UPPEER ORO doi:10.1038/nature10753 D D D D P E ntracellular C1 W P P C EC1 D Q R H C D W D R C C2 D E D E C R Q Q W P W W R P P EC2 EC3 P C C P W P W W P C W H R C R E C3 P R R P P P C Extracellular embrane H R C H Bound [ 3 H]QB (p) 160 140 120 100 80 60 40 20 0 0 0.5 1 1.5 2 2.5 3 3.5 4 ree [ 3 H]QB (n) 4 lysozyme Bound [ 3 H]QB (%) 0-12 -10-8 -6-4 -2 0 log [igand] () upplementary igure 1. chematic diagram of the 2-4 and characterization of ligand binding. a, our -linked glycosylation sites in the - terminus were eliminated by converting asparagine residues (sn2, 3, 6 and 9) to aspartic acid (D). he cysteine-less 4 (green) was inserted in the third intracellular loop. b and c, igand binding studies on the 2 (black symbols) and 2-4 (red symbols) receptors. b, aturation binding was assayed by incubating solubilized f9 cellular membranes with 0.1-4 n [ 3 H]QB with or without 1 µ atropine. he result of a representative experiment performed three times is shown. he specific activity of [ 3 H]QB was 82 dpm/fmol, the reaction volume was 0.2 ml, and the amount of total membrane protein used was 2.64 µg ( 2 ) and 4.72 µg ( 2-4). Data were fit by curves derived from the assumption of a simple mass action giving d values of 0.31 n ( 2 ) and 0.35 n ( 2-4) 120 100 80 60 40 20 WWW.URE.CO/URE 1
REERCH UPPEER ORO in the experiment shown here. aximal binding capacity was estimated to be 155 p ( 2 ) and 168 p ( 2-4), which correspond to 2540 and 2760 dpm/assay tube, 31.0 and 33.6 fmol/0.2 ml, and 11.7 and 7.1 pmol/mg of membrane protein, respectively. c, Displacement of [ 3 H]QB binding by carbamylcholine (circle) or atropine (square). i values for carbamylcholine or atropine were estimated from their concentrations (C 50 ) giving a half maximal inhibition of [ 3 H]QB binding according to the equation i = C 50 /(1 + [[ 3 H]QB]/d*), where d* is the dissociation constant for [ 3 H]QB binding. he concentration of [ 3 H]QB was 2 n. i values for carbamylcholine and atropine were estimated to be 0.14 m and 2.7 n for 2 and 0.17 m and 3.0 n for 2-4, respectively, in the experiment shown here. 2 WWW.URE.CO/URE
UPPEER ORO REERCH upplementary igure 2. attice packing in the 2-4 crystals. he 2 receptor is shown in blue and 4 lysozyme is shown in red. W W W. U R E. C O / U R E 3
REERCH UPPEER ORO upplementary igure 3. Comparison of the 2 receptor structure with other monoamine PCRs. he 2 receptor is shown in blue with the ligand QB in yellow sticks (red for oxygen atoms, blue for nitrogens). Other receptors are shown in grey, with ligands in grey sticks. 4 W W W. U R E. C O / U R E
UPPEER ORO REERCH upplementary igure 4. queous channels in structures of protein coupled receptors. Each receptor is colored differently for clarity. he aqueous region passing though much of the 2 receptor is lined by allosteric site and orthosteric site residues, and is additionally bounded below by 65, 68 D69, 72, 106, 110, 114, 392, 396, W400, 430, 432, 433, 436. WWW.URE.CO/URE 5
REERCH UPPEER ORO upplementary igure 5 Electron density in the ligand binding pocket. n o - c ligand omit map is contoured at 3 σ in grey in panels a and b, while panel c also shows a 2 o - c map in green for binding pocket residues contoured at 1.5 σ. 6 WWW.URE.CO/URE
UPPEER ORO REERCH 250 kda 150 kda 100 kda 75 kda 50 kda 37 kda upplementary igure 6 D-PE and EC analysis of 2-4. he receptor appears to aggregate in the presence of D, accounting for higher molecular weight bands on a gel (left). ize exclusion analysis (right) is a non-denaturing technique, and receptor appears monodisperse by this assay. WWW.URE.CO/URE 7
REERCH UPPEER ORO upplementary igure 7 Crystals of 2-4 shown under partially crossed polarizing filters. Crystals were typically 20 microns in the longest dimension, as shown here, although in some cases larger crystals were seen. 8 WWW.URE.CO/URE
UPPEER ORO REERCH upplementary able 1. he residues in the QB binding sites of 2 R and these equivalents in other aminergenic receptors (1). 2 (2) 1, 3-5 H 1, 2 α 1-D α 2-C β 1-3 D1,5 D2-4 Helix D103 3.32 D D D D D D D 104 3.33 / 107 3.36 /C C C C 108 3.37 111 3.40 Helix W155 4.57 W / / EC2 181 EC2 /Q / // / Helix 187 5.39 / / / / 190 5.42 /D 195 5.47 Helix W400 6.48 W W W W W W W 403 6.51 404 6.52 Helix 426 7.39 / C429 7.42 C 430 7.43 W 1) bbreviations of the receptors are as follows. 2 : muscarinic 2 acetylcholine receptor, 1, 3-5 : muscarinic 1, 3-5 acetylcholine receptors, H 1, 2 : histamine H 1, 2 receptors, α 1-D : α 1-D -adrenergic receptors, α 2-C : α 2-C -adrenergic receptors, β 1-3 : β 1-3 -adrenergic receptors, D1,5: dopamine D1, 5 receptors, D2-4: dopamine D2-4 receptors. 2) uperscripts indicate Ballesteros Weinstein numbering for conserved PCR residues 1. WWW.URE.CO/URE 9
REERCH UPPEER ORO upplementary able 2. Data collection and refinement statistics. Data collection a umber of crystals 23 pace group P 2 1 Cell dimensions a, b, c (Å) 78.2, 47.3, 88.1 α, β, γ ( ) 90.0, 109.7, 90.0 Resolution (Å) 27.5 3.0 (3.05 3.00) R merge (%) 18.5 (45.4) <>/<σ> 6.1 (1.4) Completeness (%) 94.1 (79.2) Redundancy 3.5 (2.4) Refinement Resolution (Å) 27.5 3.0 o. unique reflections 11702 (593 in test set) R work /R free (%) 22.7 / 27.6 nisotropic B tensor B 11 = 18.2 / B 22 = 2.6 / B 33 = -20.8 / B 13 = 9.8 verage B-factors (Å 2 ) 2 muscarinic receptor 53.3 (R)-( )-3-QB 46.0 4 lysozyme 57.8 olvent 42.8 R.m.s. deviation from ideality Bond length (Å) 0.002 Bond angles ( ) 0.57 Ramachandran statistics b avored regions (%) 98.8 llowed regions (%) 1.2 Outliers (%) 0 a Highest shell statistics are in parentheses. b s defined by olprobity 2. 10 WWW.URE.CO/URE
UPPEER ORO REERCH upplementary able 3. elect contacts between QB and 2 receptor QB 2 receptor Distance (Å) a mine sp103 carboxylate 3.3 Carbonyl sn404 H 2 3.2 Hydroxyl sn404 carbonyl 2.7 Quinuclidine cage yr426 phenyl 3.9 Quinuclidine cage yr403 phenyl 3.9 Quinuclidine cage yr430 phenyl 3.8 Quinuclidine cage yr104 phenyl 3.6 Quinuclidine cage rp400 indole 4.0 Pro-R phenyl hr187 Cγ 3.9 Pro-R phenyl Phe181 phenyl 3.7 Pro- phenyl yr104 phenyl 4.3 Pro- phenyl la194 Cβ 3.9 Pro- phenyl er107 Cβ 3.9 Pro- phenyl rp155 indole 3.7 a Distances reported are the closest contact between any side chain atom and any ligand atom in the indicated functional groups. b n this structure, the pro-r phenyl ring is positioned closer to the extracellular surface, while the pro- phenyl points toward the cytoplasmic side. iterature Cited 1. Ballesteros, J.. & Weinstein, H. ntegrated methods for the construction of three-dimensional models and computational probing of structurefunction relations in protein coupled receptors. eth. eurosci. 25, 366-428 (1995). 2. Chen,. B. et al. olprobity: all-atom structure validation for macromolecular crystallography. cta Crystallogr. D Biol. Crystallogr. 66, 12-21, (2010). WWW.URE.CO/URE 11