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SUPPLEMENTARY INFORMATIONdoi:10.1038/nature12393

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Supplementary Tables

Supplementary Table 1. Binding study of wild-type GCGR and crystallized BRIL-GCGR(ECD/C) construct with NNC0640.

GCGR construct IC50* (nM)

Spana,*

(% of wild-type)

Expression levelb,*

(% of wild-type)

Wild-type 189.9 ± 29.9 100 100

BRIL-GCGR(ECD/C) 182.5 ± 38.9 45.5 ± 4.9 48.7 ± 10.8 a Specific 3H-NNC0640 binding (span) is defined as the window between the highest (1.26 nM unlabeled ligand) and lowest binding (100 µM unlabeled ligand). b Expression level was determined by Western blot with an anti-V5 antibody. * Values shown are means ± SEM of at least 3 independent experiments.

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Supplementary Table 2. Data collection and refinement statistics of GCGR.

Structure BRIL-GCGR(ΔECD/ΔC) Data Collection

Number of crystals 14

Space group P212121 Cell dimensions a, b, c (Å) 56.62, 66.65, 163.33 Number of reflections measured 56,418 Number of unique reflections 9,067 Resolution (Å) * 3.3 × 3.4 × 3.3 (3.42-3.30) Rmerge (%)* 10.5 (88.2) CC1/2

(%)§,* 99.4 (76.6) Mean I/σ(I)* 22.6 (1.5)

Completeness (%)* 93.9 (90.2) Redundancy* 6.2 (6.2)

Refinement Resolution (Å)a Number of reflections (test set)b

50 – 3.4 8,981 (430)

Rwork / Rfree (%) 28.4 / 33.9

Number of atoms Receptor BRIL Other

2,230 819 7

Mean Overall B value (Å2) WILSON B Receptor BRIL Other

110 127 127 73

R.m.s. deviations Bond lengths (Å) Bond angles (°)

0.010 1.04

Ramanchandran plot statistics (%)c Favored regions Allowed regions Disallowed regions

92.1 7.4 0.5

* Highest resolution shell is shown in parenthesis.

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§ Defined by Karplus & Diederichs1. a The diffraction data are anisotropic with resolution cutoff in a*, b*, and c* axes, respectively. Diffraction data were included to 3.3 Å in the a* and c* directions, with an overall effective and reported resolution of 3.4 Å. b Number of reflections after anisotropic truncation. c As defined in MolProbity.


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Supplementary Table 3. Correspondence between class A Ballesteros-Weinstein and class B Wootten residue numbering. Human GCGR and bovine rhodopsin (PDB 1U19) are used as representative members of class B and A GPCRs, respectively (Supplementary Fig. 5).

GCGR (class B) GPCR residuea

Rho (class A) GPCR residuea

S1521.50 G51 (BW 1.46)

L1561.54 N55 (BW1.50)

H1772.50 L76 (BW 2.43)

F1842.57 D83 (BW 2.50)

E2453.50 L131 (BW 3.46)

L2493.54 R135 (BW 3.50)

W2724.50 W161 (BW 4.50)

A3145.46 P215 (BW 5.50)

N3185.50 I219 (BW 5.54)

G3596.50 L262 (BW 6.45)

V3646.55 P267 (BW 6.50)

G3937.50 A299 (BW 7.46)

A3977.54 P303 (BW 7.50)

aSuperscripts refer to the Wootten numbering in which the single most conserved residue among class B GPCRs2 is designated X.50. The Ballesteros-Weinstein numbering in which the single most conserved residue among class A GPCRs3 is designated (BW X.50). X is the transmembrane helix number. All other residues on that helix are numbered relative to this conserved position.



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Supplementary Table 4. Ligand binding pocket volume estimates for different GPCRs. Several different measurements are given with the pockets delimited by the extracellular membrane border (as predicted by OMP server (opm.phar.umich.edu) for PDB 3EML)4,5, and extracellular membrane border shifted up by 1 or 2 Å. For GCGR measurements, missing side chains in the crystal structures were modeled and optimized. Volume measurements are in units of Å3.

GPCR PDB Accession

EC membrane (Å3)

EC membrane +1 Å (Å3)

EC membrane +2 Å (Å3)

GCGR -- 1368 1537 1726

-OR 4DJH 905 1030 1507

CXCR4 3ODU 693 935 1096

NTSR1* 4GRV 331 377 439

2AR 2RH1 622 709 810

Rho 1U19 533 533 533

*Though the NTSR1 pocket accommodates a large peptide ligand, the pocket is located closer to the extracellular loop region than in other solved GPCR structures and, therefore, the volume calculations appear low relative to the other receptors where the distance is measured below the surface of the membrane6.



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Supplementary Table 5. Binding study of GCGR mutants with glucagona,b.

GCGR region

GCGR residuec

Mutation Mutant IC50 (nM)*

Spand,* (% of wild-type)

Expression levele,*

(% of wild-type)

Full-length

Wild-typef 20.0 ± 1.0 100 100

ECD D30 L 17.0 ± 9.8 82.7 ± 6.9 71.9 ± 17.1

F31 W 13.2 ± 7.6 146.1 ± 20.0 147.9 ± 25.5 W36 A NB NB 343.5 ± 41.8 D63 A NB NB 200.7 ± 95.6 K64 E 20.1 ± 11.6 14.6 ± 1.5 24.0 ± 5.9 P86 S NB NB 299.2 ± 25.7

R116 L 39.0 ± 22.5 81.7 ± 12.9 18.7 ± 7.3 Q122 E 14.9 ± 8.6 159.4 ± 14.7 56.7 ± 17.5 Q122 A 27.6 ± 5.3 101.9 ± 3.8 125.0 ± 8.0 M123 E 20.5 ± 11.9 124.1 ± 24.8 105.3 ± 26.9

Stalk G125 K 16.0 ± 9.2 155.9 ± 17.5 122.6 ± 26.4

E126 R 10.3 ± 5.9 147.9 ± 64.6 168.7 ± 27.1 E127 K 23.9 ± 6.0 31.4 ± 2.8 203.0 ± 14.7 I128 Q 13.5 ± 3.2 75.3 ± 5.8 93.9 ± 13.6 E129 R 12.2 ± 7.1 152.4 ± 25.8 81.9 ± 19.9 Q131 R 29.8 ± 8.9 39.6 ± 8.8 82.5 ± 7.7 K132 S 15.0 ± 3.2 118.8 ± 20.9 46.2 ± 8.0 E133 P 59.1 ± 11.6 13.9 ± 2.2 186.1 ± 20.5 A135 P NB NB 146.9 ± 22.0

Helix I Y1381.36 L 12.7 ± 1.7 103.1 ± 10.4 64.8 ± 12.0

S1401.38 F 28.6 ± 16.5 83.3 ± 3.8 77.8 ± 11.1 F1411.39 L 18.7 ± 10.8 169.4 ± 15.5 45.5 ± 13.3 Q1421.40 Y 42.9 ± 5.4 26.1 ± 0.8 80.7 ± 11.8 V1431.41 I 16.0 ± 9.2 225.2 ± 57.2 83.4 ± 17.4 M1441.42 I 10.8 ± 6.3 118.0 ± 23.4 58.7 ± 11.3 Y1451.43 A NB NB 173.8 ± 12.3 Y1451.43 N 215.4 ± 14.1 15.8 ± 1.1 114.4 ± 13.2 Y1491.47 A NB NB 1.2 ± 4.7 Y1491.47 H 128.6 ± 9.0 20.4 ± 0.2 108.9 ± 11.5 S1501.48 A 14.5 ± 8.4 82.7 ± 19.0 57.9 ± 7.5 L1531.51 F 21.8 ± 12.6 76.8 ± 23.0 14.2 ± 7.5 G1541.52 S 20.6 ± 11.9 130.2 ± 23.4 55.2 ± 13.6

Helix II F1842.57 A 19.1 ± 1.0 145.3 ± 23.9 33.9 ± 9.2

K1872.60 R 21.3 ± 1.4 117.1 ± 25.2 81.9 ± 14.1 K1872.60 Q 41.4 ± 2.6 74.9 ± 13.6 11.9 ± 10.8 K1872.60 L NB NB 7.4 ± 5.4 K1872.60 A NB NB 7.9 ± 4.5 S1892.62 L 14.7 ± 5.4 121.4 ± 39.9 99.9 ± 6.0 V1912.64 A 107.1 ± 10.2 12.8 ± 0.3 124.9 ± 8.6 L1922.65 F 1.9 ± 0.4 82.4 ± 27.8 3.5 ± 1.5 I1942.67 A 16.3 ± 2.6 65.7 ± 4.7 34.3 ± 3.7 I1942.67 K NB NB 65.9 ± 7.7 D1952.68 N 111.1 ± 20.9 49.6 ± 16.3 50.5 ± 11.9



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GCGR region

GCGR residuec



Expression levele,*

(% of wild-type)

Helix II D1952.68 A 170.9 ± 58.1 12.3 ± 2.6 54.1 ± 9.8 L1982.71 V 56.4 ± 5.7 26.0 ± 4.4 175.9 ± 6.4 L1982.71 A NB NB 168.1 ± 26.2

ECL1 T200 W 10.3 ± 3.2 223.0 ± 30.8 160.5 ± 11.7

R201 M 50.0 ± 1.8 49.7 ± 8.4 75.7 ± 9.8 R201 A 131.8 ± 17.1 17.7 ± 4.2 39.8 ± 10.5 R201 D NB NB 200.1 ± 25.2 Y202 A NB NB 144.1 ± 20.4 Q204 T 16.9 ± 3.2 108.7 ± 24.4 77.3 ± 16.1 K205 A 15.1 ± 3.2 184.8 ± 11.2 108.0 ± 22.4 D208 Q NB NB 122.1 ± 17.6 D208 R 100.8 ± 8.3 14.2 ± 2.6 150.9 ± 5.0 D209 Q 16.8 ± 3.2 140.4 ± 21.1 154.2 ± 36 V212 W 8.5 ± 0.7 198.6 ± 36.1 52.3 ± 8.7 W215 L NB NB 75.9 ± 12.8 S217 A 18.7 ± 4.1 110.7 ± 24.6 115.6 ± 11.9 D218 Y 30.6 ± 5.6 43.4 ± 7.6 184.4 ± 26.5

Helix III R2253.30 A NB NB 52.6 ± 7.9

A2283.33 V 56.8 ± 14.5 53.2 ± 7.3 119.6 ± 17.8 A2283.33 F 46.7 ± 7.1 26.3 ± 5.6 153.8 ± 20.0 M2313.36 A 19.3 ± 3.8 81.0 ± 4.2 48.8 ± 5.3 M2313.36 T 14.2 ± 2.5 90.4 ± 7.7 11.9 ± 1.4 Q2323.37 A 148.3 ± 5.4 17.8 ± 1.8 147.1 ± 6.9 Q2323.37 L NB NB 2.2 ± 0.4 Q2323.37 H 26.3 ± 3.7 99.6 ± 7.1 155.9 ± 16.1 Y2333.38 A NB NB 4.6 ± 6.5 I2353.40 V 21.6 ± 7.2 117.6 ± 10.8 108.0 ± 8.5 V2363.41 A 21.6 ± 1.1 34.3 ± 6.1 8.4 ± 16.2 N2383.43 A 82.1 ± 21.1 26.1 ± 3.5 22.2 ± 6.4 Y2393.44 A 20.6 ± 2.1 98.5 ± 9.2 50.3 ± 22.7 C2403.45 Y 23.0 ± 7.1 113.9 ± 39.0 102.7 ± 4.1

Helix IV F2784.56 C 16.2 ± 0.7 92.3 ± 11.1 82.4 ± 37.8

W2824.60 H 9.2 ± 2.6 46.8 ± 11.9 5.3 ± 5.2 K2864.64 L NB NB 102.2 ± 15.8

ECL2 E290 A NB NB 86.8 ± 9.2

N291 D 40.3 ± 11.5 56.5 ± 6.6 63.0 ± 12.1 N291 A 62.4 ± 23.0 26.1 ± 6.9 8.9 ± 2.0 C294 A NB NB 32.1 ± 9.4 C294 S NB NB 47.6 ± 12.7 W295 A NB NB 54.5 ± 13.2 W295 L 28.1 ± 4.5 12.7 ± 1.7 39.0 ± 4.4 W295 F 29.0 ± 1.8 22.3 ± 4.3 54.6 ± 1.6 W295 H NB NB 38.0 ± 3.1 S297 A 22.8 ± 7.2 171.1 ± 23.4 66.9 ± 16.7 S297 R 31.4 ± 6.0 122.1 ± 18.4 93.6 ± 19.3 N298 D 82.4 ± 12.7 24.4 ± 5.1 105.7 ± 12.8 N298 A 191.9 ± 34.6 18.7 ± 1.4 118.1 ± 24.8 N298 Q 211.1 ± 26.3 13.0 ± 1.0 78.2 ± 4.2



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GCGR region

GCGR residuec



Expression levele,*

(% of wild-type)

Helix V D2995.31 S 28.7 ± 0.4 119.9 ± 23.3 137.4 ± 19.8

N3005.32 A 109.5 ± 45.4 33.0 ± 1.7 131.5 ± 54.4 G3025.34 N 33.8 ± 8.4 132.0 ± 7.9 74.7 ± 15.9 F3035.35 A 101.1 ± 11.7 47.5 ± 7.8 95.2 ± 13.9 F3035.35 Y 25.3 ± 8.8 155.2 ± 6.9 68.3 ± 21.3 W3045.36 A 280.7 ± 39.5 14.8 ± 1.8 125.1 ± 3.8 W3045.36 L 93.0 ± 17.5 28.8 ± 2.5 136.3 ± 15.0 W3045.36 Q NB NB 174.1 ± 15.8 W3055.37 L 35.7 ± 6.1 85.4 ± 22.6 68.1 ± 3.7 W3055.37 A 73.1 ± 30.7 82.4 ± 5.0 45.1 ± 11.7 I3065.38 A 82.3 ± 17.3 19.0 ± 2.4 49.2 ± 2.3 L3075.39 I 29.5 ± 9.3 103.6 ± 25.8 130.1 ± 12.0 R3085.40 Q 46.5 ± 8.2 55.1 ± 4.8 45.6 ± 5.6 R3085.40 A 88.1 ± 20.4 24.8 ± 0.8 35.6 ± 7.8 V3115.43 I 60.8 ± 5.4 26.3 ± 2.8 8.6 ± 4.7

Helix VI H3616.52 A 43.7 ± 16.0 90.9 ± 16.4 33.8 ± 3.4

E3626.53 A 133.5 ± 34.3 24.2 ± 9.7 67.0 ± 18.2 E3626.53 Q 213.8 ± 47.9 27.3 ± 3.0 83.3 ± 15.2 E3626.53 D 8.3 ± 1.7 130.6 ± 7.6 7.7 ± 1.1 F3656.56 A 67.7 ± 17.4 36.8 ± 7.9 80.1 ± 0.3 F3656.56 I 550.0 ± 65.7 12.6 ± 1.5 49.5 ± 5.6 A3666.57 M 16.5 ± 4.3 265.6 ± 34.2 101.8 ± 15.5 T3696.60 M 11.2 ± 3.6 190.6 ± 11.0 34.8 ± 5.8

ECL3 Q374 R 13.7 ± 3.5 179.4 ± 15.9 62.9 ± 11.1

R378 A 129.5 ± 36.2 31.8 ± 7.1 108.0 ± 15.4

Helix VII

K3817.38 Q 36.8 ± 6.7 24.1 ± 2.8 3.7 ± 7.4 K3817.38 L 43.5 ± 4.7 41.8 ± 5.5 13.3 ± 9.3 K3817.38 A 73.3 ± 12.0 62.0 ± 14.5 165.7 ± 25.6 L3827.39 A NB NB 53.6 ± 7.1 L3827.39 V 107.2 ± 12.5 25.7 ± 1.1 108.4 ± 6.6 F3837.40 A 6.3 ± 1.0 204.6 ± 35.5 52.7 ± 8.6 F3847.41 T 71.6 ± 6.3 74.4 ± 13.6 54.2 ± 12.7 L3867.43 F NB NB 61.5 ± 4.1 L3867.43 A 182.5 ± 19.8 16.5 ± 2.1 156.1 ± 3.1 L3867.43 V 47.4 ± 5.9 61.5 ± 10.4 155.7 ± 4.5 F3877.44 S 46.8 ± 13.2 58.2 ± 12.3 60.5 ± 12.3 S3897.46 A 30.0 ± 2.7 92.9 ± 8.3 49.4 ± 9.2 S3897.46 T 32.1 ± 10.5 111.0 ± 10.7 86.9 ± 12.1

a Previous mutation studies to investigate peptide ligand binding have been reported for several class B GPCRs including GCGR7-13, GLP1R2,14-17, GIPR18,19, rSCTR20-24, and VPAC1 (refs 25-27), shown in Supplementary Table 6. b Mutants are colored coded according to Fig. 4. Mutants with <30% wild-type expression are colored grey. c Superscripts refer to the Wootten residue numbering2 in which the single most conserved residue among class B GPCRs is designated X.50, where X is the transmembrane helix number. All other residues on that helix are numbered relative to this conserved position. See Supplementary Table 3 for comparison

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between class B2 and class A3 Ballesteros-Weinstein numbering based on structural alignment of GCGR and rhodopsin (Supplementary Fig. 6). d Specific 125I-glucagon binding (span) is defined as the window between the highest (0.02 nM unlabeled ligand) and lowest binding (5 µM unlabeled ligand). If the span was <10% of the wild-type GCGR, IC50 is reported as no binding (NB). e Expression level was determined by flow cytometry with an anti-GCGR antibody. f The mutagenesis studies were performed in four separate rounds of experiments. For each round of experiments, IC50 measurements for wild-type GCGR were repeated and are shown as an average of all values generated with a standard error of the mean (SEM). * The values shown are means ± SEM of at least 3 independent experiments.



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between class B2 and class A3 Ballesteros-Weinstein numbering based on structural alignment of GCGR and rhodopsin (Supplementary Fig. 6). d Specific 125I-glucagon binding (span) is defined as the window between the highest (0.02 nM unlabeled ligand) and lowest binding (5 µM unlabeled ligand). If the span was <10% of the wild-type GCGR, IC50 is reported as no binding (NB). e Expression level was determined by flow cytometry with an anti-GCGR antibody. f The mutagenesis studies were performed in four separate rounds of experiments. For each round of experiments, IC50 measurements for wild-type GCGR were repeated and are shown as an average of all values generated with a standard error of the mean (SEM). * The values shown are means ± SEM of at least 3 independent experiments.



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Supplementary Table 6. Mutations of other class B GPCRs. Overview of 274 point mutations in total of 243 residues reported in 24 studies for human glucagon receptor (GCGR), glucagon-like peptide-1 receptor (GLP1R), gastric inhibitory polypeptide receptor (GIPR), vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide receptor 1 (VPAC1), and rat secretin receptor (rSCTR).

Region Receptor Residuea Mutation Reference

ECD GLP1R L32 A 14

GLP1R T35 A 14

GLP1R V36 A 14

GCGR Q42 C 9

GCGR H45 C 9

GCGR L50 C 9

GCGR T54 C 9

GCGR L56 C 9

GCGR T61 C 9

GLP1R E68 A 14

GLP1R E68 K 28

GLP1R Y69 A 14

GCGR T71 C 9

VPAC1 Q80 A 25

GLP1R Y88 A 14

GLP1R L89 A 14

GLP1R P90 A 14

GCGR W87 C 9

GCGR K90 C 9

GCGR R94 C 9

GCGR K98 C 9

GCGR K98 D 9

VPAC1 S104 A 27

VPAC1 S104 A 25

VPAC1 T106 A 25

VPAC1 T106 A 27

VPAC1 D107 A 27

VPAC1 E108 A 25

VPAC1 E108 A 27


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GCGR R108 C 9

VPAC1 H112 A 25

GCGR R111 C 9

GCGR Q113 A 7

GCGR Q113 N 7

GCGR Q113 E 7

GCGR Q113 C 9

VPAC1 P117 A 25

VPAC1 P119 A 25

VPAC1 I120 A 25

GLP1R R121 A 14

GLP1R L123 A 14

VPAC1 D125 A 25

Stalk VPAC1 D126 A 25

GLP1R E127 A 14

GLP1R E127 Q 14

GLP1R E128 A 14

GLP1R E128 Q 14

VPAC1 L131 A 25

VPAC1 D132 A 25

VPAC1 Q134 A 25

VPAC1 Q135 A 25

Helix I VPAC1 K1431.40 A 25

VPAC1 T1441.41 A 25

GCGR Y1451.43 A 13

rSCTR Y1241.43 A 21

rSCTR Y1241.43 H 21

VPAC1 T1471.44 A 25

GCGR Y1491.47 A 13

GLP1R Y1521.47 A 15

rSCTR Y1281.47 H 21

rSCTR Y1281.47 A 21

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GCGR S1521.50 A 13

GLP1R S1551.50 A 2

ICL1 GCGR C171 A 9

Helix II GLP1R H1762.50 A 29

GIPR H1692.50 R 19

GLP1R H1802.50 A 2

rSCTR H1562.50 A 24

GLP1R N1822.52 A 17

GLP1R S1862.56 A 2

GCGR F1842.57 A 13

GCGR K1872.60 R 8

rSCTR R1662.60 D 20

rSCTR R1662.60 L 20

rSCTR R1662.60 Q 20

GLP1R R1902.60 A 15

GLP1R R1902.60 A 17

GLP1R R1902.60 A 2

GIPR R1832.60 A 18

VPAC1 R1882.60 L 26

VPAC1 R1882.60 Q 26

GCGR S1902.63 A 13

rSCTR N1702.64 A 20

VPAC1 F1812.66 A 25

GCGR I1942.67 K 8

GLP1R K1972.67 A 15

GLP1R K1972.67 A 17

GIPR R1902.67 A 18

rSCTR K1732.67 I 20

rSCTR K1732.67 Q 20

VPAC1 K1952.67 I 26

VPAC1 K1952.67 Q 26

GLP1R D1982.68 A 15



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GLP1R D1982.68 A 17

rSCTR D1742.68 (b) N 20

rSCTR D1752.68 (b) C 22

rSCTR A1762.69 C 22

GCGR L1972.70 C 10

rSCTR V1772.70 C 22

GCGR L1982.71 C 10

rSCTR L1782.71 C 22

GCGR R1992.72 C 10

GLP1R K2022.72 A 17

ECL1 GCGR T200 C 10

rSCTR S180 C 22

GCGR R201 A 12

GCGR R201 D 12

GCGR R201 C 10

rSCTR S181 C 22

GCGR Y202 C 10

rSCTR D182 C 22

GCGR S203 C 10

rSCTR D183 C 22

GCGR Q204 C 10

rSCTR V184 C 22

GCGR K205 C 10

rSCTR T185 C 22

GCGR I206 C 10

rSCTR Y186 C 22

GCGR G207 C 10

rSCTR D188 C 22

GCGR D209 A 12

GCGR D209 H 12

GCGR D209 C 10

GLP1R H212 A 17

rSCTR A189 C 22



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GCGR L210 C 10

rSCTR H190 C 22

GCGR S211 C 10

rSCTR R191 C 22

GCGR V212 C 10

rSCTR A192 C 22

GLP1R D215 A 17

GCGR S213 C 10

rSCTR G193 C 22

GCGR T214 C 10

GCGR W215 C 10

rSCTR K195 C 22

GCGR L216 C 10

rSCTR L196 C 22

GCGR S217 C 10

rSCTR V197 C 22

GCGR D218 C 10

GCGR D218 Y 12

rSCTR M198 C 22

GCGR G219 C 10

rSCTR V199 C 22

GLP1R D222 A 17

GCGR A220 C 10

rSCTR L200 C 22

rSCTR F201 C 22

rSCTR Q202 C 22

Helix III GCGR V2213.26 C 10

GCGR A2223.27 C 10

GCGR G2233.28 C 10

GCGR C2243.29 S 10

GCGR C2243.29 S 9

GLP1R R2273.30 A 17

GIPR Q2203.33 A 18



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GIPR T2233.36 A 18

GCGR Q2323.37 A 13

GLP1R Q2343.37 A 15

GIPR Q2243.37 A 18

GCGR Y2333.38 A 13

GCGR N2383.43 A 13

GLP1R N2403.43 A 17

GLP1R N2403.43 A 2

GIPR N2303.43 A 18

GCGR Y2393.44 A 13

GIPR Y2313.44 A 18

GIPR Y2313.44 F 18

GCGR C2403.45 S 9

GLP1R E2473.50 A 17

GLP1R E2473.50 A 2

Helix IV GCGR F2784.56 A 13

GLP1R G2854.61 A 16

GLP1R I2864.62 A 16

GLP1R V2874.63 A 16

GLP1R K2884.64 A 16

GLP1R Y2894.65 A 16

GCGR C2874.65 S 9

ECL2 GLP1R L290 A 16

rSCTR F258 C 22

GLP1R Y291 A 16

rSCTR L259 C 22

GLP1R E292 A 16

rSCTR E260 C 22

GLP1R D293 A 16

rSCTR D261 C 22

GLP1R E294 A 16

rSCTR V262 C 22



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GLP1R G295 A 16

rSCTR G263 C 22

GCGR C294 S 9

GLP1R C296 A 16

GLP1R W297 A 16

rSCTR W265 C 22

GLP1R T298 A 16

rSCTR D266 C 22

GLP1R R299 A 16

GLP1R R299 K 7

rSCTR I267 C 22

GLP1R N300 A 16

rSCTR N268 C 22

Helix V GLP1R S3015.31 A 16

rSCTR A2695.32 C 22

GLP1R N3025.32 A 16

rSCTR N2705.32 C 22

GLP1R M3035.33 A 16

rSCTR A2715.33 C 22

GLP1R N3045.34 A 16

rSCTR S2725.34 C 22

GLP1R Y3055.35 A 16

rSCTR I2735.35 C 22

rSCTR W2745.36 C 22

GLP1R L3075.37 A 16

rSCTR W2755.37 C 22

rSCTR I2765.38 C 22

GIPR R3005.40 A 18

GCGR F3125.44 A 13

GLP1R N3205.50 A 2

GCGR F3195.51 A 13

GCGR F3205.52 A 13

ICL3 GLP1R V327 A 29



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GLP1R I328 A 29

GLP1R V331 A 29

Helix VI GLP1R T3536.42 A 2

rSCTR T3226.42 P 24

GCGR V3606.51 T 11

GLP1R H3636.52 A 15

GLP1R H3636.52 A 2

GIPR H3536.52 A 18

GLP1R E3646.53 A 15

GCGR V3646.55 I 11

GIPR F3576.56 A 18

rSCTR F3376.56 C 22

rSCTR A3386.57 C 22

rSCTR F3396.58 C 22

rSCTR S3406.59 C 22

GCGR T3696.60 M 11

rSCTR P3416.60 C 22

ECL3 rSCTR E342 C 22

rSCTR A343 C 22

rSCTR D344 C 22

rSCTR M345 C 22

rSCTR E346 C 22

GCGR Q374 R 11

GCGR S379 F 11

Helix VII GCGR A3807.37 I 11

rSCTR I3477.37 C 22

GCGR K3817.38 A 13

rSCTR Q3487.38 C 22

rSCTR L3497.39 C 22

GCGR F3837.40 A 13

rSCTR F3507.40 C 22



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GCGR F3847.41 A 13

GCGR F3847.41 T 11

rSCTR F3517.41 C 22

GCGR D3857.42 E 11

rSCTR E3527.42 C 22

GLP1R E3877.42 A 15

VPAC1 E3747.42 A 25

rSCTR L3537.43 C 22

VPAC1 L3757.43 A 25

GCGR F3877.44 A 13

GCGR S3907.47 A 13

VPAC1 S3797.47 A 25

GLP1R S3927.47 A 2

GLP1R Q3947.49 A 2

GIPR Q3847.49 A 18

GIPR Q3847.49 N 18

GLP1R M3977.52 L 30

GCGR C4017.58 S 9

GIPR Y3927.57 N 18

GLP1R Y4027.57 A 2

GLP1R N4067.61 A 2 a Superscripts refer to the Wootten residue numbering in which the single most conserved residue among class B GPCRs2 is designated X.50, where X is the transmembrane helix number. All other residues on that helix are numbered relative to this conserved position. See Supplementary Table 3 for comparison between class B2 and class A3 Ballesteros-Weinstein numbering based on structural alignment between GCGR and rhodopsin (Supplementary Fig. 6) b Different residue number assigned in different papers.



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Supplementary Figures

Supplementary Figure 1. Glucagon peptide and NNC0640 ligand. a, amino acid sequence of glucagon peptide b, GCGR antagonist NNC0640: 4-[1-(4-Cyclohexylphenyl)-3-(3-methanesulfonylphenyl)ureidomethyl]-N-(2H-tetrazol-5-yl)benzamide.

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Supplementary Figure 2. Snake plot representation of BRIL-GCGR(ECD/C) fusion construct. The two cysteines forming the conserved GPCR disulfide bond in the 7TM domain of GCGR are shown in yellow with a dashed magenta line illustrating the disulfide bond. The solid red line after residue 432 indicates the C-terminal truncation site.



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Supplementary Figure 3. Crystals and diffraction of BRIL-GCGR(ECD/C). a, Representative image of GCGR crystals in LCP. b, Representative diffraction of GCGR crystals.



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Supplementary Figure 4. Packing and structure of BRIL-GCGR(ECD/C). a, The unit cell is shown as a blue box. Two views of the crystal packing are shown with 90 rotation relative to each other. The zoomed view on the lower left is a space-filled cartoon depiction of the interaction between two antiparallel molecules of BRIL-GCGR(ECD/C), highlighting the insertion of helix VIII into the adjacent molecule. b, The conserved GPCR disulfide bond is depicted in yellow sticks. The extracellular (EC) and intracellular (IC) lipid bilayer boundaries are shown as brown and cyan ovals, respectively.



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Supplementary Figure 5. Comparison of the 7TM bundle of GCGR with class A GPCRs. a and b, Extracellular (EC) and intracellular (IC) views of the superimposed 7TM bundles of GCGR (blue) and class A GPCRs (grey; same PDBs as Fig. 1b). Distribution of distances between the tips of pairs of helices in a and b (black dashed lines) are plotted in the bar graphs (red and grey bars indicate distances for GCGR and class A GPCRs, respectively). The distance between the EC tips of GCGR helices II and VI is the largest among GPCR structures, and the distance between the EC tips of helices III and VII is among the largest, except for kappa (k-OR) and mu (m-OR) opioid receptors. At the intracellular (IC) side, the distances between the helical tips of GCGR are within the same range as those in available class A structures, except for an extensive inward shift of the IC tip of helix VII.



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Supplementary Figure 6. Structural alignment of GCGR and rhodopsin and comparison of Ballesteros-Weinstein numbering in class A and class B GPCRs. a, Structural superimposition of the human GCGR (blue) and bovine rhodopsin (PDB 1U19; gray), obtained with the iterative structure alignment algorithm in ICM-Pro (Molsoft LLC). Inserts show details



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of superimposition around the most conserved positions in each helix within class A GPCRs (black BW X.50 residues in Ballesteros-Weinstein numbering for class A)3. Corresponding residues of GCGR are shown and the blue number in superscript denotes their class B numbering according to Wootten et al, 2013 (ref. 2) b, Sequence alignment derived from the structural superimposition. Transmembrane helices in both structures are shown below the sequences, where red bars correspond to canonical -helices and blue bars to -helices. Both class B3 (X.50 in blue) and class A2 numbering (BW X.50 in black) positions in each helix are marked by arrows with the corresponding Ballesteros-Weinstein and Wootten number. Note that in helix IV, the Trp residue is most conserved in both GPCR classes, and therefore 4.50 positions coincide.

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Supplementary Figure 7. Location of the Ta6Br12 cluster used in the SAD phasing of BRIL-GCGR(ECD/C) fusion construct at 6 Å. BRIL-GCGR(ECD/C) is shown as ribbon with GCGR as light blue and BRIL as green. At 6 Å, the cluster is resolved as a single electron density blob, therefore the whole cluster was centered in the resolved density, and the orientations of its individual atoms are unknown. The cluster is located in a solvent cavity between BRIL and helix VIII of GCGR, and all the atoms in the cluster can be accommodated without clashes to BRIL-GCGR(ECD/C).



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Supplementary Figure 8. Initial experimental SIRAS electron density map at 4Å resolution contoured at 1.5 (0.08e/Å3) and overlaid with final GCGR model.

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Supplementary Figure 9. 2Fo-Fc (blue mesh) omit map of helix I in the GCGR structure contoured at 1.0, 0.10e/Å3.



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Supplementary Figure 10. Electron density of GCGR 7TM structural motifs from Figure 3. 2Fo-Fc (black mesh) map of residues in the structural motifs of GCGR as shown in Figure 3 contoured at 0.9, 0.09e/Å3.



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Supplemental References

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2 Wootten, D., Simms, J., Miller, L. J., Christopoulos, A. & Sexton, P. M. Polar transmembrane interactions drive formation of ligand-specific and signal pathway-biased family B G protein-coupled receptor conformations. Proceedings of the National Academy of Sciences of the United States of America 110, 5211-5216, doi:10.1073/pnas.1221585110 (2013).

3 Ballesteros, J. A. & Weinstein, H. in Methods in Neurosciences Vol. Volume 25 (ed C. Sealfon Stuart) 366-428 (Academic Press, 1995).

4 Jaakola, V. P. et al. The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist. Science 322, 1211-1217, doi:10.1126/science.1164772 (2008).

5 Lomize, M. A., Pogozheva, I. D., Joo, H., Mosberg, H. I. & Lomize, A. L. OPM database and PPM web server: resources for positioning of proteins in membranes. Nucleic acids research 40, D370-376, doi:10.1093/nar/gkr703 (2012).

6 White, J. F. et al. Structure of the agonist-bound neurotensin receptor. Nature 490, 508-513, doi:10.1038/nature11558 (2012).

7 Koth, C. M. et al. Molecular basis for negative regulation of the glucagon receptor. Proceedings of the National Academy of Sciences of the United States of America 109, 14393-14398, doi:10.1073/pnas.1206734109 (2012).

8 Perret, J. et al. Mutational analysis of the glucagon receptor: similarities with the vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide (PACAP)/secretin receptors for recognition of the ligand's third residue. The Biochemical journal 362, 389-394 (2002).

9 Prevost, M. et al. Mutational and cysteine scanning analysis of the glucagon receptor N-terminal domain. J Biol Chem 285, 30951-30958, doi:10.1074/jbc.M110.102814

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determine specificity for the glucagon amino terminus. J Biol Chem 278, 28005-28010, doi:10.1074/jbc.M301085200 (2003).

12 Unson, C. G. et al. Roles of specific extracellular domains of the glucagon receptor in ligand binding and signaling. Biochemistry 41, 11795-11803, doi:bi025711j [pii] (2002).

13 Cascieri, M. A. et al. Characterization of a novel, non-peptidyl antagonist of the human glucagon receptor. J Biol Chem 274, 8694-8697 (1999).

14 Underwood, C. R. et al. Crystal structure of glucagon-like peptide-1 in complex with the extracellular domain of the glucagon-like peptide-1 receptor. J Biol Chem 285, 723-730, doi:M109.033829 [pii]

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receptor involved in ligand binding and receptor activation: modelling the ligand-bound receptor. Molecular endocrinology 25, 1804-1818, doi:10.1210/me.2011-1160 (2011).

16 Koole, C. et al. Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) has a critical role in GLP-1 peptide binding and receptor activation. J Biol Chem 287, 3642-3658, doi:10.1074/jbc.M111.309328 (2012).



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17 Xiao, Q., Jeng, W. & Wheeler, M. B. Characterization of glucagon-like peptide-1 receptor-binding determinants. J Mol Endocrinol 25, 321-335, doi:JME00951 [pii] (2000).

18 Yaqub, T. et al. Identification of determinants of glucose-dependent insulinotropic polypeptide receptor that interact with N-terminal biologically active region of the natural ligand. Mol Pharmacol 77, 547-558, doi:10.1124/mol.109.060111

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confers constitutive activity. Biochemical and biophysical research communications 232, 96-100, doi:10.1006/bbrc.1997.6231 (1997).

20 Di Paolo, E. et al. Contribution of the second transmembrane helix of the secretin receptor to the positioning of secretin. FEBS letters 424, 207-210 (1998).

21 Di Paolo, E. et al. Mutations of aromatic residues in the first transmembrane helix impair signalling by the secretin receptor. Receptors & channels 6, 309-315 (1999).

22 Dong, M. et al. Mapping spatial approximations between the amino terminus of secretin and each of the extracellular loops of its receptor using cysteine trapping. FASEB J 26, 5092-5105, doi:10.1096/fj.12-212399

fj.12-212399 [pii] (2012). 23 Di Paolo, E. et al. Role of charged amino acids conserved in the vasoactive intestinal

polypeptide/secretin family of receptors on the secretin receptor functionality. Peptides 20, 1187-1193 (1999).

24 Ganguli, S. C. et al. Protean effects of a natural peptide agonist of the G protein-coupled secretin receptor demonstrated by receptor mutagenesis. The Journal of pharmacology and experimental therapeutics 286, 593-598 (1998).

25 Ceraudo, E. et al. Spatial proximity between the VPAC1 receptor and the amino terminus of agonist and antagonist peptides reveals distinct sites of interaction. FASEB J 26, 2060-2071, doi:10.1096/fj.11-196444

fj.11-196444 [pii] (2012). 26 Solano, R. M. et al. Two basic residues of the h-VPAC1 receptor second transmembrane helix are

essential for ligand binding and signal transduction. J Biol Chem 276, 1084-1088, doi:10.1074/jbc.M007696200 (2001).

27 Tan, Y. V., Couvineau, A. & Laburthe, M. Diffuse pharmacophoric domains of vasoactive intestinal peptide (VIP) and further insights into the interaction of VIP with the N-terminal ectodomain of human VPAC1 receptor by photoaffinity labeling with [Bpa6]-VIP. J Biol Chem 279, 38889-38894, doi:10.1074/jbc.M404460200 (2004).

28 Day, J. W. et al. Charge inversion at position 68 of the glucagon and glucagon-like peptide-1 receptors supports selectivity in hormone action. J Pept Sci 17, 218-225, doi:10.1002/psc.1317 (2011).

29 Mathi, S. K., Chan, Y., Li, X. & Wheeler, M. B. Scanning of the glucagon-like peptide-1 receptor localizes G protein-activating determinants primarily to the N terminus of the third intracellular loop. Molecular endocrinology 11, 424-432 (1997).

30 Dong, M., Pinon, D. I. & Miller, L. J. Site of action of a pentapeptide agonist at the glucagon-like peptide-1 receptor. Insight into a small molecule agonist-binding pocket. Bioorganic & medicinal chemistry letters 22, 638-641, doi:10.1016/j.bmcl.2011.10.065 (2012).

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