Microswitches for the Activation of the Nociceptin Receptor Induced by

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Computational Biochemistry

Microswitches for the Activation of the Nociceptin Receptor Induced by Cebranopadol: Hints from Microsecond Molecular Dynamics Stefano Della Longa, and Alessandro Arcovito J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.8b00759 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 16, 2019

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Journal of Chemical Information and Modeling

Microswitches for the Activation of the Nociceptin Receptor Induced by Cebranopadol: Hints from Microsecond Molecular Dynamics

Stefano della Longa1,* and Alessandro Arcovito2,3

1Department

of Life, Health and Environmental Sciences, University of L’Aquila, L’Aquila, 67100

Italy; 2Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Rome, 00168 Italy; 3Fondazione Policlinico Universitario Agostino Gemelli - IRCCS, Rome, 00168 Italy

KEYWORDS: NOP, NOPR, GRT-6005, Docking, QM/MM, Sketchmap, GPCR, opioid receptor

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ABSTRACT Cebranopadol (CBP) is a novel analgesic acting as agonist at the nociceptin (NOP) and -opioid (MOP) receptors, exhibiting high potency and efficacy as antinociceptive and antihypersensitive drug. The binding conformation and the dynamical interactions of CBP with the NOP receptor have been investigated by molecular docking, molecular dynamics (MD) in the microsecond time scale, and hybrid quantum mechanics/molecular mechanics (QM/MM). CBP binds to the NOP receptor as a bidentate ligand of the aspartate D1303,32 by means of both its fluoro-indole and dimethyl nitrogens. Starting from the known crystal structure of the inactive state of the receptor, in complex with the antagonist compound-24 (NOP-C24) the comparative analysis of 1 s MD trajectories of the NOP-C24 itself, NOP_free and NOP-CBP complex provides new insights on the already known microswitches related to receptor activation, in the frame of the extended ternary complex model. The agonist acts by destabilizing the inactive conformation of the NOP receptor, by inducing a conformational change of M1343,36, which allows W2766,48 to flip around its 2 dihedral, going in close proximity to the receptor hydrophobic core (T1383,40, P2275,50, F2726,44), which is known to be fundamental for the activation of the opioid receptors. A complete rational picture is also provided for the role of N1333,35 and W2766,48 undergoing critical conformational changes related to an anticooperativity effect, i.e. the well known role of sodium as negative modulator of agonist binding. Finally, the movement of residue Y3197,53 belonging to the NPxxY motif, is also induced by the binding of the agonist in the inactive state, opening a gate for a water channel just as upon receptor activation.


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INTRODUCTION Cebranopadol (CBP, (1r,4r)-6’-fluoro-N,N-dimethyl-4-phenyl-4’,9’-dihydro-3’H-spiro[cyclohexane-1,1’pyrano[3,4-b]indol]-4-amine) is a novel analgesic agonist binding to all opioid receptors, being full agonist at the nociceptin (NOP) and -opioid (MOP) receptors, and partial agonist at k-opioid (KOP) and -opioid (DOP) receptor, exhibiting high potency and efficacy as antinociceptive and antihypersensitive drug in several rat models of acute and chronic pain 1, 2. Both the MOP and NOP are inhibitory GPCRs of the opioid receptor family (OR) that lower the adenylate cyclase activity and the cytosolic cAMP levels as in the canonical way of the overall OR class. As all GPCRs investigated so far, ORs include seven transmembrane helices (TM1-7) three internal (ICL1-3) and three external (ECL1-3) loops. Activation of the MOP receptor, linked to important relative motions of the helices, is responsible for the efficacy of the most effective analgesics. The MOP has high affinity for enkephalins and beta-endorphin, and low affinity for dynorphins. On the other hand, the NOP is currently classified as a "non-opioid member of the opioid receptor family" and referred as a drug target having a broad therapeutic spectrum 3 and a wide range of physiological effects noted in the nervous system (central and peripheral), the cardiovascular system, the airways, the gastrointestinal tract, the urogenital tract and the immune system

4, 5.

The 17-mer peptide nociceptin is

the endogenous NOP agonist, however to date only the crystal structure of the NOP inactive state is known, in complex with the antagonist SB612111, and two peptidomimetic, compounds C24 and C35 6, 7

(PDB code: 5DHH, 4EA3, 5DHG). Attention is focussed on the use of cebranopadol as a selective

NOP/MOP agonist drug in pain therapies as it is thought that the NOP and MOP receptors are not colocalized in the same neurons and thus may have independent actions in at least partly distinct neuronal networks 8. Moreover there is some evidence that such mixed agonist drugs both potentiate opiate analgesia and display lower side effects

9, 10.

According to the extended ternary complex model of GPCR activation (Fig. 1A)

11, 12,

binding of an

agonist to the receptor is favoured when a G protein (or its nanobody mimic) allosterically stabilizes the active receptor state. Indeed, the state of full agonist-bound GPCR seems close to the inactive one, in the absence of either G-protein, -arrestin or other stabilizing nanobodies 13-15, thus the question arises if the agonist acts by binding and destabilizing the inactive conformation of the receptor or by stabilizing an already formed receptor-G-protein complex. Recently, the crystal structure of both the antagonist-bound inactive state 16 (pdb code: 4DKL) and the nanobody stabilized, agonist-bound active state14 (pdb code: 5C1M) of MOP have been reported: the conformational changes observed upon ACS Paragon Plus Environment


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activation are similar to those previously observed for 2AR (PDB codes: 2RH1, 3SN6), muscarinic M2 receptor (PDB codes: 3UON, 4MQT) and rhodopsin (PDB codes: 2I37, 3CAP), and similar to those more recently observed for the k-opioid receptor (KOP) (PDB codes: 4DJH, 6B73) with a large outward movement of TM6 and a smaller inward movement of TM5 and TM7 (Fig. 1B). From the static structures of inactive and activated GPCRs, and mutagenesis studies17-20, several highly conserved residues have been identified as activation microswitches, displaying clear conformational changes between the inactive and active state; among them are R3,50 in the DRY motif of the receptor, M3,36 in the orthosteric site, and N3,35, W6,48, Y7,53 which are closer to the central bulk and to the intracellular side of the receptor. However, the structural differences in the orthosteric binding pockets of active and inactive ORs, as shown by these crystallographic reports, are relatively small. Moreover, structure–activity (SAR) studies revealed that subtle changes in MOP ligands structure can convert an agonist into an antagonist, leading to the simple “message/address” concept

21-23,

the “message” and the “address” being the functional

groups of the opioid drug responsible for its efficacy and selectivity profile, respectively. For example the cyclo-propylmethyl group of the MOP antagonist BU74 (Fig. S1A, blue rectangle, colored gold), being the only different group with respect to the agonist BU72 (same figure, colored transparent yellow), and also present in the co-crystallized antagonist -FNA16, can be identified as the message region of the antagonist14, able to directly interact with important receptor residues such as W6,48 and Y7,42. However compound MP1104, containing the same group, is a strong agonist in KOP15; moreover known antagonists of NOP do not contain groups extruding towards residues W6,48 and Y7,42 (Fig. S1B) 6, 7.

These further studies have suggested that the “message/address” concept cannot be easily

generalized from MOP ligands to other drugs along the OR family. Opioid receptors display huge hydrophobic cavities, able to host many water molecules as well as one or two lipophilic molecules, with low specificity; thus receptor-ligand interactions involve a large number of residues and the overall surrounding water network. A better understanding would require the integration of experimental and computational strategies that allow to study or simulate a chosen receptor-ligand complex in an environment more similar to its natural one. Molecular docking has an important role in lead discovery and design24, 25, the “Holy Grail” being just the ability to correctly predict the structure and the binding energy of a receptor-ligand complex. Moreover, both homology modelling and molecular dynamics are acknowledged as reliable tools to investigate proteins as flexible and dynamic entities26. NOP microswitches were investigated by Daga and Zaveri 27 before the X-ray crystal structure of the inactive NOP was reported, by homology modelling of both inactive and active NOP, and short (8 ns) ACS Paragon Plus Environment

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molecular dynamics simulations,. The important role of the conserved residues N1333,35, M1343,36, W2766,48 and Y3197,53 borrowed from the template structures of rhodopsin and opsin, was found in agreement with the overall description of microswitches provided by previous structural and functional studies on GPCRs. However, in the face of new knowledge provided by the crystalline structure of the inactive NOP (in complex with compound C24) and other opioid receptors, including the discovery that a sodium ion occupies a central allosteric site28, 29, and thanks to the increased computational power, it is now possible to reconsider the general activation process of NOP in the framework of the extended ternary complex model. Microswitches can be observed "in early action" as activated by the agonist already in the inactive state of the receptor, and the interplay between the allosteric and orthosteric site during the state transition can be elucidated by comparing long MD trajectories of antagonist bound NOP, NOP free, and agonist bound NOP. In this work, a multi-flexible docking (MF-docking i.e. flexible ligand docking to multiple receptor conformations) procedure, already applied to the study of NOP antagonists30, was carried out to provide the best binding mode of cebranopadol to the inactive state of NOP. Thereafter, the representative structure of the NOP-CBP complex extracted from a microsecond MD trajectory, and it has been refined by a quantomechanics/molecular mechanics (QM/MM) optimization procedure. Moreover, a comparative analysis of 1 s MD trajectories of the NOP_free, the NOP-CBP and the NOP-C24 complexes has provided direct insight on which microswitches are activated by agonist binding, the signalling pathway propagating inside the receptor itself, the agonist-induced water penetration, and the allosteric role of sodium in the stabilization of the inactive state.

METHODS Model set-up of the NOP_free receptor and the NOP-C24 complex. The initial structure of the NOP receptor was set up starting from the X-ray crystal structure of the NOP-C24 complex (PDB code= 4EA3)6. Side-chain atoms missing in the reported structure due to structural disorder, were added and minimised “in vacuum” by using the “Modeller” and “Rotamer” modules in the Chimera UCSF package31. Compounds

C24

(1-benzyl-N-[3-(1'H,3H-spiro[2-benzofuran-1,4'-piperidin]-1'-yl)propyl]-D-

prolinamide , BAN ORL 24, ZINC-13679981 in the ZINC databank), was taken as such in the crystal structure, and protonated at its charge of +2 using the “Addh” module of Chimera UCSF package. Its atomic partial charges were recalculated according to Density Functional Theory at the B3LYP/G SV ACS Paragon Plus Environment


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(P) functional level, as previously reported30. Classical Molecular Dynamics. Classical molecular dynamics simulations were performed with the GROMACS 4.6 package32, 33 under the AMBER parm99sb force-field34 at the full atomistic level using a TIP3P water solvent and an explicit pre-equilibrated phospholipid bilayer of 128 POPC (1-palmitoyl2-oleoyl-sn-glycero-3-phosphocholine)

molecules

obtained

by

the

Prof.

Tieleman

website

(http://moose.bio.ucalgary.ca). All the molecular dynamics session were performed in a watermembrane system prepared as previously described30, 35. The protein-ligand-membrane systems were solvated in a triclinic water box (having basis vectors lengths of 7, 7.4, 9.3 nm) under periodic boundary conditions, for a total number of about 40000 atoms (6400 solvent molecules) The total charge of the system was neutralized by randomly substituting water molecules with Na+ ions and Cl- ions, to obtain neutrality with 0.15M salt concentration. Following a steepest descent minimization algorithm, the system was equilibrated in canonical ensemble (NVT) conditions for 300 ps, using a V-rescale, modified Berendsen thermostat with position restrains for both the protein-peptide complex and the lipids, and thereafter in a isothermal–isobaric ensemble (NPT) for 500 ps, applying position restraints to the heavy atoms of the protein-peptide complex, and using a Nose-Hoover thermostat36, 37 and a Parrinello-Rahman barostat38 at 1 Atm with a relaxation time of 2.0 ps. Finally, all restraints were removed, and molecular dynamics runs were performed under NPT conditions at 300 K with a T-coupling constant of 1 ps. Van der Waals interactions were modeled using 6-12 Lennard-Jones potential with a 1.2 nm cutoff. Long-range electrostatic interactions were calculated, with a cutoff for the real space term of 1.2 nm. All covalent bonds were constrained using the LINCS algorithm. The time step employed was 2 fs, and the coordinates were saved every 5 ps for analysis, which was performed using the standard GROMACS tools. Multi-Flexible Docking procedure and set-up of the NOP-CBP complex. The trans-cebranopadol (CBP) molecule (pubchem item: 11848225, formal charge= +1, see Fig. 2) was docked into NOP using multiple conformations of the receptor. Two 100 ns MD trajectories have been produced, one for the NOP_free receptor, and one for the NOP-C24 complex. Thereafter, about 30 representative frames have been extracted from each trajectory by a clustering algorithm based on the Gromos method. The Gromos method is described in Daura et al.39: the number of neighbors for each structure is counted using a RMSD cut-off, the structure with the largest number of neighbors, with all its neighbors, is taken as a cluster, and the cluster is eliminated from the pool of final chosen clusters. The process is repeated until the pool of structures is empty. A RMSD cutoff of 0.1-0.2 nm was used in order to get a number of about 30 representative frames for each trajectory when extracting the receptor frames. In this way we ACS Paragon Plus Environment

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have obtained a first set of frames (NOP_free) accounting for a flexibility of the receptor and the ligand stereochemical environment (the pharmacophore), and a second set of frames (NOP_”C24-ghost”) also accounting for flexibility of the receptor but with a restrained geometrical deformation of the C24 pharmacophore. Our overall method30 is probably less sensitive to conformational changes of the binding site than the method proposed by Tarcsay et al.40. However it was our choice to search for either binding site conformational changes possibly related to long range dynamics of the receptor (CA-rmsd), and moderate binding site conformational changes around the known crystal structure, found by clustering around conformations of the known bound ligand (C24-rmsd for inactive NOP, and BU72rmsd for active MOP). Thereafter, according to the Autodock Vina 1.1 software package41,

42,

all

rotatable bonds within the ligands were allowed to rotate freely, whereas each one of the receptor conformations was considered rigid. Autodock Vina calculates its own grid maps, clusters and ranking automatically, so that clustering the results after docking are no necessary, and further optimisation is left to other methods, e.g. via molecular dynamics. The docking calculation consists of a number of independent runs, each of which includes several steps involving a random perturbation of the conformation, followed by a local optimization using the Broyden-Fletcher-Goldfarb-Shanno (BFGS) algorithm and a selection in which the step is either accepted or not. Each local optimization involves many evaluations of the scoring function as well as its derivatives in the position-orientation-torsions coordinates. The number of evaluations in a local optimization is guided by convergence and other criteria. The number of steps in a run is determined heuristically, depending on the size and flexibility of the ligand and the flexible side chains, and the number of runs is set by the “exhaustiveness” parameter that for a search space in 30x30x30 angs cube is set to 8. Each run can produce several results: these are merged, refined, clustered and sorted automatically to produce the final result, i.e. a small number of “interesting poses” or modes (parameter num_modes=9) for each receptor structure, within an energy range of less than 3 kcal/mol (energy_range=3). After the structure of the NOP-CBP complex has been obtained this way, the same procedure as described for the NOP-C24 complex was carried out to set-up the complex in a water-membrane system to be followed by molecular dynamics.

QM/MM calculations. The QM/MM optimization was carried out starting from a pre-equilibrated system by MM, as quenching to T=0.15 K in 2 ps using the subtractive ONIOM method43, 44 as allowed under the standard GROMACS tools. The QM calculation was performed at the AM1 level, using the ORCA 3.0 package45. The QM part of the system included the CBP molecule and the side chains of the nearest neighbours residues I1273,29, D1303,32, Y1313,33, V2796,51, and Q2806,52, and the MM part including all other atoms of the protein-membrane-water system. Link atoms were placed between the ACS Paragon Plus Environment


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C and C atoms of the protein residues included in the QM part of the system, at a distance from the C atom equivalent to a single C-H bond.

Analysis. In the Result section, tables ranking the strength of non polar interactions between each residue of the receptor and the ligand are shown. This strength has been evaluated calculating the number of non-polar contacts, modelled as a coordination number via a continuous, differentiable switching function:

strength = nnpc =  ij

1  rij / r0 

1  rij / r0 

a b

with the i and j indexes running over the carbon atoms of the ligand and of a chosen aminoacid, respectively; a=6, b=12, r0=0.6 nm with a cut-off of 1 nm. The chosen value of r0 accounts for the typical carbon-carbon distance (0.4-0.45 nm) and thermal motion’s amplitude (0.15-0.2 nm). These coordination numbers (one per residue of the binding site) have been calculated either on single structures (e.g. Fig. 3C), or averaged along trajectories (e.g. Fig. 4D) A bidimensional representation of the conformational space explored by important residues during a molecular dynamics simulation has been obtained by the use of a number of tools including the Plumed package46 to generate a lower dimensional representation of the trajectory via chosen collective variables (dihedral angles for each protein residue, in our case), and the Sketchmap set of programs to furtherly perform multi-dimensional scaling (MDS)47, non-linear dimensionality reduction and mapping in a bidimensional graph48. The Sketchmap reduction algorithm is one of the methods proposed as alternative to the principal component analysis (PCA)49, and is aimed to best providing the characterization of the global, non-linear features of a multidimensional conformational space, just when the PCA analysis seems less appropriate50.

Homology model of the active state of NOP. The putative structure of the active state of NOP has been obtained by homology modelling via the I-Tasser server51 using the structure of the active MOP receptor (PDB code: 5C1M) as the main template. A C-score=1.47 has been obtained. C-score is typically in the range of [-5,2], where a C-score of higher value signifies a model with a higher confidence. The estimated TM-score was = 0.92±0.06. A TM-score > 0.5 indicates a model of correct topology whereas a TM-score < 0.17 means a random similarity. The finally estimated accuracy ACS Paragon Plus Environment

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(RMSD) was = 3.1±2.2 Å. According to the Ramachandran plot summary for selected residues from the Procheck analysis on 282 residues, 82.8% lie in the most favoured regions, 16.4% in the additionally allowed regions, and 0.8% (i.e. 2 residues) in the generously allowed regions, with no residue remaining in the disallowed regions. Artwork. 3D images of the ligand-receptor structures were obtained by the Chimera software31.



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RESULTS AND DISCUSSION MF-docking of CBP to NOP CBP (Fig. 2) is characterized by two donor nitrogens, one acceptor oxygen, two rotatable bonds and a formal charge of +1 with a protonated dimethyl nitrogen. Two CBP isomers co-exist, cis or trans for the relative position of the dimethyl nitrogen and the oxygen with respect to the cyclo hexane pseudo-plane. The trans isomer has subnanomolar affinity for both NOP and MOP (Ki=0.9±0.2 and 0.7±0.3 nM, respectively). In the MF-docking procedure, the multiple receptor conformations included the starting, crystallographic one (pdb code 4EA3), after removal of compound C24. As described in the Methods, two frame sets of the receptor were extracted from preliminary 100ns MD trajectories of the NOP-C24 complex (leading, after removing C24 from the trajectory, to the NOP “C24-ghost”, or simply the “ghost” frame set) and the NOP_free (the “free” frame set) in a water-membrane system. In Fig. 3A, the plot of the Nindole-CG_D3,32 distance vs. free energy (VINA score) for the ”ghost” (red points) and “free” (green points) frame sets of the MF- docking protocol NOP.vs.CBP (trans isomer) are displayed. Poses having the best docking score are marked as thick points, and poses on a rigid receptor are marked by blue circles. Meaningful distances are selected as allowing H-bond interaction, according to the contemporary satisfaction of the following parameters: Donor=Nindole, Acceptor=OD1/OD2_D1303,32, distance < 3.5 Å, H-Donor-Acceptor angle < 30°. Only one docking pose with a meaningful Nindole-D3,32 distance was observed, on a receptor conformation belonging to the "ghost" frame set (marked by a large semi-trasparent circle pointed by a red arrow), having a VINA score of -8.1 kJ/mol, and that was chosen for the subsequent analysis. The chosen conformation of the NOP-CBP complex is displayed in Fig 3B, left frame, CBP colored gold). CBP binds as a bidentate ligand to D1303.32, i.e. both the fluoro-indole nitrogen and the dimethyl nitrogen of CBP can contribute to form a salt bridge interaction with the negatively charged carboxyl group of D1303,32, with the fluoroindole oriented towards the extracellular side. The observed chelation to D3,32 is possible only for the CBP trans-isomer. The phenyl ring of the trans isomer does not enter in π-stacking interaction with the (Y1313,33, M1343,36) diad, as observed for the aromatic portion of some of the NOP antagonists, but protrudes in the opposite direction towards helix TM6 (residues V2766,51 and V2836,55), still having non polar interaction with M1343,36 and W2766,48. The more important residues interacting with CBP via H-bonds and non polar interactions are shown in the histograms of Fig. S3D,E in comparison with those of C24 in the crystal structure of the complex NOP-C24 (Fig. S3A,B). The importance of residue D1303,32 for the formation of the NOP-CBP complex is thus similar to all the already known complexes between opioid receptors (ORs) and either morphinan or piperidineACS Paragon Plus Environment

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based agonists/antagonists. The comparison between the poses of trans-CBP (Fig 4B, left frame) and cis-CBP (Fig 4B, right frame) displays a large deviation between them. As already rationalized by Schunk and coworkers2, the much lower affinity (97.4 nM) for the cis-conformer corresponds to the fact that it cannot form bidentate ligation with D1303,32. As the agonist has been docked to the inactive state of NOP, concerns could arise about the reliability of our result. In order to corroborate it, we have thus superimposed the chosen pose of trans-CBP to the one obtained by docking CBP to the active state of MOP (aMOP), which is known by crystallography14. In this second case, many poses satisfy the selection criterion and possess the same orientation of the agonist (Fig. S2A,B), with a VINA score lower than -9 kJ/mol and a best score of -10.2 kJ/mol. Although a more extended study of the MOP_CBP complex is beyond the scope of this article, the similarity between the poses of CBP in complex with NOP and aMOP strengthens their reliability and the ability of CBP to be a full agonist of both MOP and NOP. In Figs. 3C,D, the chosen docking pose for CBP is superimposed to those of some alternative compounds reported in the SAR study by Schunk et al.2 which are listed in Table 1: compound 2a, 3b (cis-CBP), 4a, 48a and 5a. All of them are compared with the compound 3a of the SAR study (i.e. transCBP, gold sticks). The experimental affinity of CBP for the NOP is 0.9 nM (Table 1). Among these alternative compounds, poses very similar to that of CBP, exhibiting chelation by both the indole and dimethyl nitrogens to D1303,32, are found for compounds 2a (the fluorine group of CBP substituted by a hydrogen, blue wires) and 48a (the fluorine and one of the two methyl groups of CBP substituted by a hydroxyl and a hydrogen group, respectively, orange wires) in the center frame of Fig. 3C). These two compounds bind with subnanomolar (0.1 nM, compound 2a) and nanomolar (1.0 nM, compound 48a) affinity. As organofluorine are in general not capable to form halogen bonds, the fluorine of the fluoroindole group is more likely to act as a H-bond acceptor52, and it could be expected its substitution with donors like hydrogen or hydroxyl groups to have non-negligible effects on its affinity. However according to our 3D conformation of the NOP-CBP complex, the nearest protein group to the fluorine atom of CBP is the carboxamide from Q1072,60, at a distance just lower than 4 Å, corresponding to a weak electrostatic interaction. A very similar pose is found also for compound 4a, (Fig. 3C, orange wires, which experimentally binds with 11.0 nM affinity; for this compound we have found, and cannot rule out, a number of alternative, reversed poses. On the contrary compound 5A binds different (Fig. 3D), due to substitution of the prolinamide oxygen by a third amino group, of which the donor nitrogen interacts with D1303,32. Molecular dynamics in the microsecond time scale, and quantomechanical refinement. ACS Paragon Plus Environment


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Starting from the inactive state of NOP, taken from the crystal structure of the NOP-C24 complex (pdb code: 4EA3), three 1 s MD trajectories have been carried out, for the NOP-C24 complex itself, for the NOP_free (removing C24) and the NOP-CBP complex (after MF-docking of CBP). Only for the NOP-C24 complex, a sodium atom has been manually introduced in its putative allosteric site (D972,50 N1333,35 S1373,39 S3127,46) 28, while for NOP_free and NOP-CBP, a water molecule, that is reported in the crystal structure, occupies the same position in the allosteric site. The MD runs have been performed at T=300K in a full receptor-membrane-water system with a 0.15M NaCl concentration. No relevant global motion of the TM helices was recorded in the absence of the antagonist, and no outward motion of TM6, related to activation, was observed in the presence of CBP, in agreement with the statement that in the presence of the agonist only, and in absence of a G-protein, the inactive state of the receptor is favoured15. However the C rmsd plots of Fig. 4A show departures from the starting structure that increase in the order NOP-C24 < NOP_free < NOP-CBP. In Fig. 4B, the comparison between the calculated C B-factors for NOP-C24 (blue) and NOP_free (grey) shows that in the presence of the antagonist C24 in the othosteric site and of the sodium ion in the allosteric site, the external ends of helices TM2, TM3 and TM4 are stabilized with respect to the NOP_free trajectory, while the flexibility of loops ECL1 and ECL2 is strongly reduced. The mean square fluctuations were calculated for the overall trajectory, including contributions due to both vibrational terms and long term collective motions. The sodium ion inserted in the allosteric site of NOP-C24 remains fully stable all along the trajectory (Fig. 4D); interestingly, in the NOP_free system we observed (Fig. 4E) a rapid migration of the sodium from the extracellular side towards, first, the orthosteric site then an intermediate site and finally into the allosteric site, where, in the absence of the antagonist, it has shown low stability. On the other hand (Fig. 4C) the comparison between the calculated C B-factors for NOP-C24 (blue) and NOP-CBP (red) shows that when CBP binds to the inactive state, in the absence of sodium in the allosteric site, the flexibility of the receptor, including all the TM segments, is far beyond the ones of both NOP-C24 and NOP_free. However, also in this case we observed entering of the sodium ion from the extracellular side after 800 ns (Fig. 4F). It is noteworthy that no migration from the external side was observed in another 1 s running session of NOP-C24, where no sodium was firstly inserted into the allosteric site (data not shown) and we will deepen these results in the following discussion. The representative structure of the NOP-CBP complex has been extracted after clustering by the Gromos method (see the Methods) of its trajectory. We have further refined this main conformation by QM/MM quenching as described in the Methods (Fig. 5), considering for the QM part a subsystem formed by the agonist itself and the side chains of the nearest neighbours residues I1273,29, D1303,32, Y1313,33, V2796,51, and Q2806,52 (for a list of neighbours see also Fig. S3D,E), while all other atoms ACS Paragon Plus Environment

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belonging to the receptor, the membrane, water molecules and ions are included in the MM subsystem. The structure obtained by a QM/MM quenching (gold), representing the energy minimum at low temperature, is compared with the MM representative structure (transparent yellow) in Fig. 5. After the QM/MM optimization, small changes affect the binding conformation, leading to enhanced stability, as shown by the atomic distances reported in Fig. 5, that define the salt bridge interaction between CBP and D1303,32: due to a rotation of the D1303,32 2 dihedral, the H1(Ndimethyl)-OD1/2(D1303,32) distances decreases from 2.13 to 1.83 Å, while the H2(Nindole) atom goes at H-bond distance with both OD1 and OD2 of D1303,32. A more detailed comparison (Figs 6A,D) between the C-rmsd plots of both the orthosteric and allosteric (sodium) sites for the NOP-CBP and NOP_free systems with respect to NOP-C24 (blue curves) confirms that the structural perturbation due to the presence of the agonist (NOP-CBP trajectories, red curves) is far beyond the one due to the simple removal of the antagonist from the orthosteric site and of the sodium from the allosteric site (NOP_free trajectory, black curves). According to the known crystal structure (pdb code: 4EA3), the binding of the piperidine-based antagonist C24 to NOP (Fig. 6B) includes the H-bond formation between the piperidine nitrogen and the negatively charged D1303.32, that is stabilized by -stacking of the C24 benzofurane ring system between the hydroxyphenyl ring of Y1313,33 and the thiomethyl group of M1343,36, and by another H-bond between the amide nitrogen of C24 and OE1(Q1072,60) (see also Figs. S3A,B). Penetration of the C24 ring system is allowed by a M1343,36 conformation that is more buried with respect to that of classical ORs, like KOP6. On the other hand, according to our "in silico" docking results, CBP binds different (Fig. 6C), by a bidentate chelation to D1303.32 (see also Fig. S3D), and its phenyl ring does not enter in -stacking interaction with the (Y1313,33, M1343,36) diad, instead it protrudes in the opposite direction towards helix TM6. The compared analysis of the ligand-receptor non-polar contacts along their respective trajectories (Figs. S2B,D) reveals that the total number of these contacts is much larger for the NOP-C24 complex than for the NOP-CBP complex (665 vs. 563), contributing to explain the antagonist induced stabilization; moreover the more important TM6 residue in contact with C24 is W2766,48 (40 contacts in average) whereas due to the orientation of the CBP phenyl ring, the more important TM6 residues in contact with CBP are V2836,55 (40 contacts) and V2796,51 (30 contacts), placed towards the extracellular side with respect to W2766,48, while the number of contacts of CBP with this last residue is 4-fold smaller (10 contacts). Just as a consequence of the good stability of the CBP conformation in the orthosteric location, the steric hindrance of the protruding CBP phenyl ring against residues V2836,55 and V2796,51, together to the absence of a stable, buried conformation of M1343,36, opens the way for large side chain fluctuations of the important residue W2766,48. Figs. 6B and 6C, showing the superimposition of the ACS Paragon Plus Environment


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main representative frames from their respective rmsd-based clustering of trajectories (rmsd calculated for sidechains of residues of the orthosteric site). The figures show a perspective going from the orthosteric site to the receptor core triad (T1383,40, P2275,50 and F2726,44), illustrating the degree of stabilization induced by the antagonist C24 (Fig. 6B), and of perturbation induced by the agonist CBP (Fig. 6C). An analogous comparison is done by looking at the rmsd-based clustering of the sidechains of residues around the allosteric (sodium) site. Fig. 6E illustrates the strong stability of the sodium atom within its putative site in the NOP-C24 complex, surrounded by D972,50, N1333,35, S1373,39 and two water molecules: the analysis of the distances between the sodium atom and these residues along the trajectory gives 100% persistence of these distances below 3.2 Å (Fig. S3C). Importantly, according to the highresolution crystal structure of the inactive -opioid (DOP) receptor, revealing the sodium site location53, 54,

the sodium ion and the highly conserved residue W2766,48 are linked in the inactive state by a

bridging water molecule. Thus, according to our simulation, conformational changes of W2766,48 contribute, in the presence of the agonist in the orthosteric site, to destabilization of the sodium coordination in the allosteric site, and viceversa. As mentioned above, the sodium ion was manually posed in its putative site only when starting the NOP-C24 trajectories. and there is no pathway for the ion connecting the receptor surface to the allosteric site, while for NOP_free and NOP-CBP, a sodium ion by the extracellular side was found to be able to migrate to the allosteric site (Fig. 4D-F). Even in the presence of the agonist, the sodium ion reaches the allosteric site after about 800 ns, (Fig. 4F). The representative frames of the allosteric site of the NOP-CBP clustered trajectory for t > 800 ns are depicted in Fig. 6F. The allosteric site in this case is destabilized and the sodium ion is in contact, besides D972,50, with residues from helix TM7 (G3087,41, S3127,46) instead of residues from helix TM3 (N1333,35 and S1373,39) (see also Fig. S3F). The ability of a sodium cation to reach, along the MD trajectory, the allosteric site even in the presence of CBP, having formal charge = +1, is somewhat intriguing, as in this case the migration pathway, elettrostatically and sterically hindered by the agonist, is certainly different by the one, crossing the orthosteric site, observed for NOP_free. The occupancy density of the migrating cation in the NOP-CBP trajectory in the range 800-1000 ns is depicted (blue wires) onto a representative frame of the NOP-CBP complex (Fig. S4), where CBP is represented as gold spheres, and the receptor as grey ribbons. Thus, an alternative pathway for sodium migration, running along negatively charged and polar residues, can be identified (red-colored sticks in the figure), starting from the so-called "electronic trap" of ECL2 (residues E194,196,197,199 and D195), and crossing an intermediate site near helix TM2 (residues D1102,63, Q1072,60) from which it reaches quickly the allosteric site in the metastable coordination geometry described above. ACS Paragon Plus Environment

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The earliest activation microswitch in the orthosteric site The different outcomes of the binding of the antagonist C24 and the agonist CBP within the orthosteric site are recapitulated and compared in Fig. 7. Penetration of the C24 ring system (C24 colored transparent yellow), in -stacking between Y1313,33 and M1343,36, make motions of the W2766,48 sterically hampered by both the antagonist and the stable, buried M1343,36 conformation. Further stabilization against W2766,48 motions is due to the presence of a sodium ion in the allosteric site via a bridging water molecule. On the other hand, the CBP phenyl ring protrudes towards V2836,55 and V2796,51 and in the absence of a stable conformation of M1343,36, open the way for a flipping of W2766,48, that goes in close proximity with the central receptor core (T1383,40, P2275,50 and F2726,44). Thus we suggest that the coupled conformational change of M1343,36 and W2766,48 is the first microswitch contributing to an early destabilization of the inactive state of the receptor, related to the presence of the agonist in the orthosteric site, and to the absence of sodium in the allosteric site. In Fig. S5, the conformations assumed by the residue M1343,36 along the trajectories of NOP-C24, NOP_free and NOP-CBP are detailed by using the Sketchmap program. As described in the Methods, a single conformation of the M1343,36 residue (Fig. S5A) along the MD trajectory can be represented by the value of its five dihedral angles [] as a point in a 5-dimensional space. The Sketchmap algorithm translates the 5-dimensional space of conformations in a 2-dimensional one [X1,X2], allowing to map it as a 2D histogram, i.e. a density plot in a free-energy style: the more likely conformations along the MD trajectory appear as local minima of the histogram. The 2D density plot of M1343,36 along the 1s MD trajectory of the NOP-CBP complex (Fig. S5B) shows no density around the starting conformation of the inactive state (blue circle, conformation taken from the one assumed in the crystal state, pdb code: 4EA3) and displays the absolute minimum (most likely conformation) near the one expected for an active state (green circle). The conformations expected for residues in the active state have been provided by a homology model of NOP from the I-TASSER server (see the Methods). The time plot of the M1343,36 dihedrals (Fig. S5C) show that the most important torsion leading from the inactive to the active-like conformation is due to the  a. More importantly, the comparison (Fig. S5D) between the [X1,X2] maps of M1343,36 relative to the three trajectories of NOPC24 (blue points), NOP_free (black points) and NOP-CBP (red points) shows that only in the presence of the agonist the active-like conformation of M1343,36 is reached. In the same way, the conformations assumed by the residue W2766,48 along the trajectories of NOPC24, NOP_free and NOP-CBP, characterized by its four dihedral angles [, are detailed in Fig. ACS Paragon Plus Environment


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8A-D. In this case, the inactive conformation and the expected active conformations of W2766,48 are very close each other (Fig. 8B, blue and green areas), while the most likely conformation assumed along the NOP-CBP trajectory is identified by a farther absolute minimum in Fig. 8B, corresponding to the residue flipping shown in Fig. 7, due to rotation of the  dihedral (Fig. 8C). The position of this minimum is far from the one expected as a result of the homology model of active NOP, however Fig. 8D shows that for both the NOP-C24 (blue points) and NOP_free (black points) trajectories, no flipping of the W2766,48 residue occurs: the flipped conformation is reached only in the presence of the agonist (red points). Thus it is possible that either the active conformation of W2766,48 obtained by the homology model of active NOP is not correct, or the most likely, flipped conformation found along the NOP-CBP trajectory is a transient, intermediate state, not less important as allowing the receptor activation to go ahead along its internal pathway. Thus in the following we will refer to conformational changes towards either an "active-like" or an "intermediate" state. Early activation microswitches of the allosteric site and the NPxxY motif. We have extended the Sketchmap analysis to a number of residues, pertaining sites thought to be important for receptor activation, i.e. in the orthosteric site (residues Q1072,60, D1102,63, I1273,29, D1303,32, Y1313,33, M1343,36, F1353,37, I2195,43, S2235,46, W2766,48, Q2806,52, V2836,55, ), in the allosteric (sodium) site (residues D972,50, N1333,35, S1373,39, S3127,46), in the central core (T1383,40, P2275,50 and F2726,44), in the NPxxY sequence of TM7 (residues N3157,49, P3167,50, I3177,51, L3187,52, Y3197,53), and in the DRY motif of TM3 (residues D1473,49, R1483,50, Y1493,51). The results of this extended analysis can be shortly summarized in Tables 2, 3 and Figs. 9, 10, S6 and S7. Some of the residues investigated show conformational changes leading to (or close to) their active-like states (Table 2, 2D maps of Fig. 9): they are colored green in Figs. 10, S7; some other residues, showing changes to an intermediate conformational state (Table 3, 2D maps of Fig. S6) are colored red in Figs. 10, S7. All of these residues are fully conserved along the human OR family, with the exception of the D1102,63 which is specific for each member of the family, and residue S2235,46 which is specific of NOP (alanin in other ORs). In particular, in the orthosteric site, in the presence of the agonist only, residue S2235,46 moves to an activelike state (Fig. 9D), while residues D1102,63 and F1353,37 show transient conformational changes towards a local minimum close to the active-like state, for about 20% and 15% of the NOP-CBP trajectory, respectively (Fig. 9A,B). More importantly, the main conformation of residue N1333,35, connecting the orthosteric and allosteric sites, is close to the active-like state, both in the NOP_free and NOP-CBP trajectories, while it remains stable in the inactive state in the NOP-C24 trajectory (Fig. 9C). This stability seems clearly related to the contemporary presence and stability of the sodium ion in the allosteric site: as well as in the case of residue W2766,48, conformational changes of N1333,35, due to the ACS Paragon Plus Environment

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presence of the agonist in the orthosteric site, contribute to destabilization of the sodium coordination in the allosteric site, and viceversa. Interestingly, the farthest residue from the bound agonist moving towards an active-like state is the highly conserved residue Y3197,53, located in the NPxxY site (Fig. 9E), due to rotation of both 1 and 2 dihedrals along the NOP-CBP trajectory (Fig. S8). The importance of this residue during GPCR activation has been already underlined by independent long MD and metadynamics simulations made on the adenosine A2A receptor, the 2-adrenergic receptor and rhodopsin55 showing that a hydrophobic layer of amino acid residues next to the NPxxY motif forms a gate for the opening of a continuous water channel only upon receptor activation, and that residue Y3197,53 undergoes transitions between three distinct conformations representative of inactive, G-protein activated and GPCR metastates. 2D maps of other residues moving towards specific intermediate states only in the presence of the agonist are shown in Fig. S6. Orientational changes of the Y1313,33 hydroxyphenyl ring (Fig. S6A) have been already discussed as related to direct interactions with the agonist. More interestingly, reorientation of the S1373,39 hydroxyl and the N3157,49 amide groups (Fig. S6B,C) reflects the rearrangement of an Hbond network along the pathway connecting the orthosteric site to the NPxxY motif via the allosteric site. Representative frames of the trajectories of NOP-C24 (t=430 ns) and NOP-CBP (t=108 ns, before sodium migration, and t=858 ns, after sodium migration) are compared in Fig. S7A-C. Persistence (expressed as a fraction between 0 and 1) of H-bonds and water bridges along the two trajectories are listed in the histograms of Fig. S7D, E. According to our analysis, the H-bond network in the internal, central volume of the receptor, remains fairly stable all along the NOP-C24 trajectory (Fig. S7A), including fully persistent H-bonds (persistence=1) connecting N1333,35 to the sodium ion (see also histogram of Fig. S3C), the sodium ion to D972,50, and this latter to Y3197,53 via a water bridge. Residue D972,50 is also in stable direct contact with TM7 (two H-bonds with S3127,46 and N3157,49). These two H-bonds, and the water bridge between D972,50 and Y3197,53, maintain a good stability also in the NOP_free trajectory (persistence=1, 0.95 and 0.9, respectively, data not shown), owing to the fast entry of the sodium ion from the extracellular side into the allosteric site. On the other hand, frames along the NOP-CBP trajectory, in the presence of the agonist permanently anchored to D1303,32 of the orthosteric site (the persistence of the N2(CBP)-OD1/2(D1303,32) bond is =1, that of the N1(CBP)-OD1/2(D1303,32) bond is =0.67) bear witness to a strong rearrangement of the network: in the absence of the sodium ion, the internal volume is rapidly filled by water molecules (Fig. S7B), i.e. a gate opening occurs for a water channel just as upon receptor activation55. The water occupancy along the trajectories of NOP-C24, NOP_free and NOP-CBP is displayed in Fig. S9. The above mentioned H-bond network is broken by the movement of N1333,35 and flipping of its amide group, and by the motion of the Y3197,53 into its ACS Paragon Plus Environment


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active-like conformation. Even after migration of the sodium ion, the flipped conformation of N1333,35 prevents the restoration of the sodium site coordination as seen for the inactive state of DOP53 and thought to be conserved along the OR family. Residue N1333,35 is at the boundary between the orthosteric site and the allosteric site; as already suggested by us when looking at the early events35 in the binding of nociceptin to NOP, the N1333,35 conformational change should be critical for the anticooperativity between the binding of the sodium ion and the agonist, i.e. for the well known role of sodium as negative modulator of agonist binding to ORs56, 57 and in particular to NOP58. The residues found to be microswitches early activated by cebranopadol in inactive NOP are conserved along the OR family, apart from S2235,46 which is NOP-specific, and are conserved and reported as microswitches of other (non-OR) GPCR-agonist complexes27. Thus, it is very likely that the same conformational changes would be observed for other agonists binding to NOP. However, not necessarily our result, about the role of M1343,36 as initial trigger, is transferable to other NOP agonists, because the reshaping of the binding pocket and subsequent microswitch activation could depend from other factors like long-range polar/non polar interactions and ligand-induced water penetration. Moreover, the sidechain of the microswitch S2235.46 (valine in other OR) is right in front of M1343.36, probably influencing its conformational space in a specific way. Last, the reduced thermostability of numerous NOP-ligand complexes7 suggests in several cases the existence of a multiplicity of ligand binding conformations at least for less potent ligands. Conclusions In conclusion the present "in silico" study based on molecular docking and molecular dynamics provides clues about the early microswitches related to receptor activation and propagating inside the receptor itself. We identified and monitored microswitches in a way that is independent from previous literature data and from external templates, according to long MD and dimensionality reduction of rotamers, starting from the known structure of inactive NOP. In agreement with the assumption that, in the absence of G-protein, the GPCR-agonist complex lies in an inactive-like state, the identified microswitches are observed to act just on the inactive state of NOP, and just due to the presence of the agonist, a fact providing clear improvement to the knowledge of the GPCR activation process. CBP induces a strong destabilization of the inactive structure, first inducing a conformational change of residue M1343,36, which allows residue W2766,48 to flip and go in close proximity of the receptor hydrophobic core, known to be crucial for activation of all the ORs receptors. Moreover, the rotation of residues N1333,35 and W2766,48 can explain anticooperativity effects between the allosteric (sodium) site and the binding of agonists to the orthosteric site, rationalizing the known role of sodium as negative ACS Paragon Plus Environment

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modulator of agonist binding. Last, agonist binding induce motion of the far residue Y3197,53 of the NPxxY motif, as an opening gate for a water channel just as upon receptor activation. Hopefully, the details obtained "in silico" on the activation microswitches and on an internal pathway within the receptor itself, can be helpful for future experimental studies aimed to design selective or multifunctional opioid drugs

AUTHOR INFORMATION Corresponding author *Phone (+39)08623433521 email [email protected] ORCID Stefano della Longa: 0000-0002-8157-9530 Author contributions. The manuscript was written through contributions of all the authors. All the authors have approved the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS. This work was supported in whole or part by Italian Ministry of University and Research, (LINEA D1 Università Cattolica del Sacro Cuore) and by the CINECA supercomputing centers through the grant IsC58 (n. HP10CRBSWJ)



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Table 1 Compounds chosen from the SAR study by Schunk et al. 2014, for "in silico" comparison based on MF-docking

Compound (Shunk et al. 2014) 2a** 3a (trans- cebranopadol) 3b (cis- cebranopadol) 48a 4a 5a***

Substitutions X=O, Y=NMe2, R=H X=O, Y=NMe2, R=F X=O, Y=NMe2, R=F X=O, Y=NHMe, R=OH X=O, Y=NMe2, R=F X=NH, Y=NMe2, R=H

Affinity Ki (nM) 0.1 0.9 97.4 1.0 11.0 0.2

Efficacy EC50 GTPyS (nM) 13 120 60 1.3

* Relative Efficacy of 100% is defined as maximum GTPγS binding induced by nociceptin ** compound with poor pharmacokinetic properties *** compound dropped due to side effects on the cardiac potassium channels


Relative Efficacy* 89 % 75% 101% 108%

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Table 2 Microswitches towards active-like state residue

trajectory

type

main

site

rotation

D1102,63

NOP-CBP

2

local minimum (20%)

orthosteric

N1333,35

NOP-CBP

2

most likely

allosteric

NOP_free

most likely

M1343,36

NOP-CBP

2

most likely

orthosteric

F1353,37

NOP-CBP

2

local minimum (15%)

orthosteric

S2235,46

NOP-CBP



most likely

orthosteric

1

NOP_free Y3197,53

NOP-CBP

local minimum (23%) 1 2

local minimum (35%)


NPxxY


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Table 3 Microswitches towards an intermediate state residue

trajectory

main rotation

type

site

Y1313,33

NOP-CBP

1 2

most likely

orthosteric

NOP_free

most likely

S1373,39

NOP-CBP

1

W2766,48

NOP-CBP



N3157,49

NOP-CBP

1 2

1

most likely

allosteric

most likely

orthosteric

most likely

NPxxY


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FIGURE LEGENDS Fig. 1. A) The extended ternary complex model for GPCR activation. R:receptor (inactive state); R*:receptor (active state) L:ligand (agonist/antagonist) X: G-protein or -arrestin. B) Structural changes between inactive (blue) and active (green) states, as observed for the -OR (PDB codes: 4DKL, 5C1M) Fig. 2. 3D and 2D structure of cebranopadol (trans isomer) Fig. 3. A) Multi-flexible docking protocol NOP.vs.CBP (trans isomer): plot of the Nindole-CG_D3,32 distance vs. free energy (VINA score) for the NOP_”C24-ghost” (red points) and the NOP_free (green points) frame sets. Poses on a rigid receptor are marked by blue circles. A red arrow indicates the chosen pose. B) Poses chosen for trans-cebranopadol (left) and cis-cebranopadol (right). C, D)Poses chosen for a number of compound reported in the SAR study by Schunk et al. 2014 (see Table 1): 2a (blue wires), 4a (orange wires) and 48a (brown wires) (frame C); 5a (green wires) (frame D). The pose of CBP-trans is also reported, for comparison (gold sticks). Fig. 4. A) C-rmsd plot for NOP-C24 (blue), NOP_free (black) and NOP-CBP (red). B) Comparison between the calculated C B-factors for the 1s trajectories of NOP-C24 (blue) and NOP_free (grey). C) Same comparison between NOP-C24 (blue) and NOP-CBP (red). 5D-F) Sodium migration observed along the MD trajectories. The distances between each one of the Na ions and the atom CG(D2,50) are plotted as points. Each ion is identified by a specific color: D) NOPC24. At the beginning a sodium ion was positioned in its putative allosteric site where it remained fully stable for 1s. E) NOP_free. No sodium was initially positioned inside the receptor, but very soon one of them from the extracellular side reaches the orthosteric site, then an intermediate site and the allosteric site, showing meta-stability. F) NOP-CBP. Interestingly, in the 1s time scale we observed entering of the Na ion from the extracellular side even in the presence of the agonist bound to the orthosteric site of the inactive receptor. Fig. 5. 3D superimposition of the representative CBP structure after clustering of the MD trajectory (colored transparent yellow), and after QM/MM refinement (quenching) (colored gold), as described in the Methods. The observed bidentate chelation to D3,32 is clearly possible only for the CBP trans-isomer. A strong stability of the complex is confirmed by QM/MM. Fig. 6. A,D) Comparison of the C-rmsd plots for the orthosteric (A), and allosteric (D) sites of NOPC24 (blue), NOP_free (black) and NOP-CBP (red). B,C) 3D superimposition of the representative ACS Paragon Plus Environment


Page 24 of 42

C24 (B) and CBP (C) structures (gold) after rmsd-based clustering of the trajectories. E,F) 3D superimposition of the allosteric site structures along the trajectory of NOP-C24 (E) and NOP-CBP (F). The sodium ion (blue spheres) and waters (red spheres) are included in the figures. Fig. 7. 3D superimposition of the orthosteric site of NOP-CBP (ligand colored gold), and NOP-C24 complex (PDB code: 4EA3, ligand colored trasparent yellow, residues colored grey). Whereas C24 is stacked between Y1313,33 and M1343,36, CBP can open the way for flipping of W2766,48 which goes in close proximity of the receptor core (T1383,40; P2275,50; F2726,44, not shown). Fig. 8. A) Representative W2766,48 conformation. B) 2D histogram (free-energy style) of the W2766,48 conformations along the MD trajectory of the NOP-CBP complex, showing low density around the starting conformations of both the inactive and active state (blue, green areas) and high density around two alternative, intermediate states. C) Plot of the W2766,48 dihedrals along the NOP-CBP trajectory, showing that the two minima correspond to rotations along of the 2 angle. D) Superimposed 2D maps of W2766,48 conformations along the trajectories of NOP-C24 (blue), NOP_free (black) and NOP-CBP (red) showing that the intermediate states are approached only in the presence of the agonist. Fig. 9. Superimposed 2D maps of the conformations of important residues along the MD trajectories of NOP-C24 (blue), NOP_free (black) and NOP-CBP (red) showing that their active-like states are reached or approached either in the absence of the antagonist (C,D) or in the necessary presence of the agonist (A,B,E). Fig. 10. A,B) Map of the microswitches induced by CBP along the MD trajectory of the NOP-CBP complex. Green: microswitches toward an expected active state. Red: microswitches towards an intermediate state. The map outlines the possible internal signalling pathway along the receptor itself, starting from the orthosteric site and going towards the intracellular side, involving residues of the hydrophobic core, the allosteric sodium site and the NPxxY sequence.


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REFERENCES 1.

Linz, K.; Christoph, T.; Tzschentke, T. M.; Koch, T.; Schiene, K.; Gautrois, M.;

Schroder, W.; Kogel, B. Y.; Beier, H.; Englberger, W.; Schunk, S.; De Vry, J.; Jahnel, U.; Frosch, S., Cebranopadol: a Novel Potent Analgesic Nociceptin/Orphanin FQ Peptide and Opioid Receptor Agonist. J Pharmacol Exp Ther 2014, 349, 535-548.

2.

Schunk, S.; Linz, K.; Hinze, C.; Frormann, S.; Oberborsch, S.; Sundermann, B.;

Zemolka, S.; Englberger, W.; Germann, T.; Christoph, T.; Kogel, B. Y.; Schroder, W.; Harlfinger, S.; Saunders, D.; Kless, A.; Schick, H.; Sonnenschein, H., Discovery of a

Potent Analgesic NOP and Opioid Receptor Agonist: Cebranopadol. ACS Med Chem

Lett 2014, 5, 857-862. 3.

Lambert, D. G., The Nociceptin/Orphanin Receptor: a Target with Broad

4.

Chiou, L. C.; Liao, Y. Y.; Fan, P. C.; Kuo, P. H.; Wang, C. H.; Riemer, C.;

Therapeutic Potential. Nature Rev. Drug Discovery 2008, 7, 694-710.

Prinssen, E. P., Nociceptin/Orphanin FQ Peptide Receptors: Pharmacology and Clinical Implications. Curr Drug Targets 2007, 8, 117-135. 5.

Mogil, J. S.; Pasternak, G. W., The Molecular and Behavioral Pharmacology of

the Orphanin FQ/Nociceptin Peptide and Receptor Family. Pharmacol Rev 2001, 53,

381-415. 6.

Thompson, A. A.; Liu, W.; Chun, E.; Katritch, V.; Wu, H.; Vardy, E.; Huang, X.

P.; Trapella, C.; Guerrini, R.; Calo, G.; Roth, B. L.; Cherezov, V.; Stevens, R. C., Structure of the Nociceptin/Orphanin FQ Receptor in Complex with a Peptide Mimetic.

Nature 2012, 485, 395-399. 7.

Miller, R. L.; Thompson, A. A.; Trapella, C.; Guerrini, R.; Malfacini, D.; Patel,

N.; Han, G. W.; Cherezov, V.; Calo, G.; Katritch, V.; Stevens, R. C., The Importance of Ligand-Receptor Conformational Pairs in Stabilization: Spotlight on the N/OFQ G Protein-Coupled Receptor. Structure 2015, 23, 2291-2299.

8.

Monteillet-Agius, G.; Fein, J.; Anton, B.; Evans, C. J., ORL-1 and Mu Opioid

Receptor Antisera Label Different Fibers in Areas involved in Pain Processing. J

Comp Neurol 1998, 399, 373-383. 9.

Cremeans, C. M.; Gruley, E.; Kyle, D. J.; Ko, M. C., Roles Of Mu-Opioid

Receptors and Nociceptin/Orphanin FQ Peptide Receptors in Buprenorphine-Induced Physiological Responses in Primates. J Pharmacol Exp Ther 2012, 343, 72-81.

10.

Toll, L., The Use of Bifunctional NOP/Mu and NOP Receptor Selective

Compounds for the Treatment of Pain, Drug Abuse, and Psychiatric Disorders. Curr ACS Paragon Plus Environment

Page 27 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Pharm Des 2013, 19, 7451-7460. 11.

Samama, P.; Cotecchia, S.; Costa, T.; Lefkowitz, R. J., A Mutation-Induced

Activated State of the Beta 2-Adrenergic Receptor. Extending the Ternary Complex Model. J Biol Chem 1993, 268, 4625-4636. 12.

Wang, W.; Qiao, Y.; Li, Z., New Insights into Modes of GPCR Activation.

13.

Manglik, A.; Kim, T. H.; Masureel, M.; Altenbach, C.; Yang, Z.; Hilger, D.;

Trends Pharmacol Sci 2018, 39, 367-386.

Lerch, M. T.; Kobilka, T. S.; Thian, F. S.; Hubbell, W. L.; Prosser, R. S.; Kobilka, B. K., Structural Insights into the Dynamic Process of Beta2-Adrenergic Receptor Signaling. Cell 2015, 161, 1101-1111. 14.

Huang, W.; Manglik, A.; Venkatakrishnan, A. J.; Laeremans, T.; Feinberg, E. N.;

Sanborn, A. L.; Kato, H. E.; Livingston, K. E.; Thorsen, T. S.; Kling, R. C.; Granier, S.; Gmeiner, P.; Husbands, S. M.; Traynor, J. R.; Weis, W. I.; Steyaert, J.; Dror, R.

O.; Kobilka, B. K., Structural Insights into Mu-Opioid Receptor Activation. Nature 2015,

524, 315-321. 15.

Che, T.; Majumdar, S.; Zaidi, S. A.; Ondachi, P.; McCorvy, J. D.; Wang, S.;

Mosier, P. D.; Uprety, R.; Vardy, E.; Krumm, B. E.; Han, G. W.; Lee, M. Y.; Pardon, E.; Steyaert, J.; Huang, X. P.; Strachan, R. T.; Tribo, A. R.; Pasternak, G. W.; Carroll, F. I.; Stevens, R. C.; Cherezov, V.; Katritch, V.; Wacker, D.; Roth, B. L.,

Structure of the Nanobody-Stabilized Active State of the Kappa Opioid Receptor. Cell 2018, 172, 55-67. 16.

Manglik, A.; Kruse, A. C.; Kobilka, T. S.; Thian, F. S.; Mathiesen, J. M.;

Sunahara, R. K.; Pardo, L.; Weis, W. I.; Kobilka, B. K.; Granier, S., Crystal Structure

of the Mu-Opioid Receptor Bound to a Morphinan Antagonist. Nature 2012, 485, 321326. 17.

Kam, K. W.; New, D. C.-.; Wong, Y. H., Constitutive Activation of the Opioid

Receptor-Like (ORL1) Receptor by Mutation of Asn133 to Tryptophan in the Third Transmembrane Region. J. Neurochem. 2002, 83, 1461–1470.

18.

Pellissier, L. P.-.; Sallander, J.; Campillo, M.; Gaven, F.; Queffeulou, E.; Pillot,

M.; Dumuis, A.; Claeysen, S.; Bockaert, J.; Pardo, L., Conformational Toggle Switches Implicated in Basal Constitutive and Agonist Induced Activated States of 5Hydroxytryptamine-4 Receptors. Mol. Pharmacol. 2009, 75, 982–990. 19.

Mansour, A.; Taylor, L. P.; Fine, J. L.; Thompson, R. C.; Hoversten, M. T.;

Mosberg, H. I.; Watson, S. J.; Akil, H., Key Residues Defining the Mu-Opioid

Receptor Binding Pocket: a Site-Directed Mutagenesis Study. J. Neurochem. 1997, 68, ACS Paragon Plus Environment


Page 28 of 42

344–353. 20.

McAllister, S. D.; Hurst , D. P.; Barnett-Norris, J.; Lynch, D.; Reggio, P. H.;

Abood, M. E., Structural Mimicry in Class A G Protein-Coupled Receptor Rotamer Toggle Switches: the Importance of the F3.36(201)/ W6. 48(357) Interaction in Cannabinoid Cb1 Receptor Activation. . J Biol Chem. 2004, 279, 48024–48037.

21.

Pasternak, G. W.; Pan, Y. X., Mu Opioids and their Receptors: Evolution of a

22.

Schwyzer, R., ACTH: a Short Introductory Review. Ann N Y Acad Sci 1977,

Concept. Pharmacol Rev 2013, 65, 1257-1317. 297, 3-26. 23.

Portoghese, P. S., The Bivalent Ligand Approach in the Design of Highly

24.

Totrov,

25.

Pagadala, N. S.; Syed, K.; Tuszynski, J., Software for Molecular Docking: a

26.

Bermudez, M.; Mortier, J.; Rakers, C.; Sydow, D.; Wolber, G., More than a

Selective Opioid Receptor Antagonists. NIDA Res Monogr 1990, 96, 3-20. M.;

Abagyan,

R.,

Flexible

Ligand

Docking

to

Multiple

Receptor

Conformations: a Practical Alternative. Curr Opin Struct Biol 2008, 18, 178-184. Review. Biophys Rev 2017, 9, 91-102.

Look into a Crystal Ball: Protein Structure Elucidation guided by Molecular Dynamics Simulations. Drug Discov Today 2016, 21, 1799-1805. 27.

Daga, P. R.; Zaveri, N. T., Homology Modeling and Molecular Dynamics

Simulations of the Active State of the Nociceptin Receptor reveal New Insights into Agonist Binding and Activation. Proteins 2012, 80, 1948-1961.

28.

Shang, Y.; LeRouzic, V.; Schneider, S.; Bisignano, P.; Pasternak, G. W.;

Filizola, M., Mechanistic Insights into the Allosteric Modulation of Opioid Receptors by Sodium Ions. Biochemistry 2014, 53, 5140-5149. 29.

Katritch, V.; Fenalti, G.; Abola, E. E.; Roth, B. L.; Cherezov, V.; Stevens, R. C.,

Allosteric Sodium in Class A GPCR Signaling. Trends Biochem Sci 2014, 39, 233244. 30.

Della Longa, S.; Arcovito, A., "In Silico" Study of the Binding of Two Novel

31.

Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D.

Antagonists to the Nociceptin Receptor. J Comput Aided Mol Des 2018, 32, 385-400.

M.; Meng, E. C.; Ferrin, T. E., UCSF Chimera--a Visualization System for Exploratory Research and Analysis. J Comput Chem 2004, 25, 1605-1612. 32.

Berendsen, H. J. C.; van der Spoel, D.; van Drunen, R., GROMACS: a

Message-Passing

Parallel

Commun. 1995, 91, 43-56.

Molecular

Dynamics

Implementation.


Computer

Phys.

Page 29 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

33.


Hess, B.; Kutzner, C.; Lindhal, E., GROMACS 4:

Algorithms for Highly Efficient,

Load-Balanced, and Scalable Molecular Simulation. J. Chem. Theory Comput. 2008, 4, 435-447. 34.

Hornak, V., Comparison of Multiple Amber Force Fields and Development of

35.

Della Longa, S.; Arcovito, A., A Dynamic Picture of the Early Events in

Improved Protein Backbone Parameters. Proteins 2006, 65, 712-725.

Nociceptin Binding to the NOP Receptor by Metadynamics. Biophys J 2016, 111,

1203-1213. 36.

Nosé, S., A Unified Formulation of the Constant Temperature Molecular-

37.

Hoover, W. G., Canonical Dynamics: Equilibrium Phase-Space Distributions.

38.

Parrinello, M.; Rahman, A., Polymorphic Transitions in Single Crystals: a New

39.

Daura, X.; Gademann, K.; Jaun, B.; Seebach, D.; van Gunsteren, W. F.; Mark,

Dynamics Methods. J. Chem. Phys. 1984, 81, 511-519.

Phys. Rev. A 1985, 31, 1695–1697.

Molecular Dynamics Method. J. Appl. Phys. 1981, 52, 7182-7190.

A. E., Peptide Folding: when Simulation meets Experiment. Angew. Chem. Int. Ed. 1999, 38, 236-240. 40.

Tarcsay, A.; Paragi, G.; Vass, M.; Jójárt, B.; Bogár, F.; Keserű, G.-. The

Impact of Molecular Dynamics Sampling on the Performance of Virtual Screening Against GPCRs. J. Chem. Inf. Model. 2013, 53, 2990-2999.

41.

Trott, O.; Olson, A. J., AutoDock Vina: Improving the Speed and Accuracy of

Docking with a New Scoring Function, Efficient Optimization, and Multithreading. J

Comput Chem 2010, 31, 455-461. 42.

Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell,

D. S.; Olson, A. J., AutoDock4 and AutoDockTools4: Automated Docking with Selective Receptor Flexibility. J Comput Chem 2009, 30, 2785-2791. 43.

Maseras, F.; Morokuma, K., IMOMM—A New Integrated Ab-Initio Plus Molecular

Mechanics Geometry Optimization Scheme of Equilibrium Structures and TransitionStates. J. Comput. Chem. 1995, 16, 1170-1179. 44.

Svensson, M.; Humbel, S.; Froese, R. D. J.; Matsubara, T.; Sieber, S.;

Morokuma, K., ONIOM: a Multilayered Integrated MO + MM Method for Geometry Optimizations and Single Point Energy Predictions. A Test for Diels–Alder Reactions and Pt(P(t-Bu)3)2 + H2 Oxidative Addition. J. Phys. Chem. 1996, 100, 19357–19363. 45.

Neese, F., ORCA: an ab-Initio, DFT, and Semiempirical Electronic Structure

Package. University of Bonn, Germany.



46.

Page 30 of 42

Bonomi, M.; Branduardi, D.; Bussi, G.; Camilloni, C.; Provasi, D.; Raiteri, P.;

Donadio, D.; Marinelli, F.; Pietrucci, F.; Broglia, R. A.; Parrinello, M., PLUMED: a Portable Plugin for Free-Energy Calculations with Molecular Dynamics. Computer

Physics Communications 2009, 180 1961-1972. 47.

1994. 48.

Cox, T. F.; Cox, M. A., Multidimensional Scaling. Chapman and Hall: London,

Tribello, G. A.; Ceriotti, M.; Parrinello, M., Using Sketch-Map Coordinates to

Analyze and Bias Molecular Dynamics Simulations. Proc Natl Acad Sci U S A 2012, 109, 5196-5201. 49.

Garcia, A. E., Large-Amplitude Nonlinear Motions in Proteins. Phys Rev Lett

1992, 68, 2696-2699. 50.

Das, P.; Moll, M.; Stamati, H.; Kavraki, L. E.; Clementi, C., Low-Dimensional,

Free-Energy Landscapes of Protein-Folding Reactions by Nonlinear Dimensionality Reduction. Proc Natl Acad Sci U S A 2006, 103, 9885-9890.

51.

Roy, A.; Kucukural, A.; Zhang, Y., I-TASSER: a Unified Platform for Automated

52.

Biswas, B.; Mondal, S.; Singh, P. C., Combined Molecular Dynamics, Atoms in

Protein Structure and Function Prediction. Nat Protoc 2010, 5, 725-738.

Molecules, and IR Studies of the Bulk Monofluoroethanol and Bulk Ethanol To

Understand the Role of Organic Fluorine in the Hydrogen Bond Network. J Phys

Chem A 2017, 121, 1250-1260. 53.

Granier, S.; Manglik, A.; Kruse, A. C.; Kobilka, T. S.; Thian, F. S.; Weis, W. I.;

Kobilka, B. K., Structure of the Delta-Opioid Receptor Bound to Naltrindole. Nature 2012, 485, 400-404. 54.

Fenalti, G.; Giguere, P. M.; Katritch, V.; Huang, X. P.; Thompson, A. A.;

Cherezov, V.; Roth, B. L.; Stevens, R. C., Molecular Control of Delta-Opioid Receptor Signalling. Nature 2014, 506, 191-196. 55.

Yuan, S.; Filipek, S.; Palczewski, K.; Vogel, H., Activation of G-protein-coupled

Receptors Correlates with the Formation of a Continuous Internal Water Pathway. Nat

Commun 2014, 5, 4733| DOI: 10.1038/ncomms5733. 56.

Pert, C. B.; Pasternak, G.; Snyder, S. H., Opiate Agonists and Antagonists

57.

Ardati, A.; Henningsen, R. A.; Higelin, J.; Reinscheid, R. K.; Civelli, O.;

Discriminated by Receptor Binding in Brain. Science 1973, 182, 1359-1361.

Monsma, F. J., Jr., Interaction of [3h]Orphanin FQ and 125i-Tyr14-Orphanin FQ with the Orphanin FQ Receptor: Kinetics and Modulation by Cations and Guanine Nucleotides. Mol Pharmacol 1997, 51, 816-824.


Page 31 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

58.


Mahmoud, S.; Margas, W.; Trapella, C.; Calo, G.; Ruiz-Velasco, V., Modulation

of Silent and Constitutively Active Nociceptin/Orphanin FQ Receptors by Potent

Receptor Antagonists and Na+ Ions in Rat Sympathetic Neurons. Mol Pharmacol 2010, 77, 804-817.



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A)

B) orthosteric site allosteric site

Fig. 1. A) The extended ternary complex model for GPCR activation. R:receptor (inactive state); R*:receptor (active state) L:ligand (agonist/ antagonist) X: G-protein or b-arrestin. B) Structural changes between inactive (blue) and active (green) states, as observed for the µ-OR (PDB codes: 4DKL, 5C1M) ACS Paragon Plus Environment

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fluoro-indole

dimethyl 1

propyl

spiro D-prolinamide 2

phenyl

Fig. 2. 3D and 2D structure of cebranopadol (trans isomer)



B)

A) B)

C) ∂ C)

D)

48a

Page 34 of 42

∂

5a

4a

2a CBP-trans

CBP-trans

Fig. 3. A) Multi-flexible docking protocol NOP.vs.CBP (trans isomer): plot of the Nindole-CG_D3,32 distance vs. free energy (VINA score) for the NOP_”C24-ghost” (red points) and the NOP_free (green points) frame sets. Poses on a rigid receptor are marked by blue circles. A red arrow indicates the chosen pose. B) Poses chosen for trans-cebranopadol (left) and cis-cebranopadol (right). C, D)Poses chosen for a number of compound reported in the SAR study by Schunk et al. 2014 (see Table 1): 2a (blue wires), 4a (orange wires) and 48a (brown wires) (frame C); 5a (green wires) (frame D). The pose of CBP-trans is also reported, for comparison (gold sticks).


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NOP-C24

A)

D) allosteric

NOP_free

B)

E) orthosteric intermediate allosteric

NOP_CBP

C)

F) allosteric Na6875

Fig. 4. A) Cα-rmsd plot for NOP-C24 (blue), NOP_free (black) and NOP-CBP (red). B) Comparison between the calculated Ca B-factors for the 1ms trajectories of NOP-C24 (blue) and NOP_free (grey). C) Same comparison between NOP-C24 (blue) and NOP-CBP (red). 5D-F) Sodium migration observed along the MD trajectories. The distances between each one of the Na ions and the atom CG(D2,50) are plotted as points. Each ion is identified by a specific color: D) NOP-C24. At the beginning a sodium ion was positioned in its putative allosteric site where it remained fully stable for 1 µs. E) NOP_free. No sodium was initially positioned inside the receptor, but very soon one of them from the extracellular side reaches the orthosteric site, then an intermediate site and the allosteric site, showing meta-stability. F) NOP-CBP. Interestingly, in the 1 µs time scale we observed entering of the Na ion from the extracellular side even in the presence of the agonist bound to the orthosteric site of the inactive receptor.



QM/MM

Docking & MD H_N2-OD1_D130(3,32) H_N1-OD1_D130(3,32)

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2.129 2.343

H_N2-OD2_D130(3,32) H_N1-OD2_D130(3,32) H_N1-OD1_D130(3,32)

1.827 2.008 2.346

Fig. 5. 3D superimposition of the representative CBP structure after clustering of the MD trajectory (colored transparent yellow), and after QM/MM refinement (quenching) (colored gold), as described in the Methods. The observed bidentate chelation to D3,32 is clearly possible only for the CBP trans-isomer. A strong stability of the complex is confirmed by QM/MM.


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orthosteric site

allosteric site

rmsd

rmsd

A)

B)

D)

NOP-C24

E)

NOP-C24

C24

C)

NOP-CBP

F)

NOP-CBP (>800ns)

CBP Na

Fig. 6. A,D) Comparison of the Cα-rmsd plots for the orthosteric (A), and allosteric (D) sites of NOP-C24 (blue), NOP_free (black) and NOP-CBP (red). B,C) 3D superimposition of the representative C24 (B) and CBP (C) structures (gold) after rmsd-based clustering of the trajectories. E,F) 3D superimposition of the allosteric site structures along the trajectory of NOP-C24 (E) and NOP-CBP (F). The sodium ion (blue spheres) and waters (red spheres) are included in the figures. ACS Paragon Plus Environment


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CBP C24

Fig. 7. 3DFig. superimposition of the NOP-CBP (ligand colored gold), and NOP7. 3D superimposition of orthosteric the orthostericsite site of of NOP-CBP (ligand colored gold), and NOPC24 complex (PDB code: 4EA3, ligand colored yellow, residues colored C24 complex (PDB code: 4EA3, ligand colored trasparent trasparent yellow, residues colored grey). grey). 3,33 and M1343,36 3,33 , CBP cancan openopen the way flipping Whereas C24 is stacked between Y131 Whereas C24 is stacked between Y131 and M1343,36 , CBP theforway forofflipping of 6,48 which goes in close proximity of the receptor core (T1383,40; P2275,50; F2726,44). W276 6,48 3,40 5,50 W276 which goes in close proximity of the receptor core (T138 ; P227 ; F2726,44, not shown).


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W(6,48)

{ Φ, Ψ, χ1, χ2 }

Residue conformer A)

C)

sketchmap

{ X1 , X2 } 2D map

B) trp χ2 χ1 D) ψ φ

Fig. 8. A) Representative W2766,48 conformation. B) 2D histogram (free-energy style) of the W2766,48 conformations along the MD trajectory of the NOP-CBP complex, showing low density around the starting conformations of both the inactive and active state (blue, green areas) and high density around two alternative, intermediate states. C) Plot of the W2766,48 dihedrals along the NOP-CBP trajectory, showing that the two minima correspond to rotations along of the χ2 angle. D) Superimposed 2D maps of W2766,48 conformations along the trajectories of NOP-C24 (blue), NOP_free (black) and NOP-CBP (red) showing that the intermediate states are approached only in the presence of the agonist.



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A) D110(2,63)

B) F135(3,37)

D) S223(5,46)

C) N133(3,35)

E) Y319(7,53)

Fig. 9. Superimposed 2D maps of the conformations of important residues along the MD trajectories of NOP-C24 (blue), NOP_free (black) and NOP-CBP (red) showing that their activelike states are reached or approached either in the absence of the antagonist (C,D) or in the necessary presence of the agonist (A,B,E).


Page 41 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A) B) NOP-CBP Orthosteric site Hydrophobic core

(t>858ns)

Sodium site NPxxY sequence

Orthosteric site

Fig. 10. A,B) Map of the microswitches induced by CBP along the MD trajectory of the NOPCBP complex. Green: microswitches toward an expected active state. Red: microswitches towards an intermediate state. The map outlines the possible internal signalling pathway along the receptor itself, starting from the orthosteric site and going towards the intracellular side, involving residues of the hydrophobic core, the allosteric sodium site and the NPY sequence.



Inactive conformation intermediate conformation active conformation

CBP

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{ φ, ψ, χ1, χ2, χ3 }

{ X1 , X2 }

Residue conformer

2D map

M(3,36)

TOC


Microswitches for the Activation of the Nociceptin Receptor Induced by

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