Supporting Information
Materials. The synthetic cannabinoid agonist, CP-55,940 ((-)-cis-3[2-hydroxy-4-(1,1dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol) was from Tocris Bioscience
(Ellisville, MO). 3H-labeled CP-55,940 and [35S]-GTPγS were from Perkin Elmer (Waltham,
MA). The lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2oleoyl-sn-glycero-3-phosphoserine (POPS) sodium salt used for CB2 receptor reconstitution
were purchased from Avanti Polar Lipids (Alabaster, AL). CHS (cholesteryl hemisuccinate)-Tris
salt was from Anatrace (Maumee, OH). The fluorophore-labeled lipids 1,1’-dioctadecyl3,3,3’,3’-tetramethylindodicarbocyanine 4-chlorobenzenesulfonate salt (‘DiD’ solid; DilC18(5)
solid) were from Invitrogen (Carlsbad, CA). The detergents CHAPS (3[(cholamidopropyl)dimethylammonio]-1-propanesulfonate) and DDM (n-dodecyl-β-D-maltoside)
were from Anatrace. LDAO (N,N-dimethyldodecylamine N-oxide) was from Sigma-Aldrich (St.
Louis, MO).
Composition of the mineral salt medium (MSM). The MSM used in this study,
slightly modified from conditions used earlier,1 contained: 4.65 g/L Na2SO4, 14.6 g/L K2HPO4,
4.07 g/L NaH2PO4 x 2H2O, 1.2 g/L MgSO4 x 7H2O, 3.32 mg/L CaCl2 x 2H2O, 0.72 mg/L ZnSO4
x 7H2O, 0.4 mg/L MnSO4 x H2O, 69.48 mg/L EDTA, 40.1 mg/L FeCl3, 0.236 mg/L CuSO4 x
5H2O, 0.84 mg/L CoCl2 x 6H2O, 100 mg/L thiamine hydrochloride and 100 mg/L ampicillin.
The concentration of NH4Cl and glucose at the beginning of fermentation was 2.73 g/L and 10
g/L, respectively, and was adjusted during the process as needed.
Fermentation in MSM. MBP-CB2 fusion protein was prepared by fermentation of E.
coli BL21-21(DE3) harboring plasmid pAY130.2 A typical experiment was performed as
follows: small aliquots of minimal-medium-adapted cells stored frozen in 20% (v/v) glycerol
were used to inoculate 25 mL of MSM in 125 mL shake flasks. The cells were grown at 37C to
OD600 = 1-1.5, and used to inoculate 500 mL of MSM in a 2 L shake flask, grown again in MSM
overnight to OD600 = 3.0-3.5, collected by centrifugation, re-suspended in a small volume of
sterilized tap water and injected into the fermentor to yield an OD600 = 1.0 at the start of
fermentation in a 3.0 L BioFlo® 110 Bench-Top Fermentor (New Brunswick Sci. Co., Edison,
NJ) in a volume of 1 L. The cell cultivation was performed with adjustment of glucose and
ammonium chloride concentrations at the beginning and during fermentation as needed. Samples
from the culture were taken every 30 minutes to monitor cell growth and glucose concentration.
The Assure® 3 Blood Glucose Monitoring System (Arkray, USA) was used to determine glucose
content. The pH was adjusted to 7.0 by the controlled addition of 10% NaOH and 10% H3PO4.
The concentration of dissolved oxygen with a set point of 40% of saturating concentration was
controlled by a cascade of stirring speed, and flow of air. Fermentation parameters were
controlled and recorded by the NBS BioCommand® Plus software (New Brunswick Sci. Co.).
When the desired OD600 was reached, the fermentation temperature was reduced from 37 to
20°C, 5 μM of stabilizing ligand CP-55,940 was added, and the CB2 protein production induced
with 1 mM IPTG.
For expression of 15N-, 13C- uniformly labeled CB2, 15NH4Cl (99% enrichment;
Cambridge Isotope Laboratories, Andover, MA) was used as the sole source of nitrogen and Dglucose (U-13C6, 99%; Cambridge Isotope Laboratories) as the sole source of carbon. The
concentration of glucose was monitored and adjusted as needed by injecting a solution of
glucose/ammonium chloride, 4:1 w/w, to maintain a concentration of glucose in the medium of
at least 3 g/L, and an adequate concentration of inorganic nitrogen. Induction of the CB2 protein
expression was initiated when an OD600 = 10 of the cell culture was reached. Cells were
harvested 10 hours after induction.
Purification of recombinant CB2 receptor. CB2-130 fusion protein,2 uniformly labeled
13
with C and 15N, was extracted from the biomass with a mixture of detergents: dodecylmaltoside
(DDM; 1%, w/v), CHAPS (0.5%, w/v) supplemented with cholesteryl hemisuccinate (CHS;
0.1%, w/v) and ligand CP-55,940 (10 μM). This combination of detergents was chosen as the
most efficient for solubilization of the recombinant receptor and for preservation of its activity
based on earlier work by R. Grisshammer on the recombinant neurotensin receptor 3 and our
study of stability of CB2 receptor in detergent micelles of various composition.4 For purification,
the content of DDM was reduced to 0.1%, w/v. Addition of the ligand is necessary to ensure the
stability of functional receptor throughout the purification procedure.4 Fusion protein CB2-130
was purified in the presence of 10 μM of CP-55,940 by affinity chromatography on Ni-NTA
Sepharose, the expression partner removed by treatment with TEV protease, and the resulting
CB2 receptor isolated by chromatography on a StrepTactin Macroprep column (EMD Chemicals:
San Diego, CA) following the procedure described earlier.2 Purified CB2 was concentrated in a
centrifugal spin concentrator (Orbital Biosciences, Topsfield, MA) with a 30 kDa molecular
mass cut off, and the protein concentration determined with a Bio-Rad DC kit. This final step of
protein sample preparation results in a co-concentration of some of the components of the elution
buffer. Typically, the 40 μM CB2 preparation contains DDM at a concentration of 0.4-0.6%
(w/v), CHAPS 2.0-3.0% (w/v), CHS 0.4-0.6% (w/v), and 40-80 μM CP-55,940.
Reconstitution of CB2 receptor into liposomes.
Reconstitution of the purified,
13
15
uniformly C-, N-labeled CB2 into liposomes was performed by the rapid dilution method as
described elsewhere.5 Briefly, 2.8 mg of the labeled protein (3.0 mg/mL) purified in the
CHAPS/DDM/CHS micelles were mixed with 28 mg of POPC/POPS lipids (4/1, mol/mol)
solubilized at 3.3 mg/mL in 0.5% (w/v) LDAO micelles for a protein-to-lipid molar ratio of
1:580. The protein and lipids were supplemented, respectively, with a trace of fluorophorelabeled CB2 receptor (60 μg of Alexa Fluor 488-labeled CB2) and fluorophore-labeled lipid (5.6
μg of DilC18(5)) for measurement of material recovery. The solution of mixed micelles in a total
volume of 8.5 mL was diluted dropwise into 650 mL of PBS buffer at 4°C under continuous
stirring; proteoliposomes are formed upon rapid dilution to below critical micelle concentrations
(c.m.c.) of the detergents. Detergent monomers formed upon dilution were removed on a
concentrating device (Amicon 8400: Millipore, Billerica, MA) using a polyethersulfone filter
with 30 kDa molecular mass cut off operated at 1 bar of argon-gas pressure and at 4°C.
Fluorescence measurements were performed on a Synergy HT Microplate reader (BioTek,
Winooski, Vermont). Alexa Fluor was excited at 488 nm and emission detected at 528 nm. The
DilC18(5) was excited at 590 nm and emission detected at 645 nm. Cross-talk between the
fluorophores was negligible at these settings. Material recoveries were 92% for the protein (2.6
mg) and nearly 100% for the lipid (33.3 mg including CHS from the protein micellar solution).
The composition of proteoliposomes was CB2/POPC/POPS/CHS, 1/505/120/150
(mol/mol/mol/mol).5 Functional activity of CB2 receptor in E. coli membranes before purification
and of purified receptor reconstituted in liposomes was assessed by a G protein-activation assay
using recombinant, purified Gαi1 and Gβ12 as described previously. 2,5,6
Molecular simulations. The computer simulations employed in this work were designed
to test the hypothesis that the endogenous cannabinoid, 2-arachidonylglycerol (2-AG) attains
access to the CB2 receptor via the lipid bilayer. To this end, we employed microsecond time
2
scale all-atom molecular dynamics (MD) simulations of the interaction of 2-AG with CB2 via a
POPC lipid bilayer. The simulations were initiated with a pre-equilibrated inactive state model of
the CB2 receptor embedded in a bilayer containing 123 POPC and 38 2-AG molecules (nineteen
2-AG in each leaflet). These 2-AG molecules were randomly distributed in bulk lipid such that
no 2-AG was in contact with the receptor. The bilayer system was solvated by 9965 water
molecules, 14 sodium ions, and 31 chloride ions, to yield an electrically neutral system with
roughly 100 mM salt. The CHARMM27 force field for proteins and lipids was used.7-9
Production dynamics was performed on the Blue Gene/W supercomputer 10,11 located at the T.J.
Watson Research Center, typically on 4096 dual-core nodes, using Blue Matter, a simulation
package developed at IBM specifically to take advantage of the Blue Gene architecture.11,12
Results of the multi-microsecond simulation suggested that (i) 2-AG first partitions out of bulk
lipid at the transmembrane helix (TMH)6/7 interface; (ii) 2-AG then enters the CB2 receptor
binding pocket by passing between TMH6/7; (iii) the entrance of the 2-AG head group into the
CB2 binding pocket is sufficient to trigger breaking of the intracellular (IC) TMH3/TMH6 ionic
lock and the movement of the TMH6 IC end away from TMH3, both hallmarks of GPCR
activation; (iv) subsequent to protonation at D3.49/D6.30, further 2-AG entry into the ligand
binding pocket results in both a W6.48 toggle switch conformational change and a large influx of
water. To our knowledge, this was the first demonstration via unbiased molecular dynamics
(MD) that a ligand can access the binding pocket of a Class A GPCR via the lipid bilayer and the
first demonstration via MD of GPCR activation triggered by a ligand binding event. Further
details about the simulation and its results can be found in our published paper.13
3
Figure S1. 13C-MAS NMR spectra as a function of MAS frequency for proteoliposomes
containing uniformly 13C-, and 15N-labeled CB2 receptor. Bars below each spectrum indicate
locations of spinning sidebands centered about the side-chain aliphatic (10-45 ppm), side-chain
aromatic (110-140 ppm), and backbone C=O (175-180 ppm) resonance-bands. At a MAS
frequency of 15 kHz, the C and the C=O bands are mostly clear from superposition of spinning
sidebands which enabled a comparison of the distribution of measured- and predicted resonances
in those bands.
4
Figure S2. The 13C MAS NMR spectrum of uniformly 13C-, and 15N-labeled CB2 receptor in
liposomes measured at the different temperatures.
5
CB2-130
(SHIFTX)
SPARTA
SHIFTX
200
190
180
170
160
80
70
60
50
40
30
20
10
ppm
ppm
Figure S3. Predicted 13C NMR spectra of the active state of CB2 for Cand C (right), and
C=O bands (left) by application of the chemical-shift prediction programs SHIFTX and
SPARTA. A predicted spectrum for the recombinant CB2-130 with tags is shown as well.
6
Recombinant CB2-130
CB2-130 receptor used for the uniform 13C-,15N-labeling and MAS NMR measurements.
Amino-acid residues in the wild-type receptor are shown with gray background. The recombinant CB2
receptor that was cleaved from the CB2-130 fusion protein contains additional amino acids in N-, and Cterminal ends (white background). Those residues did not alter the receptor function as measured by
ligand-binding and G protein activation.
Figure S4.
7
Figure S5. The C region of the 13C NMR spectra simulated for several GPCR as well as the
Kv1.2 potassium channel and the -barrel protein OmpT along with the experimentally measured
spectrum of CB2 receptor. Spectra were predicted by the program SHIFTX using the following
crystal structures: PDB3PXO, bovine rhodopsin in the MII state; PDB3AYN, squid 9-cis
isorhodopsin; PDB2Y01, beta1-adrenergic receptor with bound dobutamine; PDB3LUT, Kv 1.2
potassium channel ;PDB1I78; OmpT.
8
N-terminal
13C

ECL2
ECL1
ECL3
VII
I
II
III
IV
V
VI
VIII
ICL1
ICL3
ICL2
Chemical-shift changes
/ ppm
C-terminal
6.0
4.5
3.0
1.5
0.0
-1.5
-3.0
-4.5
-6.0
0
50
100
150
200
Residue #
250
300
350
Figure S6. Predicted 13Cβ chemical-shift changes () upon full activation of CB2 receptor 13
(light green, helices; green, terminals and loops). Amino-acid residues with changes  greater
than ±1.5 ppm are marked in the snake plot: 2.0 ppm ≥| | ≥1.5 ppm, light blue; | |≥2.0 ppm,
dark blue.
9
N-terminal
13C=O
ECL2
ECL1
ECL3
VII
I
II
IV
III
V
VI
VIII
ICL1
ICL3
ICL2
Chemical-shift changes
/ ppm
C-terminal
6.0
4.5
3.0
1.5
0.0
-1.5
-3.0
-4.5
-6.0
0
50
100
150
200
Residue #
250
300
350
Figure S7. Predicted 13Cchemical-shift changes () upon full activation of CB2 receptor 13
(light green, helices; green, terminals and loops). Amino-acid residues with changes  greater
than ±1.5 ppm are marked in the snake plot: 2.0 ppm ≥ | | ≥ 1.5 ppm, light blue; | | ≥ 2.0
ppm, dark blue.
10
N-terminal
15NH
ECL2
ECL1
ECL3
VII
I
II
IV
III
V
VI
VIII
ICL1
ICL3
ICL2
Chemical-shift changes
/ ppm
C-terminal
15.0
10.0
5.0
0.0
-5.0
-10.0
-15.0
0
50
100
150
200
Residue #
250
300
350
Figure S8. Predicted backbone 15N-chemical-shift changes () upon full activation of CB2
receptor 13(light green, helices; green, terminals and loops). Amino-acid residues with changes
 greater than ±5.0 ppm are marked in the snake plot: 6.0 ppm ≥| |≥5.0 ppm, light blue; |
|≥6.0 ppm, dark blue.
11
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