bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
1
2
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4 Structural basis for human TRPC5 channel inhibition by two distinct inhibitors
5
6 Kangcheng Song 1,4, Miao Wei 1,4, Wenjun Guo 1,4, Yunlu Kang 1,
7 Jing-Xiang Wu 1,2,3 and Lei Chen1,2,3*
8
9 1 State Key Laboratory of Membrane Biology, Institute of Molecular Medicine, Peking
10 University, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Beijing 100871,
11 China
12 2 Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China
13 3Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China
14 4 These authors contributed equally
15 * Correspondence to Lei Chen: [email protected]
16 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
17 Abstract
18 TRPC5 channel is a non-selective cation channel that participates diverse physiological
19 processes. Human TRPC5 inhibitors show promise in the treatment of anxiety disorder,
20 depression and kidney disease. Despite the high relevance of TRPC5 to human health, its
21 inhibitor binding pockets have not been fully characterized due to the lack of structural
22 information, which greatly hinders structure-based drug discovery. Here we show cryo-EM
23 structures of human TRPC5 in complex with two distinct inhibitors, namely clemizole and
24 HC-070, to the resolution of 2.7 Å. Based on the high-quality cryo-EM maps, we uncover the
25 different binding pockets and detailed binding modes for these two inhibitors. Clemizole
26 binds inside the voltage sensor-like domain of each subunit, while HC-070 binds close to the
27 ion channel pore and is wedged between adjacent subunits. Both of them exert the inhibitory
28 function by stabilizing the ion channel in a closed state. These structures provide templates
29 for further design and optimization of inhibitors targeting human TRPC5. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
30 Introduction
31 The mammalian transient receptor potential canonical (TRPC) channels are Ca2+-permeable
32 nonselective cation channels that belong to the transient receptor potential (TRP) channel
33 superfamily (1). Among all the subfamilies of TRP channels, TRPC share the closest
34 homology to the Drosophila TRP channel, the first TRP channel cloned (2). The TRPC
35 subfamily has seven members in mammals, TRPC1 to TRPC7, while TRPC2 is a pseudogene
36 in human. According to the primary amino acid sequences, TRPC channels can be divided
37 into TRPC1, TRPC2, TRPC3/6/7 and TRPC4/5 subgroups (3). Among them, TRPC5 is
38 mainly expressed in the brain, and in liver, kidney, testis and pancreas to a lesser extent (4-6).
39 It can form homotetrameric channels or heterotetrameric channels with TRPC4 and/or
40 TRPC1 (7, 8). Upon activation, TRPC5 leads to depolarization of cell membrane and increase
2+ 2+ 41 of intracellular Ca level ([Ca ]i). TRPC5 mediates diverse physiological processes and is
42 implicated in many disease conditions such as fear, anxiety, depression and progressive
43 kidney disease (9, 10).
44 Physiologically, TRPC5 channel is regulated by several mechanisms, including cell surface
45 receptors, calcium stores, redox status and several cations. TRPC5 can be activated by
+ + 46 receptor activated Gq/11-PLC pathway upon the dissociation of Na /H exchanger regulatory
47 factor (NHERF) and activation of IP3 receptors (11-13), or through the Gi/o pathway (14). By
48 interacting with STIM1, TRPC5 functions as a store-operated channel (15, 16). Extracellular
49 application of reduced thioredoxin or reducing agents enhances TRPC5 activity (17). TRPC5
2+ 50 is also regulated by [Ca ]i in a concentration-dependent bell-shaped manner (15, 18-20) and
2+ 2+ 51 extracellular Ca ([Ca ]o) at concentration higher than 5 mM robustly activates TRPC5 (11,
52 15). bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
53 Several available pharmacological tool compounds permit the identification of TRPC5 as
54 putative drug targets for certain diseases (21-23). (-)-englerin A (EA), a natural product
55 obtained from Phyllanthus engleri, can selectively activate TRPC4/5 and therefore inhibit
56 tumor growth (24, 25), suggesting activation of TRPC4/5 might be a strategy to cure certain
57 types of cancer. In contrast, TRPC5 inhibitors show promise for the treatment of central
58 nervous system (CNS) diseases and focal segmental glomerulosclerosis (FSGS) in animal
59 models (26-28). A TRPC4/5 inhibitor developed by Hydra and Boehringer Ingelheim is in
60 clinical trial for the treatment of anxiety disorder and depression (29). Another TRPC5
61 inhibitor GFB-887 developed by Goldfinch Bio is currently in phase 1 clinical trial for the
62 treatment of kidney disease (NCT number: NCT03970122).
63 Among TRPC5 inhibitors, clemizole (CMZ) and HC-070 represent two classes with distinct
64 chemical structures. CMZ is a benzimidazole-derived H1 antagonist that can inhibit TRPC5.
65 The same class of compounds includes M084 and AC1903 (28, 30, 31). In contrast, HC-070
66 is a methylxanthine derivative which inhibits TRPC4/5 with high potency (27). Another
67 hTRPC5 inhibitor HC-608 and activator AM237 belong to this class (27, 32). The distinct
68 sizes, shapes and polarities between CMZ and HC-070 type inhibitors suggest they bind
69 hTRPC5 at different pockets which are still elusive due to the lack of structural information.
70 This greatly impede further structural-based compound optimizations. To understand the
71 inhibitory mechanism of these two distinct inhibitors against human TRPC5 (hTRPC5), we
72 embarked structural studies of hTRPC5 in complex with CMZ or HC-070. Our high
73 resolution cryo-EM maps are sufficient to identify their binding pockets and will aid further
74 drug development.
75
76
77 Results bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
78 79 Structure of human TRPC5 in complex with inhibitors
80 To reveal the binding sites of CMZ and HC-070 on hTRPC5, we expressed hTRPC5 in
81 HEK293F cells for structural studies. Similar to the mouse TRPC5 counterpart (mTRPC5)
82 (33), C-terminal truncation of hTRPC5 generated a construct (hTRPC51-764) that showed
83 higher expression level of the tetramer in comparison with the full length wild type hTRPC5
84 (Fig. S1A). Moreover, we found hTRPC51-764 retained the pharmacological properties of full
85 length hTRPC5 such as activation by EA (Fig. S1, B to E). Therefore, we used hTRPC51-764
86 protein for structural studies. Tetrameric hTRPC51-764 channel was purified in detergent
87 micelles (Fig. S1F and G). To obtain the CMZ-bound and the HC-070-bound TRPC5
88 structures, CMZ or HC-070 was added to the concentrated hTRPC51-764 protein for cryo-EM
89 sample preparation. We obtained maps of hTRPC5 in CMZ-bound state and HC-070-bound
90 state to the resolution of 2.7 Å (Fig. 1, Figs. S2 to S5; table S1). The map qualities were
91 sufficient for model building and ligand assignment (Fig. S3, D and E, and Fig. S5, D and E).
92 The overall architecture of inhibitor-bound hTRPC5 is similar to the structure of mTRPC5 in
93 apo state (33), occupying 100 Å × 100 Å × 130 Å in three-dimensional space (Fig. 1). The
94 four-fold symmetric channel has two layer architecture, composed of the intracellular
95 cytosolic domain (ICD) layer and the transmembrane domain (TMD) layer (Fig. 1). In ICD
96 layer, the N-terminal ankyrin repeats domain (ARD) is below the linker-helix domain (LHD)
97 region (Fig .1F). The TRP helix mediates interactions with TMD. The C-terminal helix 1
98 (CH1) and the C-terminal helix 2 (CH2) fold back to interact with LHD and ARD (Fig. 1F).
99 In TMD layer, the pre-S1 elbow, the voltage sensor-like domain (VSLD, S1-S4), and the pore
100 domain (S5-S6) assemble in a domain-swapped fashion. Densities of several putative lipid
101 molecules were readily observed in the TMD layer, most of which are in the grooves between
102 VSLD and the pore domains (Fig. 1, A and B). bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
103 The CMZ binding site
104 By comparing the cryo-EM map of hTRPC5 in complex with CMZ, HC-070 and apo
105 mTRPC5, we unambiguously located the binding site of CMZ and HC-070 (Fig. S3 and S5).
106 A non-protein density located within the pocket surrounded by S1-S4 and TRP re-entrant
107 loop of VSLD was observed only in the presence of CMZ and its size and shape match those
108 of CMZ compound (Fig. 2, A and B, and Fig. S3E). Therefore, we assign this density as
109 CMZ. Several residues in hTRPC5 are in close proximity with CMZ (Fig. 2C), including
110 Y374 on S1, F414, G417 and E418 on S2, D439, M442, N443 and Y446 on S3, R492 and
111 S495 on S4 and P659 on TRP re-entrant loop (Fig. 2, C and D). Among them, F414 forms a
112 π-π stacking interaction with the chlorophenyl ring of CMZ. Side chain of Y374, M442,
113 Y446 and P659 show hydrophobic interactions with CMZ. Residues related to CMZ binding
114 are highly conserved between TRPC4 and TRPC5 which suggest the same binding pocket
115 both in TRPC5 and TRPC4 (Fig. S6).
116 The HC-070 binding site
117 A non-protein density surrounded by the S5, pore helix from one subunit and S6 of the
118 adjacent subunit was observed in the presence of HC-070 (Fig. 3, A and B, and Fig. S5E).
119 Moreover, the size and shape of this density perfectly match those of HC-070, suggesting this
120 density might represent HC-070 compound. HC-070 is a clover-shaped molecule with a
121 hydroxypropyl tail, a chlorophenyl ring and a chlorophenoxy ring which extend out from the
122 methylxanthine core. hTRPC5 makes extensive interactions with HC-070 (Fig. 3, C and D).
123 The side chain of F576 on pore helix forms π-stacking interaction with the methylxanthine
124 core of HC-070. R557 on the loop linking S5 and pore helix (Linker), Q573 and W577 on
125 pore helix form polar contacts with the hydroxypropyl tail of HC-070. F599, A602 and T603
126 on S6 from the adjacent subunit also stabilize the hydroxypropyl tail. F569 and L572 interact bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
127 hydrophobically with the chlorophenyl ring of HC-070. The chlorophenoxy branch only
128 weakly interacts with C525 and this might account for its weaker density and higher
129 flexibility (Fig. S5E). Residues involved in HC-070 binding are absolutely conserved
130 between hTRPC4 and hTRPC5 but not in TRPC3/6/7 (Fig. S6), in agreement with the fact
131 that HC-070 is much more potent toward TRPC4/5 than toward TRPC3/6/7 (27).
132 Ion conduction pore and non-protein densities in cryo-EM maps
133 In TMD, pore helixes, pore loops and S6 of four protomers form the transmembrane pathway
134 for ion permeation. Calculated pore profiles showed that the constriction is formed by the
135 side chains of I621, N625 and Q629, which is the lower gate of hTRPC5 (Fig. 4, A and B).
136 The smallest radius at the gate are below 1 Å, indicating the channel is in a non-conductive
137 closed state. This is in agreement with the inhibitory function of CMZ and HC-070. A
138 putative cation density inside VSLD, surrounded by E418, E421 on S2 and N436, D439 on
139 S3, was observed in both CMZ and HC-070 maps (Fig. 4C). Strikingly, this putative cation is
140 in close proximity to CMZ in CMZ-bound hTRPC5 (Fig. 2C). Similar density was previously
141 observed in mTRPC4, mTRPC5, TRPM2, TRPM4 and TRPM8 (33-37). The identity and
142 function of this putative ion still await further investigation. In ICD, another extra density is
143 found in the ankyrin repeat 4 (AR4)-linker helix (LH1) region (Fig.4D). The ion is
144 coordinated by the side chains of H178 and C182 on AR4-LH1 loop and C184 and C187 on
145 LH1 helix (Fig. 4D). Based on the chemical environments for ion coordination, this ion
146 binding site is likely a HC3 type zinc binding site, as observed previously in a bacteria
147 glucokinase (38). Because the zinc ion is from endogenous source and co-purified with
148 hTRPC5, we speculate this is a high affinity zinc binding site that constitutively chelates zinc.
149 The residues coordinating putative zinc ions are highly conserved in TRPC ion channels (Fig.
150 S6), but the physiological function of this site still awaits further investigation. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
151 In the 2.7 Å cryo-EM maps of hTRPC5, we observed several putative lipid molecules in the
152 transmembrane domain (Fig. 1, A and B and Fig. 4E). Lipid 1 locates near the S5 and pore
153 helix from one subunit and S6 from the adjacent subunit in the CMZ-bound hTRPC5. Its
154 polar head group faces the extracellular side and forms polar contacts with R557 on Linker,
155 Q573 and W577 on pore helix, suggesting its outer leaflet localization (Fig. 4F). Its two
156 hydrophobic tails interact with several hydrophobic residues F576, V579 on pore helix, L514,
157 F520, L521, Y524 on S5 and V610, V614, L616 on S6. Similar densities were observed in
158 mTRPC4 and mTRPC5 (33, 34), suggesting the important function of lipid 1. Interestingly,
159 in the map of HC-070-bound hTRPC5, the head group of lipid 1 is replaced by HC-070,
160 while the tails of lipid 1 are still in similar position (Fig. S5F). Lipid 2 and lipid 3 are
161 observed in both CMZ-bound hTRPC5 and HC-070-bound hTRPC5 maps. Lipid 2 localizes
162 in the inner leaflet and is bound by S3, S4 and S5 from one subunit and S5, S6 from adjacent
163 subunit (Fig. 4G). Lipid 3 is in the outer leaflet of membrane and bound by S3 and S4 (Fig.
164 4H). There is one putative cholesteryl hemisuccinate (CHS) molecule which is sandwiched
165 by S1, S4 from one subunit and S5 from adjacent subunit (Fig. 4I). Densities similar to the
166 putative CHS were observed previously in corresponding positions in TRPM4 and TRPCs
167 (33, 34, 36, 39). Moreover, this putative CHS binding site was reported to be important for
168 the activation of mTRPC5 (33), suggesting its potential role in channel modulation.
169 Structure comparisons between available TRPC5 structures
170 The structure of truncated mTRPC5 in the apo state was solved to the resolution of 2.8 Å
171 previously (33). The constructs of truncated hTRPC5 and mTRPC5 used for cryo-EM studies
172 share over 99% sequence identity (Fig. S6). In agreement with the high sequence
173 conservation, the overall structure of hTRPC5 and mTRPC5 (PDB ID: 6AEI) are highly
174 similar, with root-mean-square deviation (RMSD) of 0.6 Å (0.635 Å between CMZ-bound bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
175 hTRPC5 and 6AEI; 0.617 Å between HC070-bound hTRPC5 and 6AEI), although there are
176 several structural variations in details (Fig. 5).
177 The CMZ bound and the HC-070 bound structures have some subtle conformational change
178 compared with ligand-free mTRPC5 in TMD (Fig. 5A). S3 segments on the extracellular side
179 in CMZ and HC-070 bound structures tilt for 5.47 degree compared to the ligand-free
180 mTRPC5 (Fig. 5B). Moreover, the S2-S3 linker helixes move half a helix away from the
181 central axis of the channel in inhibitor-bound structures (Fig. 5C). These subtle variations
182 may be due to different sample preparation procedures between hTRPC5 (in GDN detergent)
183 and mTRPC5 (in PMAL-C8 amphipol). However, S2 of HC-070-bound hTRPC5 structure
184 tilt 12.4 degree compared with CMZ-bound structure, probably due to inhibitor binding (Fig.
185 5C).
186 Discussion
187 Thanks to the efforts from several groups (40, 41), three inhibitor binding pockets have been
188 structurally identified in hTRPC5 and hTRPC6, namely inhibitor binding pocket (IBP) A-C
189 (Fig. 6 and Fig. S7).
190 IBP-A inside the VSLD is accessible from intracellular side (Fig. 6A). Inhibitors, such as
191 CMZ, need to penetrate the cell membrane before binding at IBP-A. CMZ, M084 and
192 AC1903 are benzimidazole derivatives which can inhibit TRPC5 with IC50 values at 1.0 - 1.3
193 μM, 8.2 μM, 14.7 μM, respectively (28, 30, 31). Their similar chemical structures suggest
194 they probably share a common binding site at IBP-A (Fig. S8, A to C). CMZ are six-fold
195 more potent against TRPC5 than TRPC4β (31). Residues in the IBP-A are highly conserved
196 between TRPC5 and TRPC4, except P659 in TRPC5 is replaced by a threonine in TRPC4
197 (Fig. S6), which might be responsible for the observed six-fold selectivity of CMZ between
198 TRPC4 and TRPC5. In addition, CMZ is ten-fold and 26-fold more potent against TRPC5 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
199 than TRPC3/6 and TRPC7, respectively (31). Residues E418, D439, M442, R492 are
200 identical among TRPC4/5/3/6/7, whereas residues Y374 in TRPC5 are substituted by Phe,
201 F414 by Met, N443 by Leu, Y446 by Phe and S495 by Tyr in TRPC3/6/7, which might be
202 responsible for the lower potency of CMZ against TRPC3/6/7. Although VSLD has lost the
203 voltage sensing function, it plays important structural and functional role in TRP channels.
204 Based on the gating model of TRPV1, channel opening is associated with relative movements
205 between VSLD and pore (42, 43) and thus, stabilization of VSLD in an inactive conformation
206 might be a common scheme to inhibit channel opening. Indeed, AM-1473, a potent
207 antagonist of hTRPC6 were found to bind at IBP-A in hTRPC6 as well (44). Moreover, IBP-
208 A is the AMTB and TC-I 2014 binding sites in TRPM8 (45), 2-APB binding site in TRPV6
209 (46) (Fig. S7A).
210 IBP-B locates in the pore domain, sandwiched between subunit interface (Fig. 6B). HC-070
211 binds at IBP-B which is close to the extracellular side. Strikingly, IBP-B is partially occupied
212 by the head group of a lipid molecule in the map of CMZ-bound hTRPC5 or apo mTRPC5
213 structures (Fig. S7B). Previous studies showed disruption of this lipid binding site in
214 mTRPC5 by mutations, such as F576A and W577A, renders mTRPC5 inactive (33). In
215 addition, mutation of W680 to alanine in hTRPC6, corresponding to W577A in hTRPC5,
216 abolished OAG activation of hTRPC6 (44). Mutations of G709/G640 in hTRPC6/3,
217 corresponding to G606 in hTRPC5 in IBP-B, blunted PLC-mediated activation of TRPC6/3,
218 altered responses to DAGs of TRPC3, and also altered the pattern of photoactivation of
219 TRPC6/3 by a photo-switchable DAG analogue, OptoDArG (47). These data collectively
220 suggest this lipid binding pocket is important for the function of TRPC channels and ligands
221 binding at IBP-B might affect TRPC channel gating. The binding of HC-070 at this site
222 stabilizes hTRPC5 in a closed state and thus inhibits channel opening. The residues involved
223 in HC-070 binding are absolutely identical between hTRPC5 and hTRPC4, but they share bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
224 only 20% similarities between TRPC4/5 and TRPC3/6/7 (Fig. S6). F576 and W577 in the
225 highly conserved LFW motif and G606 out of 13 residues in IBP-B are the identical residues
226 between hTRPC5 and TRPC3/6/7, which explains the high selectivity of HC-070 for
227 TRPC4/5 over TRPC3/6/7 (27). In addition, HC-070 shows inhibitory effect on
228 heterotetrameric hTRPC1:C5 and hTRPC1:C4 with relatively lower potency compared to
229 homotetrameric hTRPC4/5 (27), probably because of the lower sequence identity between
230 TRPC4/5 and TRPC1 (46% according to the sequence alignment) (Fig. S6). HC-608, an HC-
231 070 analogue, is more potent against hTRPC4/5 than HC-070 (27), probably due to the
232 substitution of chloride with trifluoromethoxy group on the chlorophenoxy ring of HC-070
233 (Fig. S8, D and E). Unexpectedly, another HC-070 family compound, AM237, is a TRPC5
234 activator with EC50 around 15 - 20 nM (32). Compared with HC-608, AM237 has an
235 additional chloride atom on the phenoxy ring (Fig. S8F). It is reported that AM237 activates
236 homomeric hTRPC5 channel instead of the heterotetrameric TRPC1:C5 channel (32).
237 However, AM237 inhibits EA-evoked activation of both homotetrameric and
238 heterotetrameric TRPC5 channels (32), supporting that AM237 also binds at IBP-B which is
239 on the interface between adjacent subunits. The structural mechanism of how AM237
240 activates hTRPC5 remains elusive. Recently, a hTRPC6 agonist AM-0883 was found to bind
241 at IBP-B as well (Fig. S7B) (44), suggesting IBP-B is a common modulatory ligand binding
242 site in TRPC channels.
243 IBP-C is between VSLD and pore domain, formed by S3, S4, S4-S5 linker from one subunit
244 and S5-S6 from the adjacent subunit (Fig. 6C). BTDM, a high affinity inhibitor of hTRPC6
245 binds at IBP-C (Fig. 6C and Fig. S7C) (39). When BTDM is not supplied, IBP-C is occupied
246 by a putative phospholipid (44). In TRPC4/5, mutations of G503/G504 to serine on S4-S5
247 linker lead to constitutive activation of the channels (48). In addition, vanilloid agonists
248 resiniferatoxin (RTX) and capsaicin, vanilloid antagonist capsazepine, and an endogenous bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
249 phosphatidylinositol bind at IBP-C on TRPV1 (Fig. S7C) (42, 43). These data together
250 suggest IBP-C is important for the gating of both TRPC and TRPV channels.
251 In summary, our high resolution structures of hTRPC5 reported here have uncovered the
252 binding pockets for two distinct classes of inhibitors together with their detailed binding
253 mode. Further structural comparisons with previously reported TRP channel structures have
254 revealed three common inhibitor binding pockets in TRPC channels. Our studies paved the
255 way for further mechanistic studies and structure-based drug development.
256 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
257 Materials and Methods
258 FSEC
259 The cDNA of hTRPC5 and related mutants were all cloned into a home-made BacMam
260 vector with N terminal GFP-MBP tag (49). The expression levels of various hTRPC5
261 constructs were screened by FSEC (50). Various constructs were transfected into HEK293F
262 cells using PEI. After transfection for 40 h, cells were harvested by centrifuge at 5,000 rpm
263 for 5 min. Then the cells were solubilized by 1% (w/v) LMNG, 0.1% (w/v) CHS in TBS
264 buffer (20 mM Tris-HCl, pH 8.0 at 4 ℃, and 150 mM NaCl) with 1 ug/ml aprotinin, 1 ug/ml
265 leupeptin, 1 ug/ml pepstatin for 30 min at 4 ℃. Cell supernatant were collected after
266 centrifuge at 40,000 rpm for 30 min and loaded onto superpose 6 increase (GE healthcare) for
267 FSEC analysis.
268 FLIPR calcium assay
269 AD293 cells cultured on 10 cm dish (grown in Dulbecco's Modified Eagle's Medium + 10%
270 FBS, 37 °C) at 100% confluence were digested with trypsin-EDTA (0.25%) (Gibco) and
271 diluted for 3 fold with culture medium. Then the AD293 cells were seeded on black well,
272 clear bottom, Poly-D-lysine coated 96-well plate (Costar) and cultured overnight. Then
273 hTRPC5 constructs were transfected into AD293 cells (60%~80% confluence) with PEI.
274 About 24 hours after transfection, culture medium were discarded and cells were incubated
275 with 50 μl per well of 4 μM Rhod-2/AM (AAT Bioquest) in TARODES’s buffer (10 mM
276 HEPES, 150 mM NaCl, 4 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 11 mM glucose, pH 7.4)
277 supplemented with 0.02% Pluronic F-127 (Sigma) for 5 min in dark at room temperature.
278 Then dye was removed and replaced with 50 μl of TARODE’s buffer per well. Calcium
279 signals evoked by 256 nM EA (prepared in TARODES’s buffer) were detected using FLIPR-
280 TETRA (Molecular Devices) with excitation/emission at 510–545/565–625 nm. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
281 Protein expression and purification
282 The BacMam expression system was established for large-scale expression of hTRPC51-764
283 as previously reported (40). Briefly, hTRPC51-764 was subcloned into a home-made BacMam
284 vector with N terminal GFP-MBP tag. The Bacmid was then generated by transforming this
285 construct into DH10Bac E. coli cells. Baculovirus was harvested about 7 days after
286 transfecting bacmid into Sf9 cells cultured in Sf-900 III SFM medium (Gibco) at 27 ℃. P2
287 baculovirus was produced using Bac-to-bac system. P2 virus was added to FreeStyle 293-F
288 cells at a ratio of 1:12.5 (v/v) when the cells were grown to a density of 2.0×106/ml in SMM
289 293T-I medium (Sino Biological Inc.) supplemented with 1% FBS under 5% CO2 in a shaker
290 at 130 rpm at 37℃. After virus infection for 12 hours, 10 mM sodium butyrate was added
291 and temperature was lowered to 30 ℃. Cells were harvested 48 h post-infection and washed
292 twice using TBS buffer. Cell pellets were collected and stored at -80 ℃ for further
293 purification.
294 Cell pellets were resuspended in TBS buffer supplemented with 1% (w/v) LMNG, 0.1% (w/v)
295 CHS, 1 mM dithiothreitol (DTT), 1 mM phenylmethanesulfonylfluoride (PMSF), and
296 protease inhibitors, including 1 ug/ml aprotinin, 1 ug/ml leupeptin, 1 ug/ml pepstatin. The
297 mixture was incubated at 4 ℃ for 1 h. After ultra-centrifugation at 40,000 rpm for 1 h in Ti45
298 rotor (Beckman), the supernatant was loaded onto 7 ml MBP resin. The resin was rinsed with
299 wash buffer 1 (wash buffer 2 + 1mM ATP + 10 mM MgCl2) and wash buffer 2 (TBS + 40
300 μM glycol-diosgenin (GDN) + 0.005 mg/ml SPLE + 1 mM DTT) subsequently. Proteins
301 were eluted with 80 mM maltose in wash buffer 2. The eluate was concentrated using a 100-
302 kDa cut-off concentrator (Millipore) after digestion with H3C protease at 4 ℃ overnight.
303 hTRPC5 protein was further purified by size exclusion chromatography (Superose-6, GE
304 Healthcare) in buffer containing TBS, 40 μM GDN, 0.005 mg/ml SPLE, and 1 mM Tris (2- bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
305 carboxyethyl) phosphine (TCEP). The peak fractions corresponding to tetrameric TRPC5
306 channel were collected and concentrated to A280 = 1.0 with estimated concentration of 0.7
307 mg/ml for sample preparation.
308 Cryo-EM sample preparation and data collection
309 Purified protein was mixed with 50 μM HC-070, 100 μM Ca2+, or 500 μM Clemizole (CMZ)
310 in TBS buffer containing 100 μM Ca2+, respectively. Aliquots of 2.5 μl mixture were placed
311 on GO-grids as previously reported (51). Grids were blotted for 3 s (HC-070-bound) or 5 s
312 (CMZ-bound) at 100% humidity and flash-frozen in liquid ethane cooled by liquid nitrogen
313 using Vitrobot Mark I (FEI). Grids were then transferred to a Titan Krios (FEI) electron
314 microscope that was equipped with a Gatan GIF Quantum energy filter and operated at 300
315 kV accelerating voltage. Image stacks were recorded on a Gatan K2 Summit direct detector
316 in super-resolution counting mode using SerialEM at a nominal magnification of 165,000 ×
317 (HC-070-bound, calibrated pixel size of 0.821 Å/pixel) or 13,000 × (CMZ-bound, calibrated
318 pixel size of 1.045 Å/pixel), with a defocus ranging from -1.5 to -2.0 μm. Each stack of 32
319 frames was exposed for 7.1s, with a total dose about 50 e-/ Å 2 and a dose rate of 8 e-/pixel/s
320 on detector.
321 Image processing
322 The CMZ-bound TRPC5 dataset and HC-070-bound TRPC5 dataset were processed with
323 similar workflow. The collected image stacks were motion corrected by MotionCor2 (52).
324 After motion correction, a total of 1,268/991 (CMZ/HC-070) good micrographs were
325 manually selected, then GCTF (53) was used for CTF estimation. Following 354,312/274,189
326 particles were auto-picked using Gautomatch based on the projection of hTRPC6 map
327 (EMDB: EMD-6856) for CMZ-bound or HC-070-bound hTRPC5, respectively. After two-
328 rounds reference-free two-dimensional (2D) classification, a total of 109,369/114,211 (CMZ- bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
329 bound/HC-070-bound) particles were selected for three-dimensional (3D) classification with
330 previous hTRPC6 map (EMDB: EMD-6856) low pass filtered to 18 Å as the initial model
331 using RELION-2.0 (54) with C1 symmetry. After 3D classification, classes containing
332 90,446/114,211 (CMZ-bound/HC-070-bound) particles with clearly visible α-helices in TMD
333 were selected for further homogeneous refinements in cryoSPARC (55) using C4 symmetry.
334 Reported resolutions are based on the gold-standard Fourier shell correlation (FSC) 0.143
335 criterion after correction of masking effect (56). The map was sharpened with a B factor
336 determined by cryoSPARC automatically.
337 Model building, refinement, and validation
338 The previously reported TRPC4 (PDB: 5Z96) was used as the starting model and docked into
339 the HC-070-bound or CMZ-bound hTRPC5 maps in Chimera (57), respectively. The fitted
340 model was manually adjusted in COOT (58), keeping the side chains of conserved residues
341 and substituting non-conserved residues based on the sequence alignment between mTRPC5
342 and hTRPC5. The ligands models were generated using elbow (59) module in PHENIX (60).
343 The ligands and phospholipids were manually docked into densities and refined using COOT.
344 The models were further refined against the corresponding maps with PHENIX (60).
345 Quantification and statistical analysis
346 The local resolution map was calculated using cryoSPARC. The pore radius was calculated
347 using HOLE (61).
348
349 Data availability
350 The density maps of hTRPC5 have been deposited to the Electron Microscopy Data Bank
351 (EMDB) under the accession number: EMD-XXXX for HC-070-bound hTRPC5 and EMD- bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
352 XXXX for CMZ-bound hTRPC5. Coordinates of atomic model have been deposited in the
353 Protein Data Bank (PDB) under the accession number: XXXX for HC-070-bound hTRPC5
354 and XXXX for CMZ-bound hTRPC5. All other data are available from the corresponding
355 authors upon reasonable request.
356
357 References
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490
491
492 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
493 Acknowledgement
494 The cDNAs of hTRPC5 were kindly provided by Xiaolin Zhang. Cryo-EM data collection was
495 supported by Electron microscopy laboratory and Cryo-EM platform of Peking University with the
496 assistance of Xuemei Li, Daqi Yu, Xia Pei, Bo Shao, Guopeng Wang, and Zhenxi Guo. Part of
497 structural computation was also performed on the Computing Platform of the Center for Life Science
498 and High-performance Computing Platform of Peking University. We thank Yi Rao for sharing
499 FLIPR instruments and Shangchen Han for technical supports. This work is supported by grants from
500 the Ministry of Science and Technology of China (National Key R&D Program of China,
501 2016YFA0502004 to L.C.), National Natural Science Foundation of China (91957201, 31622021,
502 31870833 and 31821091 to L.C., 31900859 to J.-X. W.), Beijing Natural Science Foundation
503 (5192009 to L.C.), Young Thousand Talents Program of China to L.C and the China Postdoctoral
504 Science Foundation (2016M600856, 2017T100014, 2019M650324, and 2019T120014 to J.-X.W.). J.-
505 X. W. is supported by the Boya Postdoctoral Fellowship of Peking University and the postdoctoral
506 foundation of the Peking-Tsinghua Center for Life Sciences, Peking University (CLS).
507 Author Contribution
508 Lei Chen initiated the project. Kangcheng Song, Miao Wei and Wenjun Guo purified proteins and
509 prepared the cryo-EM samples. Kangcheng Song, Miao Wei, Wenjun Guo, Yunlu Kang and Jing-
510 Xiang Wu collected the cryo-EM data. Kangcheng Song and Miao Wei processed the cryo-EM data,
511 built and refined the atomic model with the help of Wenjun Guo and Lei Chen. Kangcheng Song
512 performed the FLIPR experiments. All authors contributed to the manuscript preparation.
513 Competing Interests
514 The authors declare no conflict interest.
515
516 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
517 Figure Legend
518 Fig. 1. Overall structure of human TRPC5. (A, B) Cryo-EM density maps of CMZ-bound
519 hTRPC5 shown in side view (A) and top view (B). Subunit A, B, C, and D are colored in
520 purple, light blue, orange and gray, respectively. Lipids are colored in gold. The approximate
521 boundary of the cell membrane is indicated by gray lines. TMD, transmembrane domain;
522 ICD, intracellular cytosolic domain. (C) Ribbon diagram of a single subunit with secondary
523 structure elements represented in different colors. ARD, ankyrin repeats domain; LHD,
524 linker-helix domain; CH1, C-terminal helix 1; CH2, C-terminal helix 2.
525
526 Fig. 2. CMZ binding site in hTRPC5. (A) Densities of CMZ and nearby residues. The map is
527 contoured at 4.6 σ (gray mesh). CMZ and hTRPC5 are shown in sticks, colored in sky blue
528 and purple, respectively. (B) Overview of the CMZ binding site in hTRPC5. The dashed
529 region demarcates the binding pocket of CMZ. (C) Close-up view of the CMZ binding site.
530 CMZ is shown as transparent surface superimposed with sticks. Side chains of residues that
531 interact with CMZ are shown as sticks. A cation near CMZ is shown as a magenta sphere. (D)
532 Cartoon representation of the interactions between CMZ and hTRPC5. S1-S4 of subunit A
533 are represented as purple ovals. Residues that interact with CMZ are labeled inside the ovals.
534
535 Fig. 3. HC-070 binding site in hTRPC5. (A) Densities within the HC-070 binding site. The
536 map is contoured at 3 σ (gray mesh). HC-070 and hTRPC5 are shown in sticks, with HC-070
537 colored in gray. Subunits A and B are colored in purple and light blue, respectively. (B)
538 Overview of the HC-070 binding site in hTRPC5. The dashed region marks the binding
539 pocket of HC-070. (C) Close-up view of the HC-070 binding site. HC-070 is shown as bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
540 transparent surface superimposed with stick-model. Side chains of residues that interact with
541 HC-070 are shown as sticks. (D) Cartoon representation of the interactions between HC-070
542 and hTRPC5. S5 of Subunit A and S6 of subunit B are represented as purple and light blue
543 ovals, respectively. Residues that interact with HC-070 are labeled inside the ovals.
544 Fig. 4. Ion conduction pore and other non-protein densities in hTRPC5 maps. (A) Side view
545 of the pore region of non-conductive hTRPC5 (CMZ-bound). Subunit A and subunit C are
546 shown as ribbon and colored the same as in Fig.1. Subunits B and D are omitted for clarity.
547 Ion conduction pathway along the pore is shown as dots with key residues labeled, calculated
548 by HOLE. Purple, green and red dots define pore radii of >2.8 Å, 1.4–2.8 Å and <1.4 Å,
549 respectively. (B) Calculated pore radius of CMZ-bound hTRPC5 and HC070-bound hTRPC5
550 are shown vertically. (C) Close up view of the putative cation binding site in TMD. Densities
551 of the cation and the related residues are contoured at 3.2σ and shown as grey mesh. The side
552 chain of the residues that interact with the cation are shown as sticks. (D) Close up view of
553 the putative zinc binding site in hTRPC5. Densities of the zinc ion and interacting residues
554 are contoured at 3.1σ and shown as grey mesh. (E) Other modeled non-protein densities in
555 TMD in CMZ-bound hTRPC5 are shown as sticks and colored the same as in Fig. 1. (F-I)
556 Close-up views of lipid1 (F), lipid2 (G), lipid3 (H) and CHS (I) binding sites. Subunit A and
557 B are colored in purple and light blue, respectively. Residues that interact with lipids are
558 shown as sticks. Insets show cryo-EM densities of these lipids, contoured at 5σ, 4.7σ, 3σ,
559 5.1σ, respectively.
560
561 Fig. 5. Structure comparison between CMZ-bound TRPC5, HC-070-bound TRPC5 and apo
562 mTRPC5 in TMD. (A) The overall conformational variation in TMD between these three
563 structures. All the structures are shown as cartoon, with apo mTRPC5 colored in grey, CMZ- bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
564 bound TRPC5 in light blue and HC-070-bound TRPC5 in purple. (B) Close up view of the
565 conformational change of S3 in both CMZ-bound TRPC5 and HC-070-bound TRPC5
566 structures compared to the apo mTRPC5 structure. The tilt angle of S3 (444-457 aa) was
567 measured when these TRPC5 structures are aligned. (C) Close up view of the structure
568 comparison of S2 between these three structures. The tilt angle of helix (419-425 aa) was
569 measured when the structure of CMZ-bound TRPC5 and HC-070-bound TRPC5 are aligned.
570 Fig. 6. Inhibitor binding pockets of TRPCs in TMD. (A) CMZ binding pocket in hTRPC5 is
571 shown in side view and bottom view. The TRPC5 structure is shown as cylinder with CMZ
572 compound shown as spheres and colored in sky blue. Each subunit of TRPC5 is colored the
573 same as Fig. 1. (B-C) HC-070 binding pocket in hTRPC5 structure and BTDM binding
574 pocket in TRPC6 structure are shown in side view and bottom view. HC-070 is shown as
575 spheres and colored in gray. The densities of BTDM are shown as red mesh.
576 Fig. S1. Functional characterization of full length hTRPC5 and truncated hTRPC5. (A) FSEC
577 profile of full length hTRPC5 and hTRPC5(1-764). (B-E) EA activation effect on PBM-NGFP-
578 MBP-hTRPC5(1-764), non-tag hTRPC5(1-764), non-tag full length TRPC5 and untransfected
579 cells detected by FLIPR. (F) Representative size-exclusion chromatography of hTRPC5
580 purified in GDN micelles. (G) SDS-PAGE gel of hTRPC5 eluted from size-exclusion
581 chromatography. The position of TRPC5 is labeled and fractions that were pooled for cryo-
582 EM analysis are denoted by asterisks.
583 Fig. S2. Cryo-EM image analysis of CMZ-bound hTRPC5. (A) Representative raw
584 micrograph recorded on K2 Summit camera. (B) Representative 2D class averages of CMZ-
585 bound hTRPC5. (C) Flowchart for Cryo-EM data processing of CMZ-bound hTRPC5. (D)
586 Fourier shell correlation (FSC) curves of the two independently refined maps for unmasked
587 (blue line, 3.5 Å) and corrected (purple line, 2.7 Å). Resolution estimation was based on the bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
588 criterion of FSC 0.143 cut-off. (E) Angular distribution of CMZ-bound hTRPC5. This is a
589 standard output from cryoSPARC.
590 Fig. S3. Cryo-EM map of CMZ-bound hTRPC5. (A-C) Cryo-EM map of CMZ-bound
591 hTRPC5 colored by local resolution, shown in top view (A), side view (B), and cross-section
592 (C). The position of cross-section is shown as dashed line in (A). (D) Cryo-EM density map
593 (contoured at 3.6σ, gray mesh) with atomic models superimposed. (E) Cryo-EM density of
594 CMZ shown in different views (contoured at 3.5σ). (F) Cryo-EM densities of CMZ-binding
595 pocket in CMZ-bound TRPC5 map (contoured at 5σ). (G) Cryo-EM densities in HC-070-
596 bound TRPC5 and apo mTRPC5 maps corresponding to the CMZ-binding pocket contoured
597 at the same σ as in (F).
598 Fig. S4. Cryo-EM image analysis of HC-070-bound hTRPC5. (A) Representative raw
599 micrograph recorded on K2 Summit camera. (B) Representative 2D class averages of HC-
600 070-bound hTRPC5. (C) Flowchart for cryo-EM data processing of HC-070-bound hTRPC5.
601 (D) Fourier shell correlation (FSC) curves of the two independently refined maps for
602 unmasked (blue line, 3.4 Å) and corrected (purple line, 2.7 Å). Resolution estimation was
603 based on the criterion of FSC 0.143 cut-off. (E) Angular distribution of HC-070-bound
604 hTRPC5. This is a standard output from cryoSPARC.
605 Fig. S5. Cryo-EM map of HC-070-bound hTRPC5. (A-C) Cryo-EM map of HC-070-bound
606 hTRPC5 colored by local resolution, shown in top view (A), side view (B) and cross-section
607 (C). The position of cross-section is shown as dashed line in (A). (D) Cryo-EM density map
608 (contoured at 3σ, gray mesh) with atomic model superimposed. (E) Cryo-EM density map of
609 HC-070 shown in different views (contoured at 3σ). (F-H) Cryo-EM densities of HC-070-
610 binding pocket in (F) HC-070-bound hTRPC5 map (contoured at 3.6σ), (G) CMZ-bound
611 hTRPC5 map (contoured at 2σ), and (H) apo mTRPC5 map (contoured at 3.1σ). bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
612 Fig. S6. Sequence alignment of the TRPC channels. Conserved residues are colored in gray.
613 Secondary structures are indicated as cylinders (α helices) and lines (loops). Unmodeled
614 residues are indicated as dashed line. Residues of CMZ binding site, HC-070 binding site,
615 VSLD’s cation binding site and zinc binding site in CTD are indicated with blue boxes, black
616 boxes, red boxes and purple boxes respectively.
617 Fig. S7. Ligand binding sites in TRP family. (A) Different ligand binding in IBP-A among
618 TRPC5, TRPC6, TRPM8 and TRPV6. One subunit of different TRPCs are shown in cartoon
619 and colored in purple. Different ligands are shown as sticks and in various color. (B)
620 Different ligand binding in IBP-B among TRPCs. One subunit and the adjacent subunit are
621 colored in purple and light blue, respectively. (C) Different ligand binding in IBP-C among
622 TRPC6 and TRPV1. The density of BTDM is shown in mesh and other modeled ligands are
623 shown as sticks.
624 Fig. S8. Chemical structure comparison between different TRPC5 ligands. (A-C) Chemical
625 structure comparison between benzimidazole derivatives inhibitors: CMZ (A), M084 (B) and
626 AC1903 (C). (D-F) Chemical structure comparison between xanthines derivatives ligands:
627 inhibitors HC-070 (D), and HC-608 (E) and activator AM237 (F). bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is Fig. 1 made available under aCC-BY-ND 4.0 International license.
A B C 100 Å S1-S4 Out TRP re-entrant Pore C helix TMD B Pre-S1 S5 S6 elbow CCDD In 90° TRP helix 30 Å 1 LHD B AB A D CH1 ICD A ARD
CH2
side view top view bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint Fig. 2 (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. A B C
S3 S4 Y446 Y446 S3 S4 S3 S1 S2 S1 M442 M442 180° N443 N443 S4 Pre-S1 CMZ S1 Y374 F414 Y374 CMZ elbow S2 S2 P659 D439 R492 S495 S495 E418 E418
TRP helix Cation Cation
D F414 N443
D439 Y446 Cl E418 M442 N G417 R492 N N
P659 Y374 S495 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
Fig. 3 A B C
Pore R557 F569 R557 Pore Pore helix F569 helix A602 helix A602 S6 Q573 Q573 F599 S6 S5 F599 180° S6 L572 L572 S6 T603 W577
S5 S5 L528 L528 F576 T607 HC-070 HC-070 T607 C525 C525
D
C525 T607 F576 T603
Cl G606 O A602 N N R557 L528 O N OH N W577 Q573 O Cl F559 L572
F569 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint Fig. 4 (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. A B C D Pore radius (Å) 2 4 6 0
S3 10 S2 N584 LH1 P2 N584 D439 20 P1 G581 G581 E418 C181 30 N436 S5 S6 E421 H172 C178 I621 I621I621 40 I621 CMZ-bound S2-S3 N625 N625 C176 Distance along pore (Å) pore along Distance HC-070-bound 50 linker Q629 Q629
60
E F G Lipid1 R557 Pore Lipid3 F569 helix Lipid3 Q573 Lipid3 F599 S6 W577 A602 S5 S4 S5 Lipid1 T603 S6 L528 Lipid1 V579 F576 T607 Y524 V610 Lipid2
V527 L521 CHS S3 Lipid2 L616 S5 V615 F520
L514 S5
I H S5 S4 S1
Lipid3
S3 S6 S4 CHS
Lipid2 TRP helix bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Fig. 5
A mTRPC5 B C HC-070-bound hTRPC5 CMZ-bound hTRPC5
S2 S3 S2 S4 S4 S1
Pore ° .47 helix 5 45° B S5 S1 S2 S3 S4 S6 S2-S3 linker
S3
12.4°
C TRP helix TRP helix bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is Fig. 6 made available under aCC-BY-ND 4.0 International license.
A B C
Inhibitor binding pocket A Inhibitor binding pocket B Inhibitor binding pocket C
CMZ HC-070 BTDM
Pore helix S3 S2 S4 S4 S6 S6 Side view S5 S5 S1
90° 90° 90°
S2 Pore S3 helix
S1 S4 S6 S4 S6 S5 S5
Bottom view S5 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Fig.S1
A B GFP-MBP-hTRPC5(1-764) 1000 1.4 tetramer TRPC5 (1-764 a.a.) 750 1.3 TRPC5 (1-973 a.a.) monomer 1.2 500 GFP-MBP
uorescence (μV) uorescence GFP
1.1 250 GFP GFP 1.0 0 0.9 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.4 0 50 100 150 200 250 300 Elution volumn (ml) Time (s)
C Non tag hTRPC5(1-764) D Non tag full length 2.0 1.8
1.8 1.6 1.6 1.4 1.4 1.2 1.2
1.0 1.0
0.8 0.8 0 50 100 150 200 250 300 0 50 100 150 200 250 300 Time (s) Time (s)
Untransfected E 1.8 F GFP-MBP 200
1.6 160
1.4 120
1.2 hTRPC5 80 Void tetramer Volume 1.0 40
0.8 0 0 50 100 150 200 250 300 0 4 8 12 16 20 24 Time (s) Elution volumn (ml)
G kDa * *
97 hTRPC5 66
43
31
22
14
10 11 12 13 14 15 Elution volumn (ml) bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint Fig. S2 (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. A B
50 nm
C Motion correction Motion correction 431 movies 403 micrographs 993 movies 865 micrographs & selection & selection Autopicking Autopicking
107,907 particles 246,405 particles 2D classi cation in Relion 2D classi cation in Relion
39,635 particles 69,734 particles
3D classi cation in Relion 3D classi cation in Relion
30,299 particles 60,147 particles
Local CTF correction
90,357 particles Homogeneous re nement in cryoSPARC with C4 symmetry
2.7 Å
D E 1.0 π/2 No mask 0.8 Masked & corrected 10 2 π/4 0.6 0 0.4 10 1 3.5 Å 2.7 Å 0.2 FSC = 0.143 -π/4
0.0 -π/2 10 0 DC 15 Å 7.3 Å 4.9 Å 3.7 Å 2.9 Å 2.4 Å -π -3π/4 -π/2 -π/4 0 π/4 π/2 3π/4 π bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint 9@ABÿDE(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 123
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A B
50nm
C Motion correction Autopicking 1,110 movies 991 micrographs 274,189 particles & selection 2D classi cation in Relion
114,211 particles
3D classi cation in Relion
30,795 particles 31,411 particles 27,578 particles 24,427 particles
Local CTF correction Homogeneous re nement in cryoSPARC with C4 symmetry
2.7 Å
D E 1.0 π/2 No mask 0.8 Masked & corrected π/4 10 2 0.6 0 0.4 1 3.4 Å 2.7 Å 10 -π/4 0.2 FSC = 0.143
-π/2 0.0 10 0 DC 15 Å 7.3 Å 4.9 Å 3.7 Å 2.9 Å 2.4 Å 2.1 Å 1.8 Å -π -3π/4 -π/2 -π/4 0 π/4 π/2 3π/4 π bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is ÿ made available under aCC-BY-ND 4.0 International license. 89@
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BC0D0H7I26ÿ# !"C15CRS0H7I26ÿ# !"C1 P7ÿQ !"C1 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint Fig. S6 (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
AR1 AR1 AR2
AR2 AR3 AR3 AR4
AR4 AR4 LH1 LH2 LH3 LH4
LH4 LH5 LH6 LH7 LH8
LH9 Pre-S1 Pre-S1 S1
S1 S2 S2-S3 linker S3 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint Fig. S6 continued(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. S3 S4
S5 Pore helix
S6 TRP helix
CH1 CH2
764 764 757 757 782 813 905 830 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint Fig. S7 (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
A TRPC5 TRPC6 TRPM8 TRPM8 TRPV6
CMZ AM-1473 AMTB TC-I 2014 2-APB
B TRPC5 TRPC5 TRPC6
HC-070 lipid1 AM-0883
C TRPC6 TRPV1 TRPV1 TRPV1
BTDM Phosphatidylinositol RTX capsazepine bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is Fig. S8 made available under aCC-BY-ND 4.0 International license.
A B C
Cl
H N N N
NH NH O
N N N N
clemizole M084 AC1903
D E F Cl
Cl O O
N O N O N O N F N F N
O FF O FF O N OH N OH N OH N N N
O O O Cl Cl Cl
HC-070 HC-608 AM237 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.21.052910; this version posted April 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.
Supplementary Table. 1 Cryo-EM data collection, refinement and validation statistics
CMZ-bound HC-070-bound hTRPC5 hTRPC5 PDB ID XXXX XXXX EMDB ID EMD-XXXX EMD-XXXX
Data collection and processing Magnification 130,000 × 165,000 × Voltage (kV) 300 300 Electron exposure (e–/Å2) 50 50 Defocus range (μm) -1.5 to -2.0 -1.5 to -2.0 Pixel size (Å) 1.045 0.821 Symmetry imposed C4 C4 Initial particle images (no.) 354,312 274,189 Final particle images (no.) 90,446 114,211 Map resolution (Å) 2.74 2.74 FSC threshold 0.143 0.143 Map resolution range (Å) 250-2.7 250-2.7
Refinement Initial model used (PDB code) 5Z96 5Z96 Model resolution (Å) 250-2.7 250-2.7 FSC threshold 0.143 0.143 Model resolution range (Å) 250-2.7 250-2.7 Map sharpening B factor (Å2) -119.0 -118.1 Model composition Non-hydrogen atoms 23,200 23,256 Protein residues 2,756 2,776 Ligands 28 24 B factors (Å2) Protein 117.47 130.08 Ligand 104.17 40.16 R.m.s. deviations Bond lengths (Å) 0.005 0.013 Bond angles (°) 1.035 1.402
Validation MolProbity score 2.02 2.94 Clashscore 8.38 29.62 Poor rotamers (%) 3.58 7.42 Ramachandran plot Favored (%) 97.21 95.34 Allowed (%) 2.79 4.66 Disallowed (%) 0.00 0.00