) and thus movement of 386 a hydrated ion through the channel is possible (Fig. 3c) . We therefore conclude that in the 387 presence of 4α-PDD, hTRPV4 is stabilized in an open ion-channel conformation. The altered pore properties result from large conformational changes to the TMD region such 414 that the relative orientation of the S1-S4 bundle and the pore-forming region are strikingly 415 different in the open and closed states. Compared to the closed state, the open state S1-S4 416 helical bundle is rotated approximately 90º (clockwise, as observed from extracellular side) 417 about the S4 helical axis (Fig. 4a, Suppl. Movie S2) . The internal arrangement of helices 418 within the S1-S4 domain itself does not change between open and closed conformations (rmsd 419 -27 -of 1.7Å for residues 467-594). In contrast, interactions between the S1-S4 helical bundle and 420 several flanking structural channel elements change significantly. Most strikingly, upon 421 channel activation the TRP helix orientation changes by approximately 60º, such that it then 422 forms an integral part of the 4α-PDD binding pocket. The TRP helix is positioned directly 423 between the S1 N-and S2 C-termini, orthogonal to the plane of the two helices, and is 424 therefore ideally positioned to form direct interactions with 4α-PDD in the open conformation 425 (Fig. 4b, Suppl. Movie S3) . In contrast, the TRP helix is located further away from the S1/S2 426 helices in the closed state and does not contribute directly towards the 4α-PDD binding site. sharing RMSDs in the range of 2-3 Å. Thus, whilst TRPV4 displays a non-archetypal closed, 446 or inactive conformation, its active conformation extends the TRP channel members for which 447 the active conformation TRP channel paradigm is conserved (Extended Data Fig. 6) . its favorable docking score, a putative binding mode was identified that is consistent with 481 both our mutagenesis data and published 4α-PDD structure-activity relationships [32] (Fig. 5) . 482
In this model, the core diterpenoid moiety binds at the interface between the S1, S2 and TRP 483 helices, where it forms specific hydrogen bonds with protein residues within the binding site, 484 most notably with N474 (Extended Data Fig. 7a) . The long and highly flexible lipophilic 485 alkyl chains extend out of the pocket into the areas embedded within the hydrophobic 486 membrane environment. This is consistent with published SAR studies indicating that the acyl 487 chains are involved in positioning the diterpenoid core for binding, rather than interacting 488 with the binding site [32] . Furthermore, the sequence conservation between the different -32 -thermo TRPs within this pocket shows divergent residues lining the site (Extended Data Fig.  490 7b), consistent with 4α-PPD activity with TRPV4, but not TRPV2 and TRPV3 [11] . 491 -33 - The high-resolution cryo-EM structure of human TRPV4 in complex with 4α-PDD provides 515 first insights into the agonistic molecular mode-of-action. Despite only moderate activity of 516 the agonist in both, cellular electrophysiology and FRET assays, as well as in biophysical 517 assays measured in detergent solubilized protein, the concerted mode-of-action of the four 518 binding sites within the homo-tetrameric assembly results in large conformational changes to 519 the ion-channel architecture that collectively result in channel pore opening. A combination of 520 complementary structural and biochemical data allowed location of the allosteric 4α-PDD 521 binding site within the S1-S4 bundle. This allosteric agonist binding site is perfectly located to 522 facilitate the structural change from the closed to the open conformation through interactions 523 with the TRP helix. Several structurally diverse small molecule effectors have now been 524 observed to bind at this allosteric binding site in several members of the TRP-family of ion-525 channels (Fig. 6) . Intriguingly, whilst compound binding at this site in the thermo-TRP family 526 elicits agonistic effects, binding to the related TRPV5 and TRPV6 proteins results in 527 antagonist effects. Obviously, small differences in the chemical structure of the small 528 molecule at the binding site modulate very different overall structural response that leads to 529 activation or inhibition of the channel. This concept is well known in the area of GPCR 530 research and observed in CCK2, CCR2, 5HT, D2, opioid receptors and many more. For 531 example, replacing the N-methyl group on potent m-opioid agonist morphine (Ki=0.53nM) 532
with N-allyl resulted in a potent antagonist nalorphine (Ki=0.36nM). Furthermore, addition of 533 an aliphatic side chain to a Ghrelin receptor modulator changed it from a 36nM agonist into a 534 -36 -20nM antagonist [43] . Consequently, the ligand binding site exploited by 4α-PDD in hTRPV4 535 exemplifies this activity switch for an ion-channel and more examples might be identified in 536 the future due to the increasing structural information for this class of drug targets. Further 537 high-resolution studies with potent agonists and antagonists at this binding site within 538 hTRPV4 are required to further understand the mechanism of activity response and structural 539 requirements. 540
The structural and biochemical data presented here are a valuable tool to support the further 542 exploration of TRPV4 biology and its pharmacological responses, as well as the rational 543 design of effective drug molecules. The interpretation of further functional characterization 544 within the context of a three-dimensional target understanding promises to provide detailed 545 insights into the processes regulating TRPV4 activity. Structural data has now identified 546 several activity-modulating sites common to various TRP ion channel family members. 547
Overall, this knowledge provides a great opportunity to support structure-guided design 548 towards potent, selective and optimized novel drug molecules in a broad spectrum of human 549 diseases. 