Supporting Information O’Connor et al. 10.1073/pnas.1510117112 Peptide Synthesis Hepes (pH 7.5), 300 mM NaCl, 40 mM (wt/vol) n-dodecyl-β-D- Synthesized peptides were produced through a solid-phase strategy maltopyranoside (DDM; Anatrace), 8 mM cholesteryl hemi- and purified by semipreparative HPLC on an Aquapore column succinate (CHS; Sigma), and 250 μM naltrexone for 3 h at 4 °C. (C8, 10 × 220 mm, 20 μm; Brownlee Labs). The purity of the final Solubilized membranes were isolated by ultracentrifugation at product was assessed by analytical reverse-phase liquid chroma- 180,000 × g for 45 min. Purification of His-tagged proteins was tography with a linear gradient from 5 to 80% over 56 min. Sol- initiated by incubation in 20 mM imidazole (pH 7.4) and 800 – vent A consisted of 0.1% TFA in H2O milli-Q and solvent B mM NaCl with 2 mL TALON IMAC resin (Clontech) for 6 18 h consisted of 0.1% TFA in acetonitrile. Molecular weights were (“overnight”). Unbound proteins were removed by centrifuga- confirmed by MALDI-TOF Voyager DE STR (Applied Bio- tion (700 × g) at 4 °C followed by batch-washing of beads in 25 systems). Fmoc-protected amino acids were purchased from column volumes (CV) of wash buffer I [1 mM DDM, 0.2 mM Novabiochem. The starting Fmoc-Lys(Boc)-HMP resin was syn- CHS, 10 mM Hepes (pH 7.4), and 150 mM KCl]. Unbound thesized by standard method in our laboratory. First couplings proteins were effectively removed after an additional 3 × 5CV were controlled by ninhydrin. wash buffer II [1 mM DDM, 0.2 mM CHS, 10 mM Hepes (pH 7.4), 20 mM imidazole, and 150 mM KCl]. The receptor was 15N-(GFLI)-Dynorphin (1–13) Peptide. The coupling was achieved with eluted in 5 CV of elution buffer [25 mM Hepes (pH 7.4), 150 mM the COMU-DIEA [(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)- KCl, 1 mM DDM, 0.2 mM CHS, and 200 mM imidazole] with 15N- dimethylamino-morpholinomethylene)] methanaminium hexa- dynorphin (1–13), or dynorphin, added to a final concentration of fluorophosphate– diisopropylethylamine] method. Fmoc15N-Gly- 25 μM to the elution fractions. Imidazole was removed and a OH, Fmoc15N-Phe-OH, Fmoc15N-Leu-OH, and Fmoc15N-Ile-OH deuterated MES buffer exchanged by gravity-flow size exclusion were synthesized by standard method in our laboratory. COMU using the PD-10 miniTrap G-25 column (GE Healthcare) with a was purchased from Iris Biotech GMBH. buffer containing 1 mM Mesd (pH 6.1), 150 mM KCl, 1 mM DDM, and 0.2 mM CHS. 15N-dynorpin was added to the desalted 15N-(GFLI)-15N,13C-R-Dynorphin (1–13) Peptide. The coupling was proteinfractiontoafinalconcentrationof25μM. The N-terminal achieved with the HOAT-DIC [1-hydroxy-7-azabenzotriazole–N, expression cassette was removed by treatment with His-tagged TeV N′-diisopropylcarbodiimide] method. Fmoc15N-Gly-OH, Fmoc protease (50 μL,5mg/mL)at4°Cfor1–3 h and incubation with 15N-Phe-OH, Fmoc15N-Leu-OH, and Fmoc15N-Ile-OH were syn- TALON IMAC resin for 6–18 h. Protein purity was judged as thesized by standard method in our laboratory. Fmoc-Arg(Pbf)- greater than 95% by SDS/PAGE (Fig. S1) and protein quality 13 15 OH (U- C6,U- N4)was purchased from AnaSpec. HOAT was judged as monodisperse by analytical size-exclusion chromatogra- purchased from PerseptiveBioSystems. phy. The purified KOR sample was concentrated to a final con- centration of ∼30 μMin100μLofH2O, 10% D2O, 40 mM Mesd Expression of KOR in Sf9 Cells (pH 6.1), 150 mM KCl, 100 μM DSS, 8 mM DDM, and 1.6 mM The wild-type (OPRK, Uniprot accession no. P41145) human kappa CHS. The receptor concentration was estimated by UV absor- opioid receptor gene was subcloned into a modified pFastBac1 bance at 280 nm using a theoretical molar extinction coefficient of − − vector with a truncated N terminus (ΔN42). An N-terminal ex- 48,400 M 1·cm 1. The KOR solution was then added to 15N-dy- pression cassette included hemagglutinin signal sequence followed norpin, 1 mM in the same buffer, to get the desired KOR:dynor- by FLAG epitope, 10x-His, and TeV protease recognition site. The phin molar ratio of 1:100 and appropriate line broadening effect. mutation I135L was introduced to increase expression and stability. The N-terminal sequence was identical to that used to solve the Radioactive Ligand Binding Experiments 2012 structure in complex with JDtic. Recombinant baculoviruses Saturation binding was performed with washed Sf9 membranes were generated with the Bac-to-Bac system (Invitrogen) and used using [3H]diprenorphine in the presence and absence of 10 μM to infect Sf9 insect cells at a density of 2 × 106 cells per mL at a JDTic. The binding assays were done in 96-well plates with a multiplicity of infection of 5 as previously described (10). Expres- final volume of 125 μL per well: 25 μL radioligand (0.16–20 nM), sion and trafficking was assessed by fluorescent detection of the 25 μL binding buffer (for total binding) or 25 μL JDTic (for FLAG epitope. Infected cells were grown at 27 °C for 48 h before nonspecific binding), and 75 μL washed Sf9 membranes ex- harvesting, with resulting cell pellet stored at −80 °C. pressing the KOR construct, IMPT1280. The binding buffer consists of 50 mM Tris·HCl, 10 mM MgCl2, and 0.1 mM EDTA, KOR Purification and Reconstitution in DDM/CHS Micelles pH 7.4 (or 5.0 or 6.0), at room temperature. Approximately KOR was purified for NMR in a manner similar to preparations 0.5 μg of total membrane protein was added to each well and the used for X-ray crystallography, briefly outlined here (10). Lysis was reaction incubated for 1 h in the dark at room temperature. The performed by a combination of thawing the frozen cell pellet, hy- reaction was stopped by vacuum filtration onto cold 0.3% pol- potonic shock, and gentle shearing forces accomplished via dounce yethyleneimine-soaked 96-well glass fiber filter mats using the homogenization in the presence of EDTA-free complete protease 96-well Filtermate harvester (Perkin-Elmer). The filter was then inhibitor mixture tablets (Roche), followed by ultracentrifugation at washed three times with cold standard wash buffer (50 mM 200,000 × g for 35 min. The resulting pellet containing the mem- Tris·HCl, pH 7.4, at 4 °C) and a wax scintillation mixture melted brane fraction was homogenized in the presence of 1 M NaCl on the filter and radioactivity counted in a MicroBeta2 counter followed by ultracentrifugation (twice) to complete membrane (Perkin-Elmer). Total binding and nonspecific binding results isolation. Preceding solubilization, the membrane fraction was re- were analyzed to determine the Kd and Ki values. Competition suspended and incubated in a solution containing 250 μMnal- binding assays were performed under similar conditions; how- − trexone, 2 mg·mL 1 iodoacetamide, 800 mM NaCl, and 50 mM ever, a constant dose of 1 nM [3H]diprenorphine was used with Hepes (pH 7.5) and incubated for 1 h at 4 °C. the competing ligand dynorphin (1–13) ranging from 0–10 μM. Solubilization of membranes was accomplished by a 1:2 di- The counts were pooled and fitted to a three-parameter logistic lution with a membrane solubilization buffer containing 50 mM function for competition binding to determine Ki. O’Connor et al. www.pnas.org/cgi/content/short/1510117112 1of7 Observation of Fast Exchange Rate mobile C-terminal residue is not affected by the peptide’s binding In the fast exchange limit, the observed chemical shift reflects to KOR. The NOE volumes have been integrated for all mixing τ the weighted average of bound and free states. In our experi- times m in both series of spectra. In case of interaction be- mental conditions, the bound fraction of dynorphin is ∼1%, as tween two groups of equivalent spins, the volumes have been rescaled by the factor 2nm=ðn + mÞ,wheren and m are the [dynorphin] = 1 mM and [KOR] = 10 μM. The relationship δav = numbers of spins in each group. The build-up curves for each xbδb + xfδf may be rewritten as δb − δf = (δav – δf)/xb, indicating that the ∼10 Hz of observed shifts are due to ∼1,000-Hz NOE have been fitted to the function given by the expression ðτ Þ = ð−ρ τ Þ · ð − ð− σ τ ÞÞ (∼1 ppm) shifts between the bound and free state. Because the I m a exp S m 1 exp 2 S m ,wherea is the signal ρ σ observed shifts are on the order of 103 Hz and proportional to intensity, S the relaxation rate in state S,and S the cross-re- = “ ” the bound fraction, we concluded that the exchange is fast on the laxation rate in state S (S averaged or free). Free refers to millisecond time scale. Strictly speaking, the infinitely fast ex- the relaxation rates observed in the presence of JDTic that change hypothesis may not apply here, and the system may be reflect the averaged relaxation between the free peptide and themultiplepossiblespeciesresulting from nonspecific in- somewhere between fast and intermediate exchange with respect σ to the millisecond time scale, which was sufficient for the ob- teractions. The averaged, observable cross-relaxation rate av servation and analysis of trNOEs. for dynorphin in the presence of KOR in DDM/CHS micelles is a weighted average of the rates in the free and specifically σ = σ + σ NMR Observation of Fast Off-Rate with a 200 nM Kd bound states: av pf f pb b,wherepf and pb are fractions of Applying the most simple model of bimolecular interaction gives the peptide in the free and bound states, respectively.