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

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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This article is a preprint version and has not been certified by peer review. 17

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preprint (which was not certified by peer review) is the author/funder.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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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

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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

150

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

183

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


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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


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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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693


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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


https://doi.org/10.1101/2019.12.23.887737


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°


https://doi.org/10.1101/2019.12.23.887737


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


https://doi.org/10.1101/2019.12.23.887737


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


https://doi.org/10.1101/2019.12.23.887737


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]


https://doi.org/10.1101/2019.12.23.887737

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


https://doi.org/10.1101/2019.12.23.887737


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


https://doi.org/10.1101/2019.12.23.887737


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


https://doi.org/10.1101/2019.12.23.887737


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


https://doi.org/10.1101/2019.12.23.887737


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


https://doi.org/10.1101/2019.12.23.887737

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









Hydrogen bond

Electrostatic interaction

Hydrogen bond

Interaction

Hydrogen bond

Hydrogen bond




https://doi.org/10.1101/2019.12.23.887737

cryo-em structure of the human pac1 receptor coupled to an ...dec 23, 2019 · em structure of the...

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