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1 Supporting information Fluorescent derivatives of AC-42 to probe bitopic orthosteric/allosteric binding mechanisms on muscarinic M1 receptors Sandrine B. Daval, Céline Valant, Dominique Bonnet, Esther Kellenberger, Marcel Hibert, Jean-Luc Galzi and Brigitte Ilien * Table of contents : Supplemental Figure S1 : Normalized absorption and emission spectra of EGFP and para-lrb-ac42 in Hepes buffer. Supplemental Figure S2 : Structural variation of the M1 receptor. Supplemental Figure S3 : Sequence alignment for M1 homology modelling. Supplemental Procedures : Solubility measurements Estimation of donor-acceptor distances through FRET Biological data analyses S1

2 Supplemental Figure S1 Normalized absorption and emission spectra of EGFP and para-lrb-ac42 in Hepes buffer. Absorption (open symbols; dashed line) and fluorescence (closed symbols; solid line) spectra for EGFP (triangles) and para-lrb-ac42 (circles) are shown together with the overlap (grey area) for normalized EGFP emission and para-lrb-ac42 absorption. Arrows at 470 nm and 510 nm point the excitation and emission wavelengths, respectively, which were selected for FRET studies. S2

3 Supplemental Figure S2 Structural variation of the M1 receptor. Left and right panels show views from the extracellular side or in the plane of the plasma membrane, respectively. The 7TMs are represented by cylinders. The side chains of key residues discussed in the manuscript are displayed as capped sticks. The M1 receptor structure modeled by homology to the D3 receptor is colored in white, the structure obtained after a short molecular dynamic simulation is colored in green. The molecular dynamics was carried out using AMBER9 (University of California, San Francisco, CA, USA) as follows. The receptor was embedded in a pre-equilibrated lipid bilayer consisting of 82 molecules of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and solvated with TIP3P water molecules (box dimensions: 88.2 Å x 93.1 Å x 83.8 Å). Energy minimization was first applied to solute molecules (by setting a positional harmonic constraint of 100 kcal/mol. Å on the receptor), then to the entire system. For the MD, the system was heated up with a linear increase of temperature from 100 to 300 K over 20 ps (periodic boundary with constant volume and no pressure control). A second equilibration stage of 100 ps at 300 K was performed using pressure and temperature control. The system was then subjected to a 1.8 ns simulation at 300 K. During all simulations, all bonds involving hydrogen atoms were frozen with the SHAKE algorithm. Interactions were calculated according to the AMBER 03 force field, using particle-mesh-ewald summation to include the long range electrostatic forces. The Van der Waals interactions were calculated using a cut-off of 8.0 Å. S3

4 Supplemental Figure S3 Sequence alignment for M1 homology modeling. Amino acids are coloured according to their physico-chemical properties. Sequence variations introduced in the recombinant proteins used for structure determination are written in lowercase (black letters). The cysteine residues involved in the conserved disulfide bridge (between the third TM and the second extracellular loop) are highlighted using a grey background. For the sake of clarity, long stretches of non conserved sequences were omitted. They are given below the alignment TM SWISSPROT ACM1_HUMAN...MNTSAPPAVSPNITVLAPGKGPWQVAFIGITTGLLSLATVTGN PDB 3PBL, chain A...dykddddgapASLSQLSSHLNYTCGAENSTGASQARPHAYYALSYCALILAIVFGN TM SWISSPROT ACM1_HUMAN LLVLISFKVNTELKTVNNYFLLSLACADLIIGTFSMNLYTTYLLMG.HWALGTLACDLWLA PDB 3PBL, chain A GLVCMAVLKERALQTTTNYLVVSLAVADLLVATLVMPWVVYLEVTGGVWNFSRICCDVFVT -TM TM SWISSPROT ACM1_HUMAN LDYVASNASVMNLLLISFDRYFSVTRPLSYR...AKRTPRRAALMIGLAWLVSFVLWAPAI PDB 3PBL, chain A LDVMMCTASIwNLCAISIDRYTAVVMPVHYQHGTGQSSCRRVALMITAVWVLAFAVSCPL. PDB 2RH1, chain A...QM TM SWISSPROT ACM1_HUMAN LFWQ...YLVGERTVLAGQCYIQFL...SQPIITFGTAMAAFYLPVTVMCTLYWRI PDB 3PBL, chain A LFGF...NTTGDPTVCSI...SNPDFVIYSSVVSFYLPFGVTVLVYARI PDB 2RH1, chain A HWYRATHQEAINCYAeETC... PDB 3EML...CLFEDV...VPMN SWISSPROT ACM1_HUMAN YRETENRARELAALQGSETPGKGGGSS...icl3 *...LVKEKKAAR PDB 3PBL, chain A YVVLKQRRRK...icl3 insert**...plrekkatq TM TM SWISSPROT ACM1_HUMAN TLSAILLAFILTWTPYNIMVLVSTFCKDC..VPETLWELGYWLCYVNSTINPMCYALCNK PDB 3PBL, chain A MVAIVLGAFIVCWLPFFLTHVLNTHCQTC.HVSPELYSATTWLGYVNSALNPVIYTTFNI SWISSPROT ACM1_HUMAN AFRDTFRLLLLCRWDKRRWRKIPKRPGSVHRTPSRQC... PDB 3PBL, chain A EFRKAFLKILSCgrplevlfq *SWISSPROT ACM1_HUMAN, icl3 SSSERSQPGAEGSPETPPGRCCRCCRAPRLLQAYSWKEEEEEDEGSMESLTSSEGEEPGSEVVIKMPMVDPEAQAPTKQPPRS SPNTVKRPTKKGRDRAGKGQKPRGKEQLAKRKTFS **PDB 3PBL, chain A, icl3 insert NIFEMLRIDEGLRLKIYKDTEGYYTIGIGHLLTKSPSLNAAKSELDKAIGRNTNGVITKDEAEKLFNQDVDAAVRGILRNAKL KPVYDSLDAVRRAALINMVFQMGETGVAGFTNSLRMLQQKRWDEAAVNLAKSRWYNQTPNRAKRVITTFRTGTWDAYGV S4

5 Supplemental Procedures Solubility measurements. Aliquots (1.5 µl) of DMSO stock solutions of AC-42, para-lrb-nbutyl, para-lrb-ac42 and ortho-lrb-ac42 were submitted to a 100 fold dilution either in Hepes buffer (10 mm Hepes, mm NaCl, 1.25 mm MgCl 2, 1.25 mm CaCl 2, 6 mm KCl and 10 mm glucose, 1 mg/ml bovine serum albumin; ph 7.4) or in CH 3 CN:H 2 O (1:1; v/v) to reach final concentrations ranging from 10 to 100µM. After mixing, samples were allowed to stand in the dark for 2 h at room temperature, in plastic test tubes used for binding assays. Following centrifugation at 15,000g for 10 min, supernatants were collected, diluted with an equivalent volume of CH 3 CN:H 2 O (1:1; v/v) in order to precipitate BSA contained in Hepes samples, and centrifuged again. Final clear supernatants were obtained and 20 µl of each of them were injected onto a Luna C18(2) column (4.6 x 50 mm; 5 µ; Phenomenex) for analytical RP-HPLC analysis. Elution was performed using a linear gradient (100% A for 0.2 min then 0 to 100% B in 2.5 min, then 100% B for 0.5 min, flow rate 2 ml/min) of solvent B (CH 3 CN 100%, 0.05% TFA, v/v) in solvent A (H 2 O 100%, 0.05% TFA, v/v). Detection was set at 280 and 565 nm. All compounds eluted as single peaks at retention times (given in min) that did not differ between assay (Hepes) or reference (CH 3 CN:H 2 O) series : AC-42 (2.03), para-lrb-nbutyl (2.00), para-lrb-ac42 (2.16) and ortho-lrb-ac42 (2.08). Peak surface areas of reference samples were calculated and plotted as a function of compound concentration to generate calibration curves. The strict linear dependency of peak surface areas with compound concentration (which was verified in all cases) allowed the absolute concentrations of assay samples to be quantified from their proper peak surface areas. Concentrations (mean values for two separate determinations) of compounds remaining in solution (following dilution in Hepes buffer), as opposed to theoretical concentrations (reference samples; 10, 30, 100 µm) were as follows : AC-42 (9.8, n.d., 98.1), para-lrb-nbutyl (2.5, 2.4, 2.2), para-lrb-ac42 (9.8, 29.5, 44.9) and ortho-lrb-ac42 (2.1, 2.7, 1.8). Estimation of donor-acceptor distances. The distance R 0 (Förster radius) for 50% transfer efficiency E, typical for each donor (EGFP)-acceptor (ligand fluorophore) pair, was calculated from: R 0 = 9790 (κ 2 n -4 Φ D J) 1/6. 63 Values for κ 2, a geometric factor accounting for the relative orientation in space of the donor emission and acceptor absorption transition dipoles, for N, the refractive index of the medium and for Φ D, the quantum yield of the EGFP donor together with calculation of J, the spectral overlap integral for the combined emission of EGFP and absorbance of fluorophore species, were as reported. 38 The distance R (Å) between EGFP and the fluorophore moiety of bound ligands was calculated using Förster s equation: R = R 0 (1/E 1) 1/6. 49 The efficiency of fluorescence energy transfer (E) was defined as the fractional decrease in EGFP fluorescence due to ligand binding and was expressed by: E = 1 F DA /F D, where F DA and F D are specific donor fluorescence emission in the presence or the absence of saturating concentrations of ligand, respectively. Specific EGFP fluorescence in the absence (F D ) or the presence (F DA ) of ligand was determined by subtracting autofluorescence of non transfected HEK cells (in the absence or presence of ligand) from total fluorescence of EGFP(Δ17)hM1 expressing cells. Biological data analyses. Nonlinear regression analyses of functional or binding data were performed using Kaleidagraph 4.0 (Synergy software, Reading, PA). Functional assays : Dose-effect curves were generated by plotting the normalized response amplitude, E, as a function of agonist or inhibitor concentration, X. Data were analysed using the equation : E = E max /(1 + (X 50 / X) nh ), to derive the maximal response E max, the drug concentration leading to halfmaximal response (EC 50 ) or inhibition (IC 50 ), and nh the slope factor. Competition-type experiments : Fractional receptor occupancy B/B 0 was plotted against the concentration X of unlabelled agent, on a logarithmic scale, and analysed using the general Hill equation : B/ B 0 = Bottom + [(1-Bottom) /(1 + (IC 50 / X) nh )], to estimate the bottom plateau, the IC 50 value and the slope factor nh. With bottom and slope values not significantly different from 0 and 1, respectively, interactions were assumed to be competitive and IC 50 values were converted into K i values by applying the Cheng and Prusoff correction. 64 S5

6 Competition curves with submaximal inhibition at high displacer concentration were analysed using the equation : B/B 0 = (L + K d ) / [L + K d.((1+(x / K X ) / (1+(X / α.k X )))], based on the allosteric ternary complex model of ligand interaction. 8 L and X are the concentrations of tracer and allosteric agent, respectively. K d and K X are equilibrium dissociation constants for the binding of the tracer and the allosteric drug at the free receptor, respectively. α denotes the cooperativity factor by which the two drugs mutually increase (α >1; negative cooperativity) or decrease (α <1; positive cooperativity) their respective equilibrium dissociation constants. Saturation experiments : Occupancy curves were generated by plotting specific tracer binding (B) at equilibrium, as a function of X, the radioactive or fluorescent tracer concentration. Data fitting to the empirical Hill equation derived for saturation : B = (B max ) /(1 + (K d / X) nh ), yields estimates of B max (density of receptor sites or maximal FRET amplitude), of K d (apparent equilibrium dissociation constant) and of nh (the slope factor). [ 3 H]-NMS dissociation kinetics : Off-rate constants (k off values) were determined from kinetic data fitting to a monoexponential time course reaction : B/B 0 = exp (-k off. t), where B 0 and B denote [ 3 H]- NMS binding at equilibrium (t = 0) and after t min of dissociation, respectively. Concentration-effect curves for the allosteric retardation of radioligand dissociation rate were generated by plotting k off obs / k off control ratios (i.e. dissociation rate constants measured in the presence and the absence of an allosteric agent) as a function of X, the alloster concentration. Fitting to the equation : k off obs / k off control = Bottom + [(1-Bottom) /(1 + (IC 50diss / X) nh )], allows the determination of the maximal amplitude E max diss (or (1-Bottom) value) of the retardation effect, of the alloster concentration IC 50diss leading to half-maximal reduction of [ 3 H]-NMS dissociation rate and of the slope factor nh. The IC 50diss parameter denotes the affinity (α.k X ) of the alloster for tracer-occupied receptors. 40 Additional References (63) Lakowicz, J. R. Principles of fluorescence spectroscopy; Kluwer Academic/Plenum Publishers: New York, (64) Cheng, Y. C.; Prusoff, W. H. Relationship between the inhibition constant (K i ) and the concentration of inhibitor which causes 50% inhibition (IC 50 ) of an enzymatic reaction. Biochem. Pharmacol. 1973, 22, S6