Cryo-EM structure of the human PAC1 receptor coupled to an ...Dec 23, 2019 · EM structure of the...
Transcript of Cryo-EM structure of the human PAC1 receptor coupled to an ...Dec 23, 2019 · EM structure of the...
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TITLE 1
Cryo-EM structure of the human PAC1 receptor coupled to an 2
engineered heterotrimeric G protein. 3
Kazuhiro Kobayashi1*, Wataru Shihoya1*‡, Tomohiro Nishizawa1*, Francois Marie 4
Ngako Kadji2, Junken Aoki2, Asuka Inoue2, Osamu Nureki1‡. 5
6
Affiliations: 7
1 Department of Biological Sciences, Graduate School of Science, The University of 8
Tokyo, Bunkyo, Tokyo 113-0033, Japan. 9
2 Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3, Aoba, Aramaki, 10
Aoba-ku, Sendai, Miyagi 980-8578, Japan. 11
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*These authors contributed equally to this work. 13
‡To whom correspondence should be addressed. E-mail: [email protected] (W.S.) 14
and [email protected] (O.N.) 15
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This article is a preprint version and has not been certified by peer review. 17
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Abstract 28
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic 29
neuropeptide hormone functioning in the central nervous system and peripheral tissues. 30
The PACAP receptor PAC1R, which belongs to the class B G-protein-coupled receptors 31
(GPCRs), is a drug target for mental disorders and dry eye syndrome. Here we present a 32
cryo-electron microscopy structure of human PAC1R bound to PACAP and an 33
engineered Gs heterotrimer. The structure revealed that TM1 plays an essential role in 34
PACAP recognition. The ECD (extracellular domain) of PAC1R tilts by ~40° as 35
compared to that of the glucagon-like peptide-1 receptor (GLP1R), and thus does not 36
cover the peptide ligand. A functional analysis demonstrated that the PAC1R-ECD 37
functions as an affinity trap and is not required for receptor activation, whereas the 38
GLP1R-ECD plays an indispensable role in receptor activation, illuminating the 39
functional diversity of the ECDs in the class B GPCRs. Our structural information will 40
facilitate the design and improvement of better PAC1R agonists for clinical 41
applications. 42
43
Main text 44
Introduction 45
Pituitary adenylate cyclase-activating polypeptide (PACAP), a 38-amino acid 46
linear peptide discovered in extracts of ovine hypothalamus1, is a multi-functional 47
peptide hormone that acts as a neurotrophic factor, neuroprotectant, neurotransmitter, 48
immunomodulator, and vasodilator2. PACAP is distributed mainly in the central nervous 49
system (CNS), but is also detected in the testis, adrenal gland, digestive tract, and other 50
peripheral organs. PACAP shares 68% amino acid sequence homology with vasoactive 51
intestinal polypeptide (VIP). PACAP and VIP stimulate three different PACAP 52
receptors: PAC1R3, VPAC1R, and VPAC2R, with different affinities. These receptors 53
share about 50% sequence identity. The affinity of PAC1R for PACAP is higher than 54
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that for VIP4, indicating that PAC1R is relatively selective for PACAP. 55
PAC1R belongs to the class B G-protein-coupled receptors (GPCRs), and 56
predominantly activates the adenylyl cyclase stimulatory G protein Gs. PAC1R is 57
widely expressed in the CNS and peripheral tissues2. PACAP/PAC1R signaling has been 58
implicated in playing essential roles in several cellular processes, including circadian 59
rhythm regulation, food intake control, glucose metabolism, learning and memory, 60
neuronal ontogenesis, apoptosis, and immune system regulation. Furthermore, 61
perturbations in the PACAP/PAC1R pathway cause abnormal stress responses 62
underlying posttraumatic stress disorder (PTSD)5, and thus PAC1R has been studied as 63
a drug target for numerous disorders. PACAP and PAC1R are expressed in lacrimal 64
glands, and induce tear secretion by increasing the aquaporin 5 (AQP5) levels in the 65
plasma membrane6. Therefore, PAC1R is also a drug target for dry eye syndrome. 66
However, the design of small molecule agonists for PAC1R has not yet been achieved, 67
limiting the clinical applications targeting PAC1R. 68
PAC1R comprises two distinct domains: an N-terminal extracellular domain 69
(ECD) and a transmembrane domain (TMD), as in the other class B GPCRs. A 70
two-step/two-domain model has been proposed for ligand binding and receptor 71
activation in the class B GPCRs7: the ECD is responsible for the initial and high-affinity 72
binding of peptide ligands, and the TMD plays a key role in both ligand binding and 73
receptor activation. A previous study suggested that PAC1R follows this model, and the 74
PAC1R-ECD is not required for receptor activation8. However, in glucagon-like peptide 75
1 receptor (GLP1R), the ECD also plays an indispensable role in receptor activation, 76
suggesting the divergent role of the ECD in the activation of class B GPCRs. Although 77
the crystal structure of the PAC1R-ECD was determined in a ligand-free conformation9, 78
little is known about the mechanism of the ligand recognition and signal transduction by 79
PAC1R. Here we present a cryo-electron microscopy (Cryo-EM) structure of the human 80
PAC1R, bound to the endogenous ligand PACAP and coupled to an engineered Gs 81
heterotrimer. The structure, combined with complementary functional analyses, 82
revealed the unique interaction between PACAP and the PAC1R-TMD and the 83
structural basis for the functional divergence between the PAC1R-ECD and 84
GLP1R-ECD. 85
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Results 86
Overall structure 87
To facilitate expression and purification, we truncated the C-terminal residues 88
418–468 of the human PAC1R. This truncation did not alter the Gs-coupling activity, as 89
measured by a NanoBiT-G-protein dissociation assay10 (Supplementary Fig. 1a, b, and 90
Table 1). For the cryo-EM analysis, we used the mini-Gs protein, an engineered 91
minimal G protein developed for structural studies11. The C-terminal truncated PAC1R 92
was purified in the presence of PACAP and subsequently incubated with the mini-Gs 93
heterotrimer (mini-Gs, β1, and γ2) and the nanobody Nb35, which stabilizes the 94
GPCR-Gs complex. The reconstructed complex was purified by gel filtration. 95
Vitrified complexes were imaged using a Titan Krios microscope equipped 96
with a VPP (Supplementary Fig. 2). The 3D classification revealed two different classes, 97
one containing a single complex (monomer class) and the other containing two 98
complexes with inverted molecular packing (dimer class), which probably formed 99
during the sample preparation. The structures of these two classes were determined at 100
4.5 Å and 4.0 Å resolutions, respectively, with the gold-standard Fourier shell 101
correlation (FSC) criteria. Since the cryo-EM density suggested almost identical 102
conformations in these classes, we built the atomic model of the receptor, ligand, and 103
G-protein based on the higher resolution dimer class cryo-EM map. The local resolution 104
of the map reached about 3.7 Å in the core region, including the TM helices of the 105
receptor and the α5 helix of the Gαs Ras-like domain (Fig. 1a, b, Table 2, and 106
Supplementary Fig. 3a). The molecular packing of the two complexes in the dimer class 107
is solely mediated through a weak hydrophobic contact between V3185.48 and M3225.52 108
(Wooten numbering in superscript) in TM5, and the ECD and G-protein are not engaged 109
in this interaction (Supplementary Fig. 3b), indicating that the dimerization minimally 110
affects the conformation of the Gs-complexed PAC1R structure. 111
The PAC1R-TMD adopts the typical architecture of the activated class B 112
GPCR conformation12–16, characterized by a sharp kink at TM6 (Fig. 1c). One notable 113
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difference is observed in TM7, which is kinked at the highly conserved G3937.50 in the 114
other class B GPCRs. PAC1R has an additional glycine G3897.46 near G3937.50, and thus 115
TM7 unwinds and bends around G3897.46 in the current structure (Fig. 1d). However, 116
the G3897.46A mutation, which would facilitate the α-helical formation of the unwound 117
TM7, did not alter the Gs-coupling activity (Supplementary Fig. 1a, b, and Table 1). 118
This result suggests that this unwinding in TM7 is not related to the PAC1R function. 119
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Interaction between PACAP and PAC1R-TMD 121
We observed an unambiguous density extending from the TMD, which allowed 122
us to assign the secondary structure and side-chain orientations of PACAP (Fig. 2a). The 123
N-terminus of the peptide ligand PACAP is directed toward the TMD core, as in the 124
other class B GPCR structures. The H1 to L27 residues of PACAP form a continuous 125
α-helix and protrude from the transmembrane binding pocket. By contrast, the residues 126
after G28 are disordered, consistent with the fact that the C-terminal truncated variant 127
PACAP1-27 has the same affinity as PACAP. Notably, the N-terminal four residues (H1 128
to G4) form a continuous α-helix, together with the I5 to L27 residues, while these 129
residues were disordered in the previous nuclear magnetic resonance (NMR) structure 130
of PACAP1-27 bound to detergent micelles17. These residues are recognized by 13 131
residues of the receptor, and G4 of PACAP closely contacts the W3065.36 side chain of 132
the receptor (Fig. 2b, c). These interactions stabilize the α-helical structure at the 133
N-terminus of PACAP, which is essential for receptor activation. 134
The N-terminal 17 residues of PACAP create an extensive interaction network 135
with TM 1-3, 5, 7, and ECL2 of the receptor (Fig. 2b, c). The details are summarized in 136
Supplementary Table 1. Notably, PACAP forms numerous interactions with the 137
extracellular portion of TM1, involving the aromatic residues of PACAP (F6, Y10, and 138
Y13), PAC1R (Y1501.36, Y1571.43, and Y1611.47), and four hydrogen-bonding 139
interactions (D3-Y1611.47, S9-Y1501.36, Y10-K1541.40, and Y13-D1471.33) (Fig. 2b-e). 140
These close interactions with TM1 are not observed in the other class B GPCR 141
structures (Supplementary Fig. 4a-d), and are a unique feature of the PACAP-PAC1R 142
structure. 143
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PACAP can activate three types of PACAP receptors, PAC1R, VPAC1R, and 144
VPAC2R with similar affinities4. To investigate the similarity in their ligand recognition, 145
we mapped the conserved residues on the current structure (Fig. 2d, e, and 146
Supplementary Fig. 5). Notably, the residues involved in the ligand recognition are 147
highly conserved in TM1, suggesting that TM1 plays a critical role in the PACAP 148
recognition by the receptors. 149
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Structural insight into G-protein activation 151
In the class B GPCRs, ligand binding induces the rearrangement of the central 152
polar interaction network, followed by the unwinding of TM6 at the highly conserved 153
P6.47-X-X-G6.50 motif and the opening of the intracellular cavity of the receptor for 154
G-protein coupling12,13,14. In the central region of PAC1R, we observed a similar polar 155
interaction network and the unwinding of TM6 (Fig. 3a, b). The polar interaction 156
network comprises D3 of PACAP and Y1611.47, R1992.60, N2403.43, Y2413.44, P3606.47, 157
G3636.50, H3656.52, Y3666.53, and Q3927.49 of the receptor. Notably, D3 forms a hydrogen 158
bond with Y1611.47 and an electrostatic interaction with R2602.60. R2602.60 in turn forms 159
a hydrogen bond with Y2413.44. Y3666.53, in the extracellular portion of TM6, is directed 160
toward the receptor core and participates in this network. Overall, this polar interaction 161
network extends from D3 to the carbonyl oxygens of P3606.47 and G3636.50 in TM6. A 162
previous SAR study showed that the substitution of D3 with alanine reduces both the 163
Emax value to 70% and the affinity for the receptor4. Therefore, PACAP binding directly 164
induces the rearrangement of the polar interaction network in the central region and 165
plays a key role in receptor activation, by unwinding TM6. 166
TM6 is kinked at P3606.47 and G3636.50 in the P6.50-X-X-G6.53 motif, as in the 167
other Gs-complexed class B GPCR structures. Notably, the kink at G3636.50 is sharp (~ 168
90°), whereas that at P3606.47 is to a less extent (Fig. 3b). Previous mutational studies of 169
the calcitonin receptor family members suggested the functional importance of P6.47 in 170
receptor activation18,19. However, the P3606.47A mutation to PAC1R did not alter the 171
Gs-coupling activity (Fig. 3c and Table 1). By contrast, the G3636.50A mutation 172
completely abolished the activity, suggesting that G3636.50, rather than P3606.47, is 173
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responsible for the TM6 unwinding upon receptor activation, consistent with the 174
structural observations. 175
The intracellular cavity of the receptor closely contacts the α5-helix of Gs, 176
which is the primary determinant for the G-protein coupling (Fig. 3a, d). Specifically, 177
S3546.41 in TM6 directly hydrogen bonds with the carbonyl oxygen of L393. K3345.64 in 178
TM5 forms a salt bridge with D381, and the carbonyl oxygens of L255 and V256 in 179
ICL2 hydrogen bond with Q384 and K380, respectively. These interactions are also 180
observed in other Gs-complexed class B GPCR structures13,16 (Fig. 3e, f), suggesting 181
that they are conserved structural features of the Gs-coupling in class B GPCRs. 182
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Diverged functional role of ECDs in class B GPCRs 184
The class B GPCRs have an ECD (about 120 amino acids) at the N-terminus, 185
which is commonly important for the initial, high-affinity binding to peptide hormones. 186
Although the ECD is less well resolved in our EM map, probably due to its flexibility, 187
we could fit the ECD region of the previous PAC1R-ECD crystal structure (PDB code: 188
3N94)9 onto the map by a rigid body. This model can facilitate discussions about the 189
interactions between PACAP and the ECD (Fig. 4a). The PAC1R-ECD adopts a 190
three-layer α–β–βα fold, which is conserved in the class B GPCRs. The C-terminal 191
portion of PACAP (Q16, V19, Y22, L23, and L27) interacts with the loops connecting 192
β1-β2 and β3-β4, and the N-terminal ends of α-helix 1 and α-helix 2 in the PAC1R-ECD, 193
as in other class B GPCRs (Supplementary Fig. 4e-g). Furthermore, the PAC1R-ECD 194
has an additional α-helix, α-helix 3, in the loop connecting β3-β4, which closely 195
contacts PACAP. A previous study showed that the N-terminal splice variant 196
PAC1R-short20, which lacks residues 89-109 between the α-helix 3 and β4, exhibits 197
increased affinity for PACAP. While residues 89-110 are not modeled in our EM map, 198
we suggest that the truncation affects the conformation of the α-helix 3 and enhances 199
the interaction with PACAP. 200
The ECD in class B GPCRs also plays a key role in receptor activation. Previous 201
functional analyses demonstrated that the ECD-truncated GLP1R does not respond to 202
GLP121. In the GLP1R structure, the ECD covers the top of the C-terminal portion of 203
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GLP1, to facilitate the interactions between the N-terminal portion of GLP1 and TMD 204
(Fig. 4b). However, the PAC1R-ECD tilts by ~40° as compared with the GLP1R-ECD 205
(Fig. 4c, d), and thus it only interacts with the side of the C-terminal portion of PACAP, 206
suggesting the different role of the PAC1R-ECD. 207
To investigate the function of the PAC1R-ECD, we truncated the C-terminal 208
portion of PACAP (residues 18-38) that interacts with the ECD (Fig. 5a). The truncated 209
peptide PACAP1-17 activated the receptor at the same level, as compared with PACAP in 210
the NanoBiT-G-protein dissociation assay, while its EC50 was significantly increased by 211
about 6000-fold (Fig. 5b and Table 3), suggesting that PACAP1-17 is capable of 212
functioning as a full agonist for PAC1R. Moreover, PACAP and PACAP1-17 also 213
activated the ECD-truncated PAC1R to mostly the same level (Fig. 5c, Table 3, and 214
Supplementary Fig. 1b). These results indicate that the PAC1R-ECD functions merely 215
as an affinity trap to bind and precisely localize the peptide hormone to the receptor, 216
whereas the interaction between PACAP and the PAC1R-TMD is necessary and 217
sufficient for receptor activation. This observation is consistent with the previous study, 218
which showed that the PAC1R-TMD covalently linked to the PACAP1-12 at the 219
N-terminus constitutively activates the G-protein21. By contrast, GLP17-23, which lacks 220
the C-terminal portion of GLP (Fig. 5d), completely lost the agonist activity for GLP1R 221
(Fig. 5e and Table 3). Furthermore, the ECD-truncated GLP1R was poorly expressed 222
and lacked receptor activity (Fig. 5f and Supplementary Fig. 1c). These results 223
confirmed that the GLP1R-ECD plays an indispensable role in receptor activation. 224
While the ECDs are commonly essential for ligand recognition in the class B GPCRs, 225
their contributions to receptor activation diverge among the receptors. 226
227
Discussion 228
We determined the PAC1R structure in complex with PACAP and the Gs-protein, 229
which revealed a unique interaction between PACAP and the PAC1R-TMD, involving 230
the aromatic residues in PACAP and TM1. Structural observations and functional 231
analyses indicated that the interaction between PACAP and the TMD is necessary and 232
sufficient for receptor activation, while the ECD is only required for the high-affinity 233
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binding. Our structural information will help the design of novel peptide-mimetic 234
agonists for PAC1R, to treat dry eye syndrome and mental disorders. 235
The class B GPCRs include 15 receptors in humans and are commonly activated 236
by peptide ligands. The class B receptors share similar characteristics, such as the 237
N-terminal ECD, and are distinct from the class A GPCRs, which are activated by 238
diverse ligands (e.g., peptides, amines, purines, and lipids)22. The structures of the class 239
B GPCRs suggested two different types of ligand recognition. In the structures of 240
calcitonin receptor12 and calcitonin receptor-like receptor (CLR)15, the N-terminal 241
portions of the peptide ligands bind to the TMD in α-helical conformations, while the 242
C-terminal portion binds to the ECD in an extended conformation (Supplementary Fig. 243
4d, h). The function of the calcitonin receptor family is modified by receptor 244
activity-modifying proteins (RAMPs). Essentially, CLR can receive calcitonin 245
gene-related peptide (CGRP) by the interaction between its ECD and RAMP1, 246
suggesting that the ECD plays a key role in both ligand binding and receptor activation. 247
In the structures of the glucagon receptor family members, GLP1R13,14 and glucagon 248
receptor, GCGR23,24, the peptide ligands adopt continuous α-helices and their ECDs 249
cover the ligands to facilitate the interactions with the TMDs, thus playing an 250
indispensable role in receptor activation. Although PACAP also adopts a continuous 251
α-helix, the PAC1R-ECD has no functional role in receptor activation, because PACAP 252
can interact with the TMD without the aid of the ECD. The PAC1R-ECD functions 253
merely as an affinity trap for the high-affinity binding of PACAP. Despite the structural 254
similarities in the class B GPCRs, the functional roles of the ECD are diverse. 255
256
Acknowledgements 257
We thank R. Danev and M. Kikkawa for setting up the cryo-EM infrastructure, 258
K. Ogomori for technical assistance, and K. Yamashita for model building. We also 259
thank Ayumi Inoue (Tohoku University, Japan) for technical assistance. This work was 260
supported by grants from the Platform for Drug Discovery, Informatics and Structural 261
Life Science by the Ministry of Education, Culture, Sports, Science and Technology 262
(MEXT), JSPS KAKENHI grants 16H06294 (O.N.), 17J30010 (W.S.), 30809421 (W.S.), 263
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 27, 2019. ; https://doi.org/10.1101/2019.12.23.887737doi: bioRxiv preprint
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17K08264 (A.I.), 17H05000 (T.N.) and the Japan Agency for Medical Research and 264
Development (AMED) grants: the PRIME JP18gm5910013 (A.I.) and the LEAP 265
JP18gm0010004 (A.I. and J.A.), and the National Institute of Biomedical Innovation. 266
267
Author contributions 268
K.K. expressed and purified the mini-Gs heterotrimer, and performed the 269
complex formation, grid-preparation, and cryo-EM observation. W.S. designed the 270
experiments, purified the receptor, established the preparation method for the mini-Gs 271
heterotrimer and Nb35, and refined the structure. T.N. performed the cryo-EM data 272
collection and single particle analysis. A.I., F.M.N.K., and J.A. performed and oversaw 273
the cell-based assays. The manuscript was mainly prepared by W.S., K.K., and A.I., 274
with assistance from T.N. and O.N. 275
276
Competing interests 277
The authors declare no competing interests. 278
Figures 279
Fig. 1. Overall structure of the PAC1R-mini-GSβ1γ2-Nb35 complex. 280
a, Sharpened cryo-EM map with variably colored densities (PAC1R TM: cyan, PACAP: 281
yellow, mini-Gs heterotrimer: green, red, and purple, Nb35: light blue). b, Structure of 282
the complex determined after refinement in the cryo-EM map. The model is shown as a 283
ribbon representation with the transparent map. c, Superimposition of the TMD 284
structures of PAC1R (cyan) and the other class B GPCRs determined to date (gray). d, 285
TM7 unwinding in the PAC1R structure. Residues 387 to 393 are shown as sticks. 286
287
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Fig. 2. PACAP binding site in TMD. 288
a, Sharpened map of PACAP and the TMD, viewed from the extracellular side. PACAP 289
and TMD are shown as ribbon representations with the transparent map. b, c, Detailed 290
interactions between PACAP and the TMD, shown as ribbon representations colored as 291
in Fig. 1. Contact residues are shown as sticks. The interactions with TM1, 6, and 7 are 292
shown in (b), while those with TM2, 3, and 5 are shown in (c). Hydrogen-bonding 293
interactions are indicated by black dashed lines. d, e, Sequence conservation of the 294
PACAP binding site between three types of PACAP receptors (PAC1R, VPAC1R, and 295
VPAC2R), mapped onto the PAC1R structure. Conserved and non-conserved residues 296
are colored magenta and cyan, respectively. The conserved hydrogen-bonding 297
interactions are shown in (d), and all of the conserved residues are shown in (e). 298
299
Fig. 3. Mechanism of receptor activation and Gs coupling. 300
a, Ribbon representation of the PAC1R-TMD, PACAP, and α5-helix of mini-Gs, viewed 301
from the membrane plane and colored as in Fig. 1. b, The residues involved in the 302
central polar interaction network are represented by sticks. Hydrogen bonding 303
interactions are indicated by black dashed lines. c, PAC1R-mediated Gs activation, 304
measured by the NanoBiT-G-protein dissociation assay. Cells transiently expressing the 305
NanoBiT-Gs along with the indicated PAC1R construct were treated with PACAP 306
(1-38), and the change in the luminescent signal was measured. d-f, Cytoplasmic views 307
of the PAC1R (cyan) with the C-terminal α5 helix of Gαs (yellow) (d), compared to the 308
GLP1-GLP1R:Gs complex (orange, PDB 5VAI) (e) and the LA-PTH-PTH1R:Gs 309
complex (pink, PDB 6NBF) (f). Hydrogen bonding interactions are indicated by black 310
dashed lines. 311
312
Fig. 4. Structural comparison of PAC1R and GLP1R. 313
a, Interaction between PACAP and the PAC1R-ECD. The PAC1R-ECD is shown as a 314
ribbon representation with the transparent sharpened map. C54 is shown as a stick 315
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model. b, c, Surface representations of GLP1R (b) and PAC1R (c). The receptors are 316
shown as ribbon representations with transparent surfaces. PACAP and PAC1R are 317
colored as in Fig. 1a. GLP1 and GLP1R are colored orange and light-green, respectively. 318
d, Superimposition of PAC1R and GLP1R, viewed from the membrane plane. 319
320
Fig. 5. Characterization of the truncated analogs of PACAP and GLP1. 321
a, Overall structure of the PACAP-bound PAC1R, viewed from the membrane plane. 322
PACAP and PAC1R are shown as ribbon representations, colored as in Fig. 1a. The 323
C-terminal portion of PACAP (18-38) is shown with increased transparency. b, c, 324
PACAP-induced Gs activation measured by the NanoBiT-G-protein dissociation assay. 325
Cells transiently expressing the NanoBiT-Gs along with PAC1R (b) or PAC1R∆ECD 326
(c) were stimulated by the indicated PACAP peptides, and the change in the luminescent 327
signal was measured. d, Overall structure of the GLP1-bound GLP1R, viewed from the 328
membrane plane. GLP1 and GLP1R are shown as ribbon representations, colored as in 329
Fig. 4b. The C-terminal portion of GLP1 (24-37) is shown with increased transparency. 330
e, f, GLP-1-induced Gs activation measured by the NanoBiT-G-protein dissociation 331
assay. Cells transiently expressing the NanoBiT-Gs along with GLP1R (e) or 332
GLP1R∆ECD (f) were stimulated by the indicated GLP-1 peptides, and the change in 333
the luminescent signal was measured. 334
335
Table 1. Pharmacological characterization of mutant PAC1Rs. 336
PAC1R--WT ΔC G389A P360A G363A
n = 4 3 4 4 4
EC50 (nM) 0.73 0.38 1.2 0.83 > 1 μM
pEC50 (mean ± SEM) 9.14 ± 0.22 9.42 ± 0.03 8.92 ± 0.20 9.08 ± 0.19 < 6
337
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Table 2. Data collection, processing, model refinement, and validation. 338
Data collection and processing FEI Titan Krios
Magnification 96,000
Voltage (kV) 300
Electron exposure (e− Å−2) 64
Defocus range (μm) −0.8 to -1.6
Pixel size (Å)a 0.861
Symmetry imposed C2
Initial particle images (no.) 980,964
Final particle images (no.) 132,808
Map resolution (Å) 4.0
FSC threshold 0.143
Map resolution range (Å) 3.7–4.9
Refinement
Initial model used (PDB code) 3N94, 6B3J
Model resolution (Å) 4.0
FSC threshold 0.143
Model resolution range (Å)
Map sharpening B factor (Å2) -162.683
Model composition
Non-hydrogen atoms 8842
Protein residues 1081
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R.m.s. deviations
Bond lengths (Å) 0.010
Bond angles (°) 1.162
Validation
Clashscore 6.46
Rotamer outliers (%) 1.88
Ramachandran plot
Favored (%) 92.14
Allowed (%) 7.86
Disallowed (%) 0
339
Table 3. Pharmacological characterization of truncated analogs of PACAP 340
and GLP1 341
PAC1R-WT PAC1R-TMD
n = 6 4
PACAP1-38 EC50 (nM) 1.3 580
pEC50 (mean ± SEM) 8.90 ± 0.10 6.24 ± 0.08
PACAP1-17 EC50 (μM) 8.1 19
pEC50, (mean ± SEM) 5.09 ± 0.09 4.72 ± 0.17
GLP1R-WT GLP1R-TMD
n = 4 3
GLP17-37 EC50 (nM) 1.2 >1000
pEC50 (mean ± SEM) 8.93 ± 0.17 < 6
GLP17-23 EC50 (μM) >100 >100
pEC50, (mean ± SEM) <4 <4
342
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343
Expression and purification of the human PAC1R 344
The N-terminal signal sequence in human PAC1R (Genbank ID: AK290046) 345
was replaced with the haemagglutinin signal peptide. The C-terminus was truncated 346
after S417. The modified receptor was subcloned into a modified pFastBac vector25, 347
with the resulting construct encoding a TEV cleavage site followed by a GFP-His10 tag 348
at the C-terminus. The recombinant baculovirus was prepared using the Bac-to-Bac 349
baculovirus expression system (Invitrogen). Sf9 insect cells were infected with the 350
virus at a cell density of 4.0 × 106 cells per milliliter in Sf900 II medium, and grown 351
for 48 h at 27 °C. The harvested cells were disrupted by sonication, in buffer 352
containing 20 mM Tris-HCl, pH 7.5, and 20% glycerol. The crude membrane fraction 353
was collected by ultracentrifugation at 180,000g for 1 h. The membrane fraction was 354
solubilized in buffer, containing 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1% LMNG, 355
0.1 %CHS, 20% glycerol, and 1 μM PACAP38, for 2 h at 4 °C. The supernatant was 356
separated from the insoluble material by ultracentrifugation at 180,000g for 20 min, 357
and incubated with TALON resin (Clontech) for 30 min. The resin was washed with 358
ten column volumes of buffer, containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 359
0.01% LMNG, 0.001% CHS, 0.1 μM PACAP38, and 15 mM imidazole. The receptor 360
was eluted in buffer, containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.01% 361
LMNG, 0.001% CHS, 0.1 μM PACAP38, and 200 mM imidazole. The eluate was 362
treated with TEV protease and dialyzed against buffer (20 mM Tris-HCl, pH 7.5, 363
500 mM NaCl). The cleaved GFP–His10 tag and the TEV protease were removed with 364
Co2+-NTA resin. The receptor was concentrated and loaded onto a Superdex200 365
10/300 Increase size-exclusion column, equilibrated in buffer containing 20 mM 366
Tris-HCl, pH 7.5, 150 mM NaCl, 0.01% LMNG, 0.001% CHS, and 0.1 μM PACAP38. 367
Peak fractions were pooled, concentrated to 5 mg ml−1 using a centrifugal filter 368
device (Millipore 50 kDa MW cutoff), and frozen in liquid nitrogen. 369
370
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Expression and purification of the mini-Gs heterotrimer 371
The gene encoding mini-Gs26, with codons optimized for an E. coli expression 372
system, was synthesized (GeneArt) and subcloned into a modified pET21a(+)-vector, 373
with the resulting construct encoding a His6 tag followed by a TEV cleavage site at the 374
N-terminus. The protein was expressed in E. coli BL21 cells. Protein expression was 375
induced by 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) for 20 h at 25 °C. The 376
harvested cells were disrupted by sonication, in buffer containing 20 mM Tris-HCl, 377
pH 7.5, 20% glycerol, 10 μM GDP, and 10 mM imidazole. The cell debris was removed 378
by centrifugation at 25,000g for 30 min. The supernatant was incubated with Ni-NTA 379
resin (Qiagen) for 30 min. The resin was washed with ten column volumes of buffer, 380
containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 10 μM GDP, and 30 mM imidazole. 381
The protein was eluted in buffer, containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 10 382
μM GDP, and 200 mM imidazole. The eluate was treated with TEV protease and 383
dialyzed against buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 10 μM GDP). 384
The TEV protease was removed by Ni-NTA resin. The protein was concentrated and 385
loaded onto a Hiload Superdex200 10/300 Increase size-exclusion column, equilibrated 386
in buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 1 μM GDP). Peak 387
fractions were pooled, concentrated to 8 mg ml−1 using a centrifugal filter device 388
(Millipore 10 kDa MW cutoff), and frozen in liquid nitrogen. 389
His6-rat Gβ1 and bovine Gγ2 were subcloned into the pFastBac Dual vector. 390
The recombinant baculovirus was prepared using the Bac-to-Bac baculovirus expression 391
system (Invitrogen). Sf9 insect cells were infected with the virus at a cell density of 392
4.0 × 106 cells per milliliter in Sf900 II medium, and grown for 48 h at 27 °C. The 393
harvested cells were disrupted by sonication, in buffer containing 20 mM Tris-HCl, 394
pH 7.5, 150 mM NaCl, 10 mM imidazole, and 20% glycerol, and clarified by 395
ultracentrifugation at 180,000g for 30 min. The supernatant was incubated with 396
Ni-NTA resin (Qiagen) for 30 min. The resin was washed with ten column volumes of 397
buffer, containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 30 mM imidazole. The 398
protein was eluted in buffer, containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 399
200 mM imidazole. The protein was concentrated and loaded onto a Superdex200 400
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10/300 Increase size-exclusion column, equilibrated in buffer containing 20 mM 401
Tris-HCl, pH 7.5, and 150 mM NaCl. Peak fractions were pooled, concentrated to 402
8 mg ml−1 using a centrifugal filter device (Millipore 10 kDa MW cutoff), and frozen 403
in liquid nitrogen. 404
The purified mini-Gs and Gβ1Gγ2 were mixed and incubated overnight on ice. The 405
sample was concentrated and loaded onto a Superdex200 10/300 Increase 406
size-exclusion column, equilibrated in buffer containing 20 mM Tris-HCl, pH 7.5, 407
150 mM NaCl, and 1 μM GDP. The fractions containing the mini-Gs heterotrimer 408
were pooled, concentrated to 8 mg ml−1 using a centrifugal filter device (Millipore 409
10 kDa MW cutoff), and frozen in liquid nitrogen. 410
411
Expression and purification of Nb35 412
The gene encoding the C-terminally His6-tagged nanobody-35 (Nb35), with 413
codons optimized for an E. coli expression system, was synthesized (GeneArt) and 414
subcloned into the pET22b(+)-vector. The protein was expressed in the periplasm of E. 415
coli C41(Rosetta) cells. Protein expression was induced by 1 mM isopropyl 416
β-D-thiogalactopyranoside (IPTG) for 20 h at 25 °C. The harvested cells were disrupted 417
by sonication, in buffer containing 20 mM Tris-HCl, pH 7.5, and 20% glycerol. The cell 418
debris was removed by centrifugation at 25,000g for 30 min. The supernatant was 419
incubated with Ni-NTA resin (Qiagen) for 30 min. The resin was washed with ten 420
column volumes of buffer, containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 30 421
mM imidazole. The protein was eluted in buffer, containing 20 mM Tris-HCl, pH 7.5, 422
500 mM NaCl, and 200 mM imidazole. The eluate was dialyzed against buffer (20 mM 423
Tris-HCl, pH 7.5, 150 mM NaCl). The protein was concentrated to 3 mg ml−1 using a 424
centrifugal filter device (Millipore 10 kDa MW cutoff), and frozen in liquid nitrogen. 425
426
Formation and purification of the PAC1R-mini-GSβ1γ2-Nb35 complex 427
Purified PAC1R was mixed with a 1.2-fold molar excess of mini-Gsβ1γ2 and a 428
1.5-fold molar excess of Nb35 in the presence of apyrase (0.1 U/ml) and the mixture 429
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was incubated on ice overnight. The sample was loaded onto a Superdex200 10/300 430
Increase size-exclusion column, equilibrated in buffer containing 20 mM HEPES-Na, 431
pH 7.5, 150 mM NaCl, 0.0075% LMNG, 0.0025% GDN, and 0.00025%CHS. Peak 432
fractions of the PAC1R-mini-Gsβ1γ2-Nb35 complex were pooled and concentrated to 8 433
mg/ml. 434
435
Sample vitrification and cryo-EM data acquisition 436
The purified complex was applied onto a freshly glow-discharged Quantifoil 437
holey carbon grid (R1.2/1.3, Cu/Rh, 300 mesh), blotted for 4 s at 4 °C in 100% humidity, 438
and plunge-frozen in liquid ethane by using a Vitrobot Mark IV. The grid images were 439
obtained with a 300kV Titan Krios G3i microscope (Thermo Fisher Scientific), 440
equipped with a GIF Quantum energy filter (Gatan), a Volta phase plate (Thermo Fisher 441
Scientific), and a Falcon III direct electron detector (Thermo Fisher Scientific). A total 442
of 2,895 movies were obtained in the electron counting mode, with a physical pixel size 443
of 0.861 Å. The data set was acquired with the EPU software, with a defocus range of 444
−0.8 to −1.6 μm. Each image was dose-fractionated to 64 frames at a dose rate of 6–445
8 e− pixel−1 per second, to accumulate a total dose of 64 e− Å−2. In total, 2,895 446
super-resolution movies were collected. 447
448
Image processing 449
The movie frames were aligned in 5 × 5 patches, dose weighted, and binned by 2 450
in MotionCor227. Defocus parameters were estimated by CTFFIND 4.128. First, 451
template-based auto-picking was performed with the two-dimensional class averages of 452
a few hundred manually picked particles as templates. A total of 980,964 particles were 453
extracted in 3.24 Å pixel−1. These particles were subjected to three rounds of 454
two-dimensional classification in RELION 3.0. The initial model was generated in 455
RELION-3.028. Subsequently, 980,964 particles were further classified in 3D without 456
symmetry. Two stable classes showed detailed features for all subunits. One contained a 457
single complex (monomer class). The other contained two complexes in an inverted 458
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molecular packing with C2 symmetry (dimer class). The particles of the monomer and 459
dimer classes were 282,622 and 132,808 particles, respectively, were then re-extracted 460
with the original pixel size of 1.35 Å pixel, and subsequently subjected to 3D refinement. 461
The resulting 3D models and particle sets were subjected to per-particle defocus 462
refinement, Bayesian polishing, and 3D refinement. The final 3D refinement and 463
postprocessing yielded maps of the monomer and dimer classes with global resolutions 464
of 4.5 Å and 4.0 Å, respectively. The comparison of the density maps of the two classes 465
suggested almost identical conformation, and therefore, we built an atomic model onto 466
the higher resolution map of the dimer class. All density maps were sharpened by 467
applying the temperature-factor, which was estimated using the post-processing in 468
RELION-3.1. The local resolution was estimated by RELION-3.1. The processing 469
strategy is described in Supplementary figure 2. 470
471
Model building and refinement 472
The initial template for the PAC1R transmembrane regions, PACAP, G-protein, 473
and Nb35 was derived from the structure of human GLP1R in complex with a 474
dominant-negative Gαs (PDB code: 6B3J), followed by extensive remodeling using 475
COOT29. Owing to the discontinuous and/or variable density in the ECD region, we 476
assigned the high-resolution X-ray crystal structure of the PAC1R (PDB code: 3N94)9 477
by a rigid body fit, and the model was rebuilt using Rosetta30 against the density, 478
manually readjusted using COOT, and refined using phenix.real_space_refine31. 479
Validation was performed in MolProbity32. The potential overfitting of the refined 480
models was tested by using a cross-validation method, as described previously. Briefly, 481
the final models were ‘shaken’ by introducing random shifts to the atomic coordinates 482
with an rms of 0.5 Å, and were refined against the first half map. These shaken refined 483
models were used to calculate the FSC against the same first half maps (FSChalf1 or 484
work), and the second half maps (FSChalf2 or free) that were not used for the refinement, 485
using phenix.mtriage. The small differences between the FSChalf1 and FSChalf2 curves 486
indicated no severe overfitting of the models. The curves representing model vs. full 487
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map were calculated, based on the final model and the full, filtered and sharpened map. 488
The statistics of the 3D reconstruction and model refinement are summarized in Table 2. 489
All molecular graphics figures were prepared with CueMol (http://www.cuemol.org) 490
and UCSF Chimera33. 491
492
NanoBit G-protein dissociation assay 493
PAC1R- and GLP1R-induced Gs activation was measured by a 494
NanoBiT-G-protein dissociation assay10, in which the interaction between a Gα subunit 495
and a Gβγ subunit was monitored by a NanoBiT system (Promega). Specifically, a 496
NanoBiT-Gs protein consisting of a large fragment (LgBiT)-containing Gαs subunit and 497
a small fragment (SmBiT)-fused Gγ2 subunit, along with the untagged Gβ1 subunit, was 498
expressed with a test GPCR, and the ligand-induced luminescent signal change was 499
measured. We used the N-terminal FLAG (DYKDDDK) tagged constructs of the human 500
PAC1R, PAC1RΔECD (148-468), GLP1R, and GLP1RΔECD (140-463). HEK293 cells 501
deficient for Gq/1134 were seeded in a 6-well culture plate at a concentration of 2 x 105 502
cells ml-1 (2 ml per well in DMEM (Nissui Pharmaceutical) supplemented with 10% 503
fetal bovine serum (Gibco), glutamine, penicillin, and streptomycin), one day before 504
transfection. The transfection solution was prepared by combining 4 µl (per well 505
hereafter) of polyethylenimine solution (Polysciences, 1 mg ml-1) and a plasmid mixture 506
consisting of 100 ng LgBiT-containing Gαs subunit, 500 ng Gβ1, 500 ng SmBiT-fused 507
Gγ2, and 200 ng test GPCR (or an empty plasmid) in 200 µl of Opti-MEM 508
(ThermoFisher Scientific). To prepare a larger volume of transfected cells, 10-cm 509
culture dishes (10 ml culture volume) were used with 5-fold scaling of the 6-well plate 510
contents. After an incubation for one day, the transfected cells were harvested with 0.5 511
mM EDTA-containing Dulbecco’s PBS, centrifuged, and suspended in 2 ml of HBSS 512
containing 0.01% bovine serum albumin (BSA fatty acid–free grade, SERVA) and 5 513
mM HEPES (pH 7.4) (assay buffer). The cell suspension was dispensed in a white 514
96-well plate at a volume of 80 µl per well, and loaded with 20 µl of 50 µM 515
coelenterazine (Carbosynth), diluted in the assay buffer. After 1 h incubation at room 516
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temperature, the titrated antagonist (Atropine, NMS, or Tiotropium), diluted in the assay 517
buffer at 10X of the final concentration, was added at a volume of 10 µl per well. After 518
2 h incubation, the plate was measured for baseline luminescence (Spectramax L, 519
Molecular Devices) and 20 µl portions of 6X test compound, diluted in the assay buffer, 520
were manually added. After an incubation for 3-5 minutes at room temperature, the 521
plate was read for the second measurement. The second luminescence counts were 522
normalized to the initial counts, and the fold-changes in the signals over the vehicle 523
treatment were plotted for the G-protein dissociation response. Using the Prism 8 524
software (GraphPad Prism), the G-protein dissociation signals were fitted to a 525
four-parameter sigmoidal concentration-response curve, from which the pEC50 values 526
(negative logarithmic values of EC50 values) were used to calculate the mean and SEM. 527
The pEC50 values for PACAP-17 were calculated by restraining the “Shared values for 528
all datasets” for the “Top” and “Bottom” parameters, using both PACAP-17 and 529
PACAP-38. 530
Flow cytometry analysis 531
Gq/11-deficient HEK293 cells34 were seeded in a 12-well culture plate at a 532
concentration of 2 x 105 cells ml-1 (1 ml per well), one day before transfection. The 533
transfection solution was prepared by combining 2 µl of the polyethylenimine solution 534
(1 mg ml-1) and 500 ng of a plasmid encoding the FLAG epitope-tagged GPCR in 100 535
µl of Opti-MEM. One day after transfection, the cells were collected by adding 100 μl 536
of 0.53 mM EDTA-containing Dulbecco’s PBS (D-PBS), followed by 100 μl of 5 mM 537
HEPES (pH 7.4)-containing Hank’s Balanced Salt Solution (HBSS). The cell 538
suspension was transferred to a 96-well V-bottom plate and fluorescently labeled with 539
an anti-FLAG epitope (DYKDDDDK) tag monoclonal antibody (Clone 1E6, FujiFilm 540
Wako Pure Chemicals; 10 μg/ml diluted in 2% goat serum- and 2 mM EDTA-containing 541
D-PBS (blocking buffer)) and a goat anti-mouse IgG secondary antibody conjugated 542
with Alexa Fluor 488 (ThermoFisher Scientific, 10 μg/ml diluted in the blocking buffer). 543
After washing with D-PBS, the cells were resuspended in 200 μl of 2 mM 544
EDTA-containing-D-PBS and filtered through a 40-μm filter. The fluorescent intensity 545
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of single cells was quantified by an EC800 flow cytometer equipped with a 488 nm 546
laser (Sony). The fluorescent signal derived from Alexa Fluor 488 was recorded in an 547
FL1 channel, and the flow cytometry data were analyzed with the FlowJo software 548
(FlowJo). Live cells were gated with a forward scatter (FS-Peak-Lin) cutoff at the 390 549
setting, with a gain value of 1.7. Values of mean fluorescence intensity (MFI) from 550
approximately 20,000 cells per sample were used for analysis. 551
552
Data Availability 553
The raw image of the PAC1R-miniGsβ1γ2-Nb35 complex after motion 554
correction has been deposited in the Electron Microscopy Public Image Archive, under 555
accession code XXXX. The cryo-EM density map and atomic coordinates for the 556
PAC1R-mini-Gs-Nb35 complex have been deposited in the Electron Microscopy Data 557
Bank and the PDB, under accession codes XXXX and ZZZZ, respectively. 558
559
Supplementary Figures 560
Supplementary Fig. 1. Functional characterization of mutant PAC1 561
receptors. 562
a, PACAP-induced Gs activation, measured by the NanoBiT-G-protein dissociation 563
assay. Cells transiently expressing the NanoBiT-Gs, along with the indicated PAC1R 564
construct, were treated with PACAP (1-38) and the change in the luminescent signal 565
was measured. b, c, Cell surface expression of the PAC1R and the GLP1R constructs. 566
Cells transiently expressing the indicated FLAG epitope-tagged PAC1R constructs (b) 567
or the GLP1R constructs (c) were labeled with an anti-FLAG tag antibody along with 568
an Alexa488-conjugated secondary antibody, and the fluorescent signals from individual 569
cells were measured by a flow cytometer. 570
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 27, 2019. ; https://doi.org/10.1101/2019.12.23.887737doi: bioRxiv preprint
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571
Supplementary Fig. 2. Cryo-EM analysis. 572
Flow chart of the cryo-EM data processing for the PACAP–PAC1R–Gs complex, 573
including particle projection selection, classification, and 3D density map reconstruction. 574
Details are provided in the Methods section. 575
576
Supplementary Fig. 3. Map/model quality. 577
a, The cryo-EM density map and model are shown for PACAP, including all seven 578
transmembrane α-helices, ECD, and α5 of Gαs. b, Dimer interface. The complexes are 579
shown as ribbon representations, colored as in Fig. 1a. The side chains of V3185.48 and 580
M3225.52 are shown as sticks. Two complexes form an anti-parallel dimer with C2 581
symmetry in the detergent micelles. This dimer does not reflect the physiological 582
condition, but is produced during the sample preparation. The molecular packing of the 583
two complexes in the dimer class is mediated through only a weak hydrophobic contact 584
between V3185.48 and M3225.52. Therefore, the dimerization minimally affects the 585
conformation of the Gs-complexed PAC1R structure. 586
Supplementary Fig. 4. Comparison of peptide binding interactions in class 587
B GPCRs. 588
a-d, Ligand binding interactions with the TMDs in the class B GPCR structures (a, 589
PAC1R, b, GLP1R, c, PTH1R, and d, CGRP). Hydrogen bonding interactions are 590
indicated by black dashed lines. PACAP forms extensive hydrogen-bonding interactions 591
with TM1, whereas GLP1 forms only a hydrophobic contact with Y1451.40. PTH also 592
interacts with TM1; however, these interactions are mainly hydrophobic. d-f, Relative 593
positions of the peptide ligands and the ECDs in the class B GPCR structures (e, 594
PAC1R, f, GLP1R, g, PTH1R, and h, CGRP). 595
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596
Supplementary Fig. 5. Sequence alignment of PAC1R, VPAC1R, and 597
VPAC2R. 598
Amino-acid sequences of the transmembrane domains of the human PAC1R (UniProt 599
ID: P41586), VPAC1R (P32241), and VPAC2R (P25101). Secondary structure elements 600
for α-helices and β-strands are indicated by cylinders and arrows, respectively. 601
Conservation of the residues between the PACAP receptors is indicated as follows: red 602
panels for completely conserved, red letters for partly conserved, and black letters for 603
not conserved. The residues involved in the PACAP binding are indicated by squares. 604
605
Supplementary Table 1. Interactions of the PACAP N-terminal helix with the 606
PAC1R-TMD. 607
Residues within 4.0 Å are shown. 608
609
Reference. 610
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Activation of Class B G Protein-coupled Receptors. J. Biol. Chem. 291, 15119–663
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preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 27, 2019. ; https://doi.org/10.1101/2019.12.23.887737doi: bioRxiv preprint
Kobayashi et al. Figure 1
Nb-35
a b
c d
ECD
PACAP
miniGαs
Gγ
Gβ
PAC1R TMD
E3927.49
P3606.47
L3616.48
F3626.49
TM6TM7
TM1
G3636.50
G3897.46
G3937.50
S3907.47
F3917.48
TM6
TM7
TM1
H8
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 27, 2019. ; https://doi.org/10.1101/2019.12.23.887737doi: bioRxiv preprint
Kobayashi et al. Figure 2
ca b
d e
PACAP
PAC1R TMD
Y1501.36
Y10S11
S10
F6
G4
T7D8
S2
S2K1541.40
K2062.67
K2062.67 K2062.67
D3D3
H1
D2985.52
D3
Y1501.36
D1471.33
N1461.32
S11Y10
T7
F2333.36
V2032.64
V2032.64
Y2413.44
V3135.43
K3105.40
I3095.39
I3095.39
R3817.38
L3827.39
L3867.43
L3867.43
I5
F6
Y13
Y10
M17
E3857.42
E3857.42
E3857.42
Y1611.47
Y1611.47
Y1611.47
Y1571.43
Y1571.43
V1531.39
K1541.40
K3787.35
Y1501.36
S2
I5
D3
V2373.40
Y2112.70D2985.52
D2985.52
D8
S11
R14
K15
G4
H1
T7
M299
N300
W3065.36
W3065.36
TM1 TM6TM7
TM1
TM5TM2
TM2TM4TM5TM5
TM4TM3
TM2
TM1
TM6
TM1
TM7
TM7
45°
45°
90°
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 27, 2019. ; https://doi.org/10.1101/2019.12.23.887737doi: bioRxiv preprint
Kobayashi et al. Figure 3
10-10
10-9
10-8
10-7
10-6
0.7
0.8
0.9
1.0
0Gs
diss
ocia
tion
(RLU
cha
nge
over
bas
al)
PACAP-38 (M)
WT
P360A
G363A
WTP360AG363A
a b c
d e f
Gs
diss
ocia
tion
(RLU
cha
nge
over
bas
al)
PACAP (1-38) [M]
Y2413.44Y2413.44
R1992.60 R1992.60
D3D3S2S2
Y1611.47Y1611.47Y3666.53Y3666.53
E3857.37E3857.37
Q3927,49
G3636.50G3636.50
P3606.47P3606.47
N2403.43N2403.43
TM5TM5
TM6TM6
TM6TM6
TM3TM3
a5Ha5Ha5Ha5Ha5Ha5H
TM5TM5
H8H8
TM3TM3
TM5TM5
H8H8H8H8
TM3TM3
TM6TM6 TM6TM6
TM7
D381
L2553.58
A2563.59
R380
Q384 D381R385
Q390
R1762.46
L394
K3345.64
V4057.60
L393
N4067.61
S3526.41
Q384
K3885.64
S4096.41
L393L394
R385
I3103.58
Q384
R380
L2553.58
K3345.64
S3546.41
V2563.59
L394
L393
E392
TM5TM5
ICL2ICL2ICL2ICL2ICL2ICL2
PAC1R GLP1RGLP1R PTH1R
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 27, 2019. ; https://doi.org/10.1101/2019.12.23.887737doi: bioRxiv preprint
Kobayashi et al. Figure 4
pEC50 (± SEM)
9.14 ± 0.22
9.08 ± 0.19
<6
EC50
0.73 nM
0.83 nM
>1 M
n =
4
4
4
b c da
V19
V23Y22
Q16
L27
40°
89-110
α1
α3
α2
β1β2
β3 β4β5
GLP1R-GLP1 PAC1R-PACAP
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 27, 2019. ; https://doi.org/10.1101/2019.12.23.887737doi: bioRxiv preprint
Kobayashi et al. Figure 5
10-10
10-9
10-8
10-7
10-6
10-5
10-4
0.7
0.8
0.9
1.0
010
-11
10-10
10-910
-810
-710
-610
-510
-4
0.7
0.8
0.9
1.0
010
-11
10-10
10-910
-810
-710
-610
-510
-4
0.7
0.8
0.9
1.0
0Gs
diss
ocia
tion
(RLU
cha
nge
over
bas
al)
GLP-1 (M)
MockFL N
NanoBiT-G-protein dissociation assay
GLP-1 (16)
GLP-1 (7-37)pEC50 (± SEM)EC50
GLP-1 (16)pEC50 (± SEM)EC50
n =
8.93 ± 0.171.2 nM
<4>100 M
4
<6>1 M
<4>100 M
3
GLP-1 (7-37)
10-10
10-9
10-8
10-7
10-6
10-5
10-4
0.7
0.8
0.9
1.0
010
-1010
-910
-810
-710
-610
-510
-4
0.7
0.8
0.9
1.0
0Gs
diss
ocia
tion
(RLU
cha
nge
over
bas
al)
PACAP (M)
Mock FL
10-10
10-9
10-8
10-7
10-6
0.7
0.8
0.9
1.0
0Gs
diss
ocia
tion
(RLU
cha
nge
over
bas
al)
PACAP-38 (M)
10-10
10-9
10-8
10-7
10-6
10-5
10-4
0.7
0.8
0.9
1.0
0
N
Mock
WT
C
G389A
Gs
diss
ocia
tion
(RLU
cha
nge
over
bas
al)
PACAP-38 (M)
WT
pEC50 (± SEM)
9.14 ± 0.22
9.42 ± 0.03
8.92 ± 0.20
EC50
0.73 nM
0.38 nM
1.2 nM
n =
4
3
4
pEC50 (± SEM)
9.14 ± 0.22
9.08 ± 0.19
<6
EC50
0.73 nM
0.83 nM
>1 M
n =
4
4
4
PACAP-38pEC50 (± SEM)EC50
PACAP-17pEC50 (± SEM)EC50
n =
8.90 ± 0.101.3 nM
5.09 ± 0.098.1 M
5
6.24 ± 0.08580 nM
4.72 ± 0.1719 M
4
P360A
G363A
10-10
10-9
10-8
10-7
10-6
10-5
10-4
0.7
0.8
0.9
1.0
0
C
8.95 ± 0.101.1 nM
4.86 ± 0.0614 M
4
Gs
diss
ocia
tion
(RLU
cha
nge
over
bas
al)
Gs
diss
ocia
tion
(RLU
cha
nge
over
bas
al)
GLP1 (7-23)
GLP1 (7-37)
PACAP (1-17)
PACAP (1-38)
d
ca b
e f
PAC1R PAC1RΔECD (148-468)
GLP1R GLP1RΔECD (140-463)
H1
M17
H7
Q23
PACAP [M]
GLP1 [M]
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 27, 2019. ; https://doi.org/10.1101/2019.12.23.887737doi: bioRxiv preprint
Kobayashi et al. Supplementary Figure 1
Mock
PAC1R-W
T
ΔC (1-41
7)
ΔECD (1
48-46
8)
P360A
G363A
G389A
0
2000
4000
6000
8000
GPC
R e
xpre
ssio
n le
vel
(mea
n flu
ores
cenc
e un
it, a
.u.)
Gs
diss
ocia
tion
(RLU
cha
nge
over
bas
al)
Mock
WT
C
G389A
10-10
10-9
10-8
10-7
10-6
0.7
0.8
0.9
1.0
0Gs
diss
ocia
tion
(RLU
cha
nge
over
bas
al)
PACAP-38 (M)
WT
pEC50 (± SEM)
9.14 ± 0.22
9.42 ± 0.03
8.92 ± 0.20
EC50
0.73 nM
0.38 nM
1.2 nM
n =
4
3
4
pEC50 (± SEM)
9.14 ± 0.22
9.08 ± 0.19
<6
EC50
0.73 nM
0.83 nM
>1 M
n =
4
4
4
P360A
G363A
10-10
10-9
10-8
10-7
10-6
0.7
0.8
0.9
1.0
0
Gs
diss
ocia
tion
(RLU
cha
nge
over
bas
al)
10-10
10-9
10-8
10-7
10-6
0.7
0.8
0.9
1.0
0Gs
diss
ocia
tion
(RLU
cha
nge
over
bas
al)
PACAP-38 (M)
WT
pEC50 (± SEM)
9.14 ± 0.22
9.42 ± 0.03
8.92 ± 0.20
EC50
0.73 nM
0.38 nM
1.2 nM
n =
4
3
4
pEC50 (± SEM)
9.14 ± 0.22
9.08 ± 0.19
<6
EC50
0.73 nM
0.83 nM
>1 M
n =
4
4
4
P360A
G363A
PACAP (1-38) (M)
0
500
1000
1500
2000
2500
GPC
R e
xpre
ssio
n le
vel
(mea
n flu
ores
cenc
e un
it, a
.u.)
GLP1R
-WT
ΔECD
(140
-463)
b c
a
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 27, 2019. ; https://doi.org/10.1101/2019.12.23.887737doi: bioRxiv preprint
Kobayashi et al. Supplementary Figure 2
PostProcessPostProcess
Extract (1.35 Å/pix) Extract (1.35 Å/pix)
Polish, Refine3D Polish, Refine3DRefine3D, CtfRefine Refine3D, CtfRefine
Motion Correction, CtfFind
Autopick, Extract (3.24 Å/pix)
Class3D
Refine3D Refine3D
4.5 Å 4.0 Å
4.05 Å
2,895 movies
980,964 particles
282,622 particles 132,808 particles
3.7
4.9
4.6
4.3
4.0
Monomer class Dimer class Monomer class Dimer class
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40-0.2
0.0
0.2
0.4
0.6
0.8
1.0
Fourier Shell Correlation
FSC = 0.143
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40-0.2
0.0
0.2
0.4
0.6
0.8
1.0
Resolution (1/Å)
Four
ier S
hell
Cor
rela
tion
FSC = 0.143
90°
0.9half1 vs modelhalf2 vs model sum vs model0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.050 0.10 0.15 0.20 0.25 0.30 0.35
0
-0.1
FSC = 0.5
Resolution (1/Å)
Resolution (1/Å)
Four
ier S
hell
Cor
rela
tion
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Kobayashi et al. Supplementary Figure 3
TM1 (147-176) TM4 (265-291) TM5 (306-336) TM6 (346-371) TM7 (376-400)
Gαs Ras α5 (360-384)Helix8 (404-416)
TM2 (183-211) TM3 (224-256)
PACAP (1-28)
a
b
TM5
TM5
TM5
TM5
M3185.48
V3225.52
V3225.52
M3185.48
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Kobayashi et al. Supplementary Figure 4
TM7TM6TM5 TM6TM5 TM5TM5 TM1TM7TM4TM4TM5 TM1
TM1 TM2TM7TM1 TM2TM7
TM7TM4TM4 TM5 TM1TM7TM5
TM1
TM7
TM2 TM2TM1TM7
α1β1
β2
β3
β4
β5
α2α1
α1
β1
β2
β3
β4
β1
β2
β3
β4α1
β1
β2
β3
β4α2
TM1 TM7
a b c d
e f g h
α3
Y1451.40
RAMP1
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 27, 2019. ; https://doi.org/10.1101/2019.12.23.887737doi: bioRxiv preprint
Kobayashi et al. Supplementary Figure 5
AT YF T MNDS AL G G G V QS GG TA YL T TNDH VP I I S R TS GG TI HF Y TIN. SL G T C R RP RK
E V FH S F F ETFFPE Y YW T T VCVT G S LQ A L H AMLPP. C LA L L VCIG G A FQ A L Y VSFFSE Y WG I V TFTM
SDMGV E EPF .H FD FD YESETGD.QDY LS AL ST SKAGN S ETF .D VD YS PEDES...KIT IL AI SV QGRN D HLE GP PI LD KAASLDEQQTM GS TG GL
AGVVHVS AALL P............M PA HS I KKEQAMCLEKIQR NE MGF RTLLPPA LTCW A............P NS HP R HLEIQE..EETKC EL RSQ RPPSPLP RWLC AGALAWALGPAGGQ AR QE D VQMIEV..QHKQC EE QLE
1 10 20 30 40
PAC1 M L C L L A M D F A L NVIPR2 M L C L L V I E F A L TVIPR1 M L C A V A L E Y L A N
50 60 70 80 90 100
PAC1 C WDN TCW G V CP F F D M I V L SSPG PG KPAHV EM L S E RI NPDQVWETETIGESDFGDSNSLDLVIPR2 C WDN TCW G V CP F F E V I V V KHKA SG RPANV ET T P K SN Y.......................VIPR1 C WDN TCW G V CP F F E M L L I T.IG SK PATPR QV V A L KL SS......................
110 120 130 140 150 160
PAC1 S CT GW P ACG Y VK YT GY SL V RN D S Y E Y V V VIPR2 S CT GW P ACG Y VK YT GY SL I KN D S F D F L M VIPR1 S CT GW P ACG Y VK YT GY SL V RS E T Y D F I AI
170 180 190 200 210 220
PAC1 L IL FRKLHCTRN IH LF SF LRA V KD L C T V F M V M I I I Y S TTAM CR N S F W AEQD NH ...FISTVIPR2 L IL FRKLHCTRN IH LF SF LRA V KD L C S I Y L L I I V V Y T ATGS CL N S L D SSSG LH PDQPSSWVIPR1 L IL FRKLHCTRN IH LF SF LRA V KD L C T A Y M I I A I A F S LVAT SL H A F L DSGE DQ S...EGS
230 240 250 260 270 280
PAC1 V CK VF YC N FWL EGLYL TLL R F Y IGWG P A M VV Y I V R I T VVIPR2 V CK VF YC N FWL EGLYL TLL R F Y IGWG P L L IM F V V R L T AVIPR1 V CK VF YC N FWL EGLYL TLL R F Y IGWG P A M VM F V A K L S V
290 300 310 320 330 340
PAC1 W R D GCWD WW I P SI VNF LFI II IL QKL PD L L D T V K VV M V V M NVIPR2 W R D GCWD WW I P SI VNF LFI II IL QKL PD A L E S V R IL I V L V NVIPR1 W R D GCWD WW I P SI VNF LFI II IL QKL PD A I E S I K IL L I L I S
350 360 370 380 390 400
PAC1 S Y RLA STLLLIPLFG HY FA P FEL GSFQG VVA LYCFLES R I V N LV L V I L T FS E VSKRER G F VIPR2 S Y RLA STLLLIPLFG HY FA P FEL GSFQG VVA LYCFLDQ K V V S IL L V Q K M VF I ISSKYQ C L VIPR1 S Y RLA STLLLIPLFG HY FA P FEL GSFQG VVA LYCFLDS R V M N MV V I P S I FF D FKPEVK V F
410 420 430 440 450 460
PAC1 N EVQ E RKWR S G Q IK N K S SK S I G A SWKV RYFAVDF HRHP LAS VNGGT LSIL S SQ RMSGLPAVIPR2 N EVQ E RKWR S G Q LK T R N SR Q L S C SRCP PSASRDY VCGS FSR SEGAL FHRG A SF QTETSVIVIPR1 N EVQ E RKWR S G Q LR Q K N TR S A G A RWHL GVLGWNP YRHP GGS ATCST VSML V PG RRSSSFQ
PAC1 DNLAT. VIPR2 ...... VIPR1 AEVSLV
TM1
TM2
TM3 TM4
TM5
TM6 TM7
H8
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 27, 2019. ; https://doi.org/10.1101/2019.12.23.887737doi: bioRxiv preprint
Kobayashi et al. Supplementary Table 1
PACAP PAC1R ConcervedHis1 Val237
Tyr241Trp306Ile309 CLys310Val313
Ser2 Glu385Leu382Leu386 C
Asp3 Tyr161 CVal203 CPhe233Leu386 C
Gly4 Asn300Trp306 C
Ile5 Lys378Arg381Leu382
Phe6 Tyr150 CVal153Lys154 CTyr157 CLeu382Leu386 C
Thr7 Lys206 CTyr211Asp298 C
Asp8 Asp298 CMet299Asn300
Ser9 Tyr150 CLys378
Tyr10 Lys154 CTyr150 CTyr211
Ser11 Lys206 CTyr211Asp298 CMet299
Arg12 Asp301Tyr13 Gln146
Asp147Arg14 Leu210
Tyr211Lys15 Met299Met17 Asp147
Hydrophobic interaction
Hydrophobic interactionHydrophobic interaction
Hydrophobic interactionHydrogen bond
Hydrophobic interaction
Hydrophobic interaction
Hydrophobic interaction
Hydrophobic interaction
Hydrophobic interaction
Hydrophobic interaction
Hydrophobic interaction
Hydrophobic interaction
Hydrogen bond
Electrostatic interaction
Hydrogen bond
Interaction
Hydrogen bond
Hydrogen bond
Hydrophobic interaction
Hydrophobic interaction
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 27, 2019. ; https://doi.org/10.1101/2019.12.23.887737doi: bioRxiv preprint