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. In our ex- periments the fractions of free and bound amounted to ∼99% and Kd = koff/kon. The experimental association rates for protein– 3 10 –1 –1 1%, respectively. Hence, σb could be calculated from the above protein pairs cover a wide range of kon, from ∼10 to 10 M ·s (64). Therefore, it is difficult to determine a priori that a certain equation. However, these values are still affected by an unknown amplification factor, resulting from experimental settings, such as Kd will be compatible with a fast off-rate and with the observa- tion of trNOEs. In our case, the NMR measurements revealed tuning quality, analog-to-digital converter resolution, and process- ing parameters. For this reason the obtained cross-relaxation rates that the interaction we observe was due to a fast exchange ligand α have been calibrated using the intraresidual HN-H NOEs, for binding condition being satisfied, suggesting that the on-rate which the distances are known (2.9 Å). The NOEs have been di- should be of the fast side. The theory of diffusion controlled on vided into groups (strong, medium, weak, and very weak) with rates of association is well described by Fersht (64). For two appropriate upper limits for distances (2.7, 3.3, 5, and 6 Å, re- molecules of the same radius in water at 25 °C, the encounter − − spectively). The lower distance limit was held at 1.8 Å in all cases. frequency is equal to 7 × 109 M 1·s 1. When one partner is larger Computations of the peptide structure involved NMR restraints- than the other one (e.g., a peptide–receptor interaction) it is + 2 driven MD simulations with simulated annealing. After the initial faster and scales with (rA rB) /rArB,. The actual rate can be structure minimization, the system was heated to 1,000 K within lower due to nonproductive binding and activation energy (due, 20 ps, followed by an MD run of 300 ps, divided into three stages. for instance, to a structural rearrangement of the receptor). It During the first 100 ps the temperature was maintained at 1,000 K. can be higher due to favorable electrostatic interaction energies, − − In the second stage the system was slowly cooled to 300 K, and which can bring the encounter frequency up to 1011 M 1·s 1. 10 −1· −1 during the last 100 ps the temperature was decreased to 100 K. In Thus, for a Kd of 200 nM and a kon of 10 M s , the koff is parallel with the temperature changes, the force constants were equal to 2,000 Hz, which is compatible with our observed fast also evolving. At the beginning they were reduced to 1% of their exchange rate on the millisecond time scale. On rates may be nominal values. During the first 20 ps they increased to 5%, during faster if the dominant binding mechanism is not a 3D random the following 20 ps they increased up to 20%, then during the next diffusion search, but rather a two-step binding mechanism. For 40 ps they were increased to 100% and kept at their maximal value instance, a first collision of dynorphin with the micelles followed to the end of the procedure. The structures were minimized again by a 2D diffusion on the surface to the receptor leads to an in- at the end of calculations. The quality of the final structures was “ ” creased kon by the so called reduction of dimensionality effect assessed from the violations of NOE distances and dihedral angles (65). Furthermore, we follow the interaction between positively and deposited in the Protein Data Bank (ID code 2N2F). charged (+5) dynorphin peptide and negatively (−6) charged extracellular surface of KOR (mostly within ECL2). This may R2 Analysis lead to a collision factor close to one, the peptide being driven For dynorphin, R2 is strongly enhanced by the receptor-bound toward to entry site by the electrostatic potential, as well as in- fraction due to the dominant contribution of J(0). Given R2KOR/JDTic creased collision rate. In a similar situation, despite a Kd of 62 nM, to represent the contribution to R2 relaxation of all of the non- trNOEs have been observed in systems where electrostatics sig- specific interactions and R2KOR the contribution to R2 of all states, 2 nificantly increased kon (47). For a similar reason, at low ionic one may write R2KOR − R2KOR/JDTic = xb·R2bound ∼ xb·(0.5 d + 1/6 strength, the barnase–barstar association rate was found to be >5 × c2)·4J(0). In this expression, J(0) dominates R relaxation at high 9 −1· −1 2 10 M s (64),andtheassociationrateofAD2toTAZ2has correlation time τc, such as observed in the KOR-bound state, d and 10 –1 –1 been estimated to be 1.7 × 10 M ·s (66). c are the dipolar and chemical shift anisotropy constants, re- spectively, and xb is the peptide-bound fraction, equal to 1% (59). Details of Structure Determination Protocols Furthermore, under the Lipari–Szabo formalism and assuming An extended structure of the peptide was created as the starting an internal motion characterized by the correlation time τi and point for a series of MD simulations coupled with the simulated 2 ð Þ = 2 · τ + ð − 2Þ · ½τ =ð + ω2τ2 ∼ 2 · τ amplitude S ,J 0 S c 1 S i 1 i S c (again annealing algorithm, in the presence of NMR distance restraints due to the large value of τc compared with τi). (Table 1). In total, 1,000 putative structures were generated, with Thus, the difference of R2 relaxation rate constants in the each MD run covering 320 ps. The best structures retained were absence and in the presence of JDTic, those with the lowest distance violations. To compare directly two 2 2 2 sets of spectra, acquired with and without JDTic, the NOE R2KOR − R2KOR=JDTic ∼ 0.5d + 1 6 c xb · 4 · τc · S , volumes in each series of spectra were divided by a scale factor, defined as the product of number of scans, receiver gain, and the is simply proportional to S2, and thus describes directly the re- volume of a reference peak. As reference, we chose the amide sidual amplitude of motion of each NH vector in the KOR- proton of K13 in dynorphin, because the signal of this highly bound state.

O’Connor et al. www.pnas.org/cgi/content/short/1510117112 2of7 Details of the Molecular Modeling Protocols pocket, to be flexible during docking. As for dynorphin, we have The initial structures used as starting points for MD simulations fixed the single bonds in the backbone of residues 5–8 to preserve were obtained from flexible docking. Each complex was placed at the helical turn in the core of the peptide and kept all of the other the center of a periodic box. To hydrate the system, the volume bonds rotatable, both in the backbone and in the side-chains. The occupied by the complex was extended by 10 Å on each side, and docking was performed on all six major KOR conformers, with 12,268 water molecules were added to the box. The final system 1,320 peptide poses generated for each conformer. The resulting × × had the dimensions of 73 100 72 Å and contained 41,727 structures showed significant diversity while having similar scoring atoms. The MD simulations were performed with the Amber 12 function values; therefore, we have filtered the results with respect software (67), using the FF03 force field for the protein and the to experimental evidence available: The aromatic residues (Y1, peptide and the TIP3P water model for the solvent. The equil- ibration of the entire system was achieved in several steps. Ini- F4) should be found within the pocket, whereas the C-terminal tially, the energy of the system was minimized by 1,000 cycles of part (K11) should face the outside of the site to be able to interact the steepest descent (SD) algorithm, with the solute held fixed, with the KOR’s extracellular loops. These restraints reduced the by constraining its Cartesian coordinates using a harmonic po- number of poses down to several dozen per conformer. In the end, − − tential with the force constant k equal to 100 kcal(mol) 1·(Å2) 1. we have selected some 10 poses per receptor conformer for vali- In the second step, the energy was minimized by 500 cycles of SD dation via MD simulations. and 1,500 cycles of the conjugate gradient algorithm, with weakly The local order parameters S2 for selected NH vectors in a −1 −2 restrained solute (k = 10 kcal·mol ·Å ). Next, a short 20-ps given trajectory have been calculated by measuring the degree of MD run was performed on weakly restrained solute with tem- dispersion of these vectors, quantified by the ratio of the volume perature varying linearly from 0 to 300 K. The integration step of the space they occupy to the volume of the sphere corre- used in this run was 1 fs. Throughout the calculations a cutoff of sponding to the isotropic reorientations. The volume of a cone 12 Å was used for electrostatic interactions. The MD simulation formed by fluctuating NH vectors is given by continued for 100 ps at constant temperature at 300 K with no restraints, with the integration step of 2 fs. Finally, a 50-ns run Zr Z2π Zθ with constant pressure of 1 bar was launched, with atomic co- ðθÞ = 2 θ θ ϕ = 2 π 3ð − θÞ ordinates saved every 10 ps. The Langevin dynamics was used to V r sin d d dr r 1 cos , −1 3 control the temperature, with γ = 1.0 ps , and the pressure was 0 0 0 controlled by the Berendsenbarostat with the pressure relaxation time τp = 2 ps. Bonds involving hydrogen were constrained with where r is the NH distance and θ is the cone semiangle. The the SHAKE algorithm. Calculations were performed using Ge- isotropic reorientations are described by θ = π, with Viso = (4/3)π Force GTX TITAN Black GPU cards, which worked at the r3. The mobility M of a fluctuating NH vector can be quantified · −1 speed of nearly 1 ns h . Trajectories obtained from MD simu- by its normalized volume V/Viso, given by (1 − cosθ)/2. Conse- lations were analyzed with the AmberTools 13 programs as well quently, the order parameter S2 can be defined as as with in-house software. Pairwise decomposition of the interaction energy, along with 1 + cos θ S2 = 1 − M = a detailed analysis of persistent intermolecular contacts, de- 2 termined the conserved contact residues between the two mol- ecules. The analysis of individual trajectories shows that the N and varies between 1 and 0 for angle θ varying between 0 and π. terminal of dynorphin and its helical core have low rmsd, whereas For each fluctuating NH vector, the temporal dispersion was the C terminal is flexible with a high rmsd value (Fig. S5), con- determined from the MD trajectory and the angle θ of the cor- sistent with the experimental findings. responding cone has been calculated as the width of the NH An initial MD simulation of KOR was performed in a periodic vector distribution. Then it was converted to the order parameter box with explicit solvent. The simulation covered 100 ns, of according to the above equation. For comparison with NMR which ∼25 ns were necessary to equilibrate the system. The frames derived order parameters, S2 was averaged over the last 20 ns in the remainder of the trajectory were used in the clustering of MD trajectories from the five best conformers (Table S1). A analysis. With the cluster radius set to 2 Å, six main-chain con- mixture of these conformations was required to reproduce cor- formers were found. We selected 16 residues inside the pocket of rectly the order parameter profile, particularly for the N-termi- the binding site whose side chains protrude to the interior of the nal residues G2 to L5.

O’Connor et al. www.pnas.org/cgi/content/short/1510117112 3of7 Fig. S1. Radioligand binding and protein purification. (A) Radioactive diprenorphine was displaced by dynorphin (1–13), giving Ki of 210 nM. Similar dis- placement profiles were observed at pH 6.1 and 7.4. Ligand binding was performed according to the Psychoactive Drug Screening Program radioligand protocol (see https://pdspdb.unc.edu/pdspWeb/) with a radioactive ligand concentration of 1 nM in binding buffer of 50 mM Tris·Cl, 0.1 mM EDTA, and 10 mM

MgCl2.(B) SDS/PAGE of the purification of His-tagged KOR (NuPAGE precast gels, Bis-Tris 4–12%, MES running buffer); lane 3: KOR after IMAC elution, lane 2: after PD-10 desalting, lane 1: after TEV cleavage and reverse IMAC.

ABR R C 1 15 1 2 H- N hetNOE 2 50 0.4

1.5 40 0 30 1 20 -0.4 0.5 10 0 0 -0.8 G2 G3 F4 L5 I8 L12 G2 G3 F4 L5 I8 L12 G2 G3 F4 L5 I8 L12

15 15 Fig. S2. N relaxation rates of dynorphin bound to KOR without (red) and with (blue) JDTic, measured at 800 MHz on N-(GFLI)–labeled dynorphin. (A)R1 −1 −1 15 1 relaxation rates (s , accuracy 1%). (B)R2 relaxation rates (s , accuracy 5%). (C) N- H NOE (accuracy 5%). Note that R1 and hetNOEs are hardly affected by the small bound fraction, whereas R2 is largely increased. These relaxation properties are congruent with simulations shown in Fig. S3.

R (s-1) R (s-1) 1 2

2.0 750

1.0 250

0.0 0 -9 -8 -7 -6 -11 -10 -9 -8 -7 -6 10 10 10 10 10 10 10 10 10 10 τ (s) τc (s) c NOE 1 τ (s) c 0 10-9 10-8 10-7 10-6 -1 R (s-1) 1 1.6 1 0.11 0.01 -2 R (s-1) 2111001000 2 -3 NOE 0.22 0.87 0.88 0.88 -4 10-11 10-10 10-9 10-8 10-7 τ c (s) Dyn DDM KOR

15 Fig. S3. N relaxation times at 600 MHz as a function of the rotational correlation times τc (x axis, logarithmic scale) related to the KOR-bound (KOR), micelle- −1 bound (DDM), and free dynorphin peptide (Dyn) states. R1 and R2 are given in seconds and derived from the equations described in ref. 59; hetNOE has no dimension. The experimental values of R2 determined in the presence of 1 mol % of KOR indicate that the rotational correlation time for the most immobilized − residues bound to KOR is above 10 7 s, much larger than that of the free or micelle-bound peptide.

O’Connor et al. www.pnas.org/cgi/content/short/1510117112 4of7 Fig. S4. (A) Overlay of two [15N, 1H]-HSQC-IPAP spectra, performed on 15N-GFLI–labeled peptide, 800 MHz, 280 K, pH 6.1: (red) KOR + dynorphin, (blue) KOR + dynorphin + JDTic. Asterisk indicates impurity. (B) Overlay of two [15N, 1H]-HSQC spectra performed on 15N-GFLI, 15N-13C-R–labeled peptide, 600 MHz, 280 K, pH 6.1: (red) KOR + dynorphin, (blue) KOR + dynorphin + JDTic. G2 cross peak is attenuated by NH exchange with water and shows up at lower contour level in all spectra.

Fig. S5. Decomposition of the dynorphin rmsd during a 50-ns molecular dynamic simulation. Contributions from the N-terminal (black), helical core (blue), and the C-terminal (red) segments are shown. Over the same trajectory, the receptor’srmsdis∼2 Å (i.e., similar to the helical core value, which is the least mobile part of the peptide over the trajectory). The C-terminus mobility is apparent after several nanoseconds, whereas the N terminus is more static over 15 50 ns; a longer time scale such as those involved in NR2 relaxation may be required to observe mobility. A much longer MD simulation (1 μs) of the KOR-2 conformation (Fig. 4) performed with NMR restraints in the central helix indicated that conformations KOR-1 to KOR-5 did not interchange within this mi- crosecond time scale.

O’Connor et al. www.pnas.org/cgi/content/short/1510117112 5of7 Fig. S6. Structural modeling of the dynorphin–KOR complex. KOR is represented in gray cartoon and dynorphin in orange sticks. Blue and red coloring on KOR show side chains with positive and negative charges, respectively. (A) Location of JDTic in the binding site. (B) KOR-1: a stable pose of dynorphin with the side chain of Tyr1 occupying a location close to the aromatic part of JDTic. (C) KOR-3: another pose with Tyr1 taking the place of JDTic. (D) KOR-2: a stable pose of dynorphin with the side chain of Tyr1 extended toward the sodium-binding site. The Protein Data Bank files corresponding to the 3D structures of the five KOR–dynorphin complexes are available in Datasets S1–S5.

Table S1. List of persistent contacts between dynorphin and KOR in the major conformations (KOR-1 to KOR-5) obtained from docking and MD simulations Dynorphin KOR-1 KOR-2 KOR-3 KOR-4 KOR-5

Interaction energy, −22.68 −21.11 −20.21 −14.96 −9.12 kcal/mol Y1 Y1(HA)-D138 (O) Y1(HA)-D138 (OD1) Y1(H1)-D138 (OD1) Y1(HH)-D105 (OD2) Y1(HH)-D105 (OD2) Y1(OH)-M142 (HB3) Y1(HB3)-N141(OD1) Y1(HB2)-N141 (OD1) Y1(HB3)-N141 (OD1) Y1(O)-N141 (HD21) Y1(HH)-V230 (O) Y1(HD1)-W287(HE3) Y1(OH)-F231 (HA) Y1(OH)-N322 (HD22) Y1(OH)-N322 (HD22) Y1(HB2)-Y320(OH) Y1(O)-I316 (HA) G2 G2(H)-D138 (O) G2(H)-D138 (OD2) G2(H)-F231(HE2) G2(O)-I316 (HA) G2(HA2)-Y139(HE1) G3 G3(H)-D138 (OD2) G3(H)-D138 (OD1) G3(HA2)-I290(HG12) F4 F4(HA)-Y312(OH) F4(HZ)-S211(HA) L5 R6 R6(HH21)-E209 (OE2) R6(HH11)-E203 (OE1) R6(O)-S211 (HB2) R6(HH12)-E209 (OE2) R6(O)-S211 (HB3) R7 R7(HH12)-D223 (OD2) R7(HH21)-D138 (OD2) R7(O)-S211 R7(HH22)-E297 (OE2) R7(HH11)-E297 (OE2) R7(HH22)-M226 (O) (HB2) R7(O)-L212 (H) I8 R9 R9(HH22)-E203 (OE1) R9(HH22)-E209 (OE1)

Characters in parentheses refer to the atoms for which contacts have been observed during at least 80% of the length of the MD trajectory. The numbering refers to the full length KOR sequence. The Protein Data Bank files corresponding to the 3D structures of these five KOR–dynorphin complexes are available in Datasets S1–S5.

O’Connor et al. www.pnas.org/cgi/content/short/1510117112 6of7 Table S2. Input for the structure calculations and validation of the bundle of 10 energy-minimized conformers used to represent the NMR structure of dynorphin (1–13) NOE distance constraints

Intraresidual 2 Sequential 42 Medium-range (2 ≤ ji − jj ≤ 4) 14 Dihedral angle constraints 4 Residual NOE violations No. >0.2 Å 7 ± 2 Maximum, Å 0.35 ± 0.04 Residual dihedral angle violations No. >2° 0 Maximum,° 0 Amber energies, kcal/mol Total −110,523 ± 233 Van der Waals 25,209 ± 85 Electrostatic −142,889 ± 260 Rmsd from ideal geometry Bond lengths, Å 0.0152 ± 0.0006 Bond angles, ° 2.08 ± 0.74 Rmsd to the mean coordinates, Å Backbone (1–4) 1.76 ± 0.53 Backbone (5–8) 0.46 ± 0.16 Backbone (9–13) 5.47 ± 1.92 Backbone (1–13) 3.57 ± 1.11 Heavy atoms (1–4) 2.66 ± 0.81 Heavy atoms (5–8) 2.14 ± 0.46 Heavy atoms (9–13) 6.26 ± 1.65 Heavy atoms (1–13) 4.38 ± 0.99 Ramachandran plot statistics, % Most favored regions 41.25 Additional allowed regions 51.25 Generously allowed regions 7.50 Disallowed regions 0 Protein Data Bank validation suite OK

Other Supporting Information Files pdb files of the KOR-1 (Dataset S1) to KOR-5 (Dataset S5) peptide-receptor complexes described in Table S1.

Dataset S1 (PDB) Dataset S2 (PDB) Dataset S3 (PDB) Dataset S4 (PDB) Dataset S5 (PDB)

O’Connor et al. www.pnas.org/cgi/content/short/1510117112 7of7