Activation Dynamics of the Neurotensin G Protein-Coupled Receptor 1

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Activation dynamics of the neurotensin G protein-coupled receptor 1 Xiaojing Cong, Sebastien Fiorucci, and Jérôme Golebiowski J. Chem. Theory Comput., Just Accepted Manuscript • DOI: 10.1021/acs.jctc.8b00216 • Publication Date (Web): 02 Jul 2018 Downloaded from http://pubs.acs.org on July 2, 2018

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Journal of Chemical Theory and Computation

Activation dynamics of the neurotensin G proteincoupled receptor 1 Xiaojing Cong,* Sébastien Fiorucci, Jérôme Golebiowski* Institute of Chemistry - Nice, UMR 7272 CNRS - University Côte d'Azur, 06108 Nice cedex 2, France KEYWORDS. G protein-coupled receptor, neurotensin receptor 1, molecular dynamics, enhanced sampling, replica exchange

ABSTRACT

A replica-exchange protocol remarkably enhances the sampling of the activation dynamics of the neurotensin receptor type 1, a G protein-coupled receptor (GPCR) and important drug target. Our work highlights the dynamic communication between conformational changes of the agonist and the G protein-binding site, via contraction-oscillation of the orthosteric pocket. It also gives insights into the mechanism by which certain mutations diminish or stimulate activation. The replica-exchange protocol effectively enhances barrier crossing where standard brute-force molecular dynamics simulations fail. It is readily applicable to other GPCRs and represents a promising approach for virtual ligand screening, using the typical features of receptor activation as a benchmark.

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INTRODUCTION

G protein-coupled receptors (GPCRs) are the largest family of cell membrane receptors and the targets for over 30% of FDA-approved drugs 1. They share a conserved structure of seven transmembrane helices (TM1–TM7) which, upon activation, undergo large-scale conformational changes from the extracellular to the intracellular side, spreading over a distance of 20-30 Å 2. Activated GPCRs recruit intracellular partners (e.g. G proteins) and trigger a cascade of complex signaling events. Recent breakthroughs in GPCR X-ray crystallography have unveiled highresolution structures for over 30 GPCRs, most of which belong to the class A (or the rhodopsinlike class) 3. Five of these class A receptors have been resolved in both inactive and active states (Table S1 in Supporting Information), which exhibit common conformational rearrangements upon activation 4: i) the intracellular half of TM6 moves outward from the TM bundle and creates a cleft to accommodate G proteins (or other signaling partners), while a water channel is formed within the receptor’s hydrophobic core; ii) the so-called transmission switch residues, which connect the orthosteric ligand-binding pocket to the hydrophobic core, switch to an active configuration; and iii) the intracellular end of TM7 moves inward as TM6 moves out. Atomisticlevel understanding of GPCR activation mechanisms and pathways is highly desirable, because of the potential for applications in GPCR functional studies and pharmaceutical research. To date, few seminal molecular simulation studies

5-7

have captured such activation events, which

presumably occur on a millisecond timescale 8. Some other microsecond-timescale molecular simulations have observed partially active-like features 9-13. Consistent with analytical studies on crystal structures 2, these simulations indicate varied activation pathways involving discrete receptor conformational states. The diversity and similarity in activation pathways/mechanisms across different GPCR families remain undescribed.


2

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Here we study the neurotensin receptor type 1 (NTSR1) with all-atom molecular dynamics (MD) simulations to shed light on GPCR activation mechanisms at the atomistic level. NTSR1 is a class A GPCR that mediates most of the effects of neurotensin (NTS), a 13-amino acid peptide that acts as a neurotransmitter and hormone in the central nervous system and in the gut displays a wide range of biological activities with important roles in cancer disorders

16,17

15

14

. It

and neurological

. Several X-ray crystal structures are available for rat NTSR1 (which has 96%

sequence identity to human NTSR1 in the TM domain). These structures could only be obtained by thermostabilization of the receptor with several mutations and/or a T4 lysozyme, and cocrystallization with a fragment of NTS (Table S2). Four of these structures are considered to be in inactive-like states despite the presence of the agonist, whereas the other four are in activeintermediate states showing some of the above-mentioned active-state structural features (Table S2). No structure is yet available for the wild-type (wt) NTSR1 in antagonist-bound or apo inactive state, or in a G protein-bound fully active state. A constitutively active mutation (cam), F358A7.42 (superscript refers to the Ballesteros-Weinstein nomenclature

18

) in TM7, has been

found to induce agonist-independent activation possibly by altering the orientation of the adjacent residue W3216.48 in the conserved transmission switch 19. Nevertheless, little is known about the pathways and mechanisms of agonist-dependent or agonist-independent activation, or about the roles of mutations in modulating its activity. Here we address these issues by studying rat NTSR1 wt with and without bound NTS (apo-wt and wt-nts, respectively), the F358A7.42 mutant in apo form (apo-cam) (Fig. S1), as well as the mutant of the inactive X-ray structure (PDB ID 4BUO)

20

bound with NTS (mut-nts). The mutant contains 11 point mutations (Table

S2) that together abolishes the receptor’s response to NTS 20. Starting from the inactive state in the X-ray structure, we performed all-atom MD simulations in explicit membrane-solvent with


3


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and without enhanced sampling by replica exchange with solute scaling (REST2) MD 21. From the technical aspect, we show how the REST2 MD effectively samples GPCR conformational changes between inactive and a distinct active-like state. The brute-force MD tends however to be trapped within one of the energy minima (inactive or active-like states). Regarding the activation mechanism, the REST2 MD is consistent with the differential experimental behavior of apo-cam and wt-nts with respect to apo-wt and mut-nts

20

. In addition to existing knowledge

of class A GPCR activation, our REST2 MD reveals new dynamic features underlying the initiation of the activation: (i) both the agonist and the cam contract remarkably the orthosteric ligand-binding pocket, inducing conformational changes of the transmission switch, which trigger preorganization of the receptor intracellular side for G protein coupling; (ii) More elaborately, the agonist swings at the pocket entrance, which communicates in distance with the transmission switch by causing a smaller-scale oscillation of the pocket; (iii) Mutations present within the pocket of the X-ray structure shifts the agonist binding pose, which impairs the agonist-induced activation.

RESULTS

Differential dynamics of the four systems. During the REST2 MD, the four receptors generally maintain the initial 3-dimensional fold (Fig. S2). Major differences emerge from the intracellular halves of TM5 and TM6 in apo-cam and wt-nts, and at the extracellular half of TM7 in apo-wt (Fig. S3). A principal component analysis (PCA) captures such differential dynamics (Fig. 1A and S4). Namely, apo-wt displays in-and-out movements of TM7 on the extracellular side relative to TM3; apo-cam and wt-nts exhibit comparable back-and-forth displacements of TM5 and TM6 on the intracellular side with respect to the rest of the TM bundle; whereas mut-


4

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wt preserves the most the initial X-ray structure with small-scale fluctuations (Fig. 1A, S4 and S5). Correspondingly, apo-wt displays a remarkable broadening of the orthosteric ligand-binding pocket due to the outward movement of TM7 on the extracellular side (Fig. 1B). Apo-cam and wt-nts exhibit frequent cleft openings between TM6 and TM3 on the intracellular side due to the substantial TM6 outward movement (Fig. 1B). This is notably the most remarkable feature of class A GPCR activation. TM5 concerts with TM6 to steer away from TM3 (Fig. S6), similar to previous findings by X-ray crystallography 22 and MD studies 9 on other GPCRs.

Figure 1. Prominent differential dynamics among the four systems that display intracellular cleft opening (pink arrows) or binding pocket widening (green arrows). (A) Projection of the REST2 MD trajectories along the first eigenvector from PCA. For clarity, only the helical TM domain is shown. Arrows indicate the directions of extreme movements. The extracellular view can be found in Fig. S4. (B) Contour maps of the intracellular cleft opening (as measured by R1673.50L3036.30 Cɑ distance) and the orthosteric pocket opening (as measured by the Cɑ center-of-mass distance between residues C1423.25–R1493.32 and T3407.24–T3547.38) sampled by the four systems


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during the REST2 MD. Black dots indicate the initial structure built from the inactive crystal structure (PDB ID: 4BUO). To assess the sampling enhancement by REST2, we performed two independent runs of microsecond-timescale standard brute-force MD of apo-wt, apo-cam and wt-nts. The two runs produced rather different results, suggesting insufficient sampling. They tend to be trapped in one of the states sampled by the REST2 MD and do not systematically capture the initiation of activation (Fig. S7-S8). Therefore, the rest of the article will focus on the REST2 MD results unless otherwise specified. Apo-cam and wt-nts exhibit active-like features. During class A GPCR activation, the most remarkable conformational changes consist of an outward movement of TM6 together with an inward movement of TM7 with respect to the TM bundle. These are also seen in X-ray crystal structures of NTSR1 mutants in intermediate active-like states (Table S2). Consistently in our simulations of apo-cam and wt-nts, the intracellular-side TM3-TM6 cleft opening is accompanied by an inward movement of the conserved NPxxY motif in TM7 (Fig. 2A and S6). Although less remarkable than those in the crystal structures (fused to a T4 lysozyme), these movements result in a distinct conformational cluster that exhibits typical active-like features (Fig. 2). Namely, the hydrophobic core of the receptor located in the intracellular half of the TM bundle becomes hydrated (Fig. S6). In apo-wt, the W3216.48 side chain is mostly parallel to the membrane bilayer, whereas in apo-cam and mut-nts it is tilted due to the mutations at F3587.42, consistent with X-ray structures (Table S2) and previous findings by Krumm et al. 19. When the agonist is present in the wt orthosteric pocket, W3216.48 regularly flips between the parallel and the tilted orientations (Fig. 2B and S9). W3216.48 (together with residues F3176.44, P2495.50 and A1573.40) is part of the conserved transmission switch motif in non-olfactory class A GPCRs,


6

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which is known to change configuration upon activation. In particular, F3176.44 and P2495.50 are conserved in 91% and 77% of class A GPCRs, respectively, and they move toward each other as F3176.44 switches from the TM bundle core toward TM5 upon activation. These two residues are conserved in the neurotensin receptor family and show a similar pattern in the activeintermediate state crystal structure (PDB ID 5T04)

19

. Here, F3176.44 and P2495.50 adopt the

active-like configuration most frequently in apo-cam, but also in wt-nts (Fig. 2B and S10). In apo-wt they gradually rearrange into a distinct configuration where F3176.44 points away from P2495.50, which appears to be associated with the ligand-binding pocket width (Fig. 2B, S9 and S10). PCA-based cluster analysis shows that this typical state accounts for ~51% of the apo-wt trajectory, and is likely the dominant state of the receptor in its apo form (Fig. S11). In mut-nts, F3176.44 and P2495.50 remains mostly in the initial inactive configuration (Fig. 2B, S9 and S10). The active-like state of apo-cam and wt-nts accounts for about 84% and 28% of their trajectory frames, respectively (Fig. S11). In both receptors, this state shows nearly identical TM bundle conformations, Cɑ RMSD < 1.2 Å from the cluster averages, indicating it is a common state in the agonist- and cam-induced activation pathways.


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Figure 2. NTSR1 apo-cam and wt-nts show activation features while apo-wt and mut-nts do not. (A) Cartoon presentation and contour maps illustrating the intracellular cleft opening (R1673.50L3036.30 Cɑ distance) and the TM7 inward movements (Cɑ center-of-mass distance between residues T1563.39-R/L1673.50 and N3657.49-Y3697.53 (the NPxxY motif) in apo-cam and wt-nts, in contrast to apo-wt and mut-nts. Corresponding values in crystal structures are labeled (inactive: 4BUO, intermediate active like: 4XES and 5T04). (B) Cartoon presentation and density distributions of the orthosteric pocket width (Cɑ center-of-mass distance between residues C1423.25-R1493.32 and T3407.23-T3547.37); the transmission-switch residue W3216.48 side-chain dihedral angle; and the transmission-switch residues P2495.50-F3176.44 side-chain (center-ofmass) distance. The Cɑ atoms considered for the distance calculations are shown in balls. The above findings suggest that the F358A7.42 mutation affects the apo receptor structure in a similar fashion to NTS binding. Compared with apo-wt, the mutation and the agonist both reduce


8

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the TM3-TM7 distance in the extracellular half (Fig. 2 and S9). W3216.48 located between TM3 and TM7 becomes tilted (upward or downward), matching the narrower interhelical space (Fig. 2 and S9). W3216.48 likely activates the transmission switch, which then induces the allosteric intracellular TM6 outward movement to create a cleft. Our MD simulations demonstrate that the F358A7.42 mutation affects W3216.48 dynamics and alters the transmission switch configuration in a similar manner to NTS, confirming previous assumptions from X-ray crystallography and MD simulation studies 19. In mut-nts, however, mutations within the intracellular portion of the receptor diminish its activity

20

. Consistently, we observe no allosteric movement triggering

activation during the simulation despite the tilted W3216.48. In silico reversal mutation of apo-cam into apo-wt recovers inactive-state features. To verify the above findings and the capacity of the REST2 MD in barrier crossing between inactive-active states, we carried out a control simulation of apo-wt starting from the apo-cam active-like state (with the typical active-like features). The system is expected to transition toward the inactive state once the mutation is restored. As soon as the simulation begins, W3216.48 side chain shows a strong preference for the orientation parallel to the membrane bilayer, such as that found in the previous apo-wt simulation (Fig. 3). The orthosteric pocket width then quickly increases to recover the range seen in the previous simulation. These observations confirm that the orientation of W3216.48 is controlled by the nature of residue 3587.42 and that the opening of the orthosteric pocket is indeed characteristic of the apo-wt receptor. Later in the simulation, the receptor tends to recover inactive-state features. The intracellular portion of TM6 shifts back to the TM bundle, reducing the cleft present in the initial structure and dehydrating the hydrophobic core. The intracellular portion of TM5 moves toward TM3 and TM7 moves outward (Fig. S12). In correlation with the cleft closure, the transmission


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switch residues, P2495.50 and F3176.44, move away from each other (Fig. 3). The receptor thus slowly converges toward inactive states.

Figure 3. In silico reversal mutation of apo-cam to apo-wt recovers structural characteristics of the apo-wt and inactive-state features. Initialized in an active-like state, the receptor deactivates through loosely coupled communications between the orthosteric pocket and the intracellular transmission cleft. (A) Projection of the trajectory along the first two eigenvectors (PCs) from the PCA, demonstrating the closure of the intracellular cleft and the opening of the orthosteric pocket. (B) Time series plots of selected structural features over the course of the simulation. Frames that recover the typical values in the initial apo-wt trajectory are shaded in dark gray.


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Dynamic communication between the agonist and the transmission switch. We find that the alternation of wt-nts between inactive and active-like states is correlated with an oscillation of the agonist conformation. Namely, the NTS residue Y11NTS swings at the orthosteric pocket entrance (Fig. 4A), which correlates with the flipping of W3216.48 located 20 Å deep down (Table S3, Fig. 4 and S13). Y11NTS and W3216.48 communicate via an oscillation of the pocket width (Table S3, Fig. 4 and S13). Because Y11NTS initially binds between TM3 and TM7, when it swings out, the two helices approach each other and reorient W3216.48 in between (Fig. 4). In mut-nts, however, two mutations in direct contact with NTS shift the agonist’s C-terminus away from the TM2-TM3-TM7 cavity (Fig. 4B). This reduces the TM3-TM7 interhelical distance and hinders the pocket oscillation with the swinging Y11NTS, abolishing the agonist’s long-range effects on W3216.48 (Table S3, Fig. 4 and S13).

Figure 4. Communication between NTS and the transmission switch is impaired by mutations in mut-nts compared to wt-nts. (A) Extracellular view of the agonist residue Y11NTS swinging


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between the initial (cyan) and the alternative (orange) positions. Within the binding pocket this causes oscillation of the TM3-TM7 interhelical distance in wt-nts (illustrated in green/orange) but not in mut-nts (not shown). (B) Mutations in the mut-nts pocket shifts NTS with respect to its position in wt-nts (green in wt-nts and red in mut-nts). Mutated sites are shown in balls and sticks. For clarity, part of the receptor is not shown. Density distributions of the associated features: the Y11NTS swinging (side-chain dihedral χ1), the pocket oscillation (TM3-TM7 distance as defined in Fig. 2 caption), the shift of NTS binding pose (Cα center-of-mass distance between I12NTS–L13NTS and E1242.60–F1292.65), and the transmission switch residue W3216.48 flipping (side-chain dihedral χ2).

DISCUSSION

Overall, the REST2 MD clearly enhances barrier crossing and samples better the conformational changes of each system. It captures back-and-forth transitions between inactiveand active-like states of apo-cam and wt-nts in merely 2 µs of accumulated simulations (65 ns × 32 replicas). The brute-force MD, however, tends to be trapped in only one of these states (Fig. S14), unable to overcome the energy barrier in 2–3 µs. The active-like states show some of the typical structural features of class A GPCR activation, i.e. reconfiguration of the transmission switch, hydration of the receptor’s hydrophobic core and the opening of a cleft at the intracellular G protein-binding site. The cleft opening is less remarkable than that in the crystal structures of intermediate active-like state (Fig. 2A), in which the cleft is likely enlarged by the presence of a T4 lysozyme fused to the intracellular ends of TM5 and TM6. In the absence of G proteins, we observe modest but significant outward movements of TM6 intracellular end in apo-cam and wt-nts, accompanied by inward movements


12

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of the NPxxY motif in TM7 (Fig. 2A). These are likely to pre-organize the receptor prior to G protein coupling. Further conformational rearrangements, from such an active-like state to the fully active state, are probably an induced-fit process that is initiated by G protein coupling, as indicated by several studies 23-25. In agreement with previous work by Krumm et al.

19

, we find that in apo-cam, the F358A7.42

mutation modifies the W3216.48 orientation, similar to but independent of the effect of NTS binding. Here, we provide a more complete view of the cam- and NTS-induced activation mechanism. Both the cam and the agonist significantly reduce the orthosteric pocket width compared to apo-wt. Moreover, NTS alters the W3216.48 orientation in a long range by oscillating the pocket width. This long-range effect is abolished as mutations in the pocket of mut-nts shifts the binding pose of NTS. The contraction and oscillation of the orthosteric pocket communicate with the intracellular side through the transmission switch, resulting in nearly identical active-like states of apo-cam and wt-nts. Therefore, the two receptors likely adopt a common activation pathway via a similar active-like state, which is probably intrinsic to the receptor structure. Interestingly, site-directed mutagenesis has shown for a number of class A GPCRs that elevated agonist-independent (constitutive) activity often enhances the receptors’ response to agonists 26,27, indicating common pathways underlying the two types of activation. The control simulation of apo-wt initialized in the active-like state confirms the above findings. The receptor rapidly recovers the pocket width and the W3216.48 orientation that is observed in the putative resting state of apo-wt and gradually deactivates. The orthosteric pocket and the intracellular half appear to be loosely coupled, via the transmission switch whose role as a connector in (de)activation has been repeatedly demonstrated by MD simulations of other class A GPCRs

5-7,28

. Based on these findings we propose a model of activation (Fig. 5) triggered by


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orthosteric pocket contraction-oscillation and W3216.48 rotation, which activates the transmission switch. The transmission switch further induces conformational rearrangements of the intracellular half of the receptor. Interestingly, pocket contraction in the presence of agonist (relative to antagonist) has been seen in crystal structures of several other class A GPCRs

29-31

.

An in-situ study on the M3 muscarinic receptor has also demonstrated shorter distance between the extracellular ends of TM3 and TM7 upon agonist binding

32

. Thus, pocket contraction

associated with agonist binding is likely a common phenomenon across different GPCR families. Therefore, the activation model proposed here may be also applicable to other GPCRs. It reveals one of the possible communication mechanisms between the ligand- and G protein-binding sites.

Figure 5. Schematic view of the activation mechanism: conformational rearrangements at the orthosteric pocket propagate toward the intracellular side via the transmission switch residues, which act as connectors. This leads to TM6 outward displacement and the opening of a cleft on the intracellular side as a preorganization of the receptor to accommodate the G protein.


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Starting from a single structure of the receptor, the REST2 MD protocol unambiguously differentiates the constitutively activate mutant and the agonist-bound wt receptor, from the poorly active apo-wt form and mut-nts, by monitoring typical activation structural features as a benchmark. A similar protocol has been shown previously by one of us to effectively sample the activation of a µ-opioid receptor cam 7. To the best of our knowledge, these are the first applications of the REST2 technique on GPCR activation. In contrast, two independent runs of microsecond-timescale brute-force MD are unable to achieve the sampling. Particularly in the case of wt-nts, they fail to sample the swinging of Y11NTS and the associated pocket oscillation and W3216.48 flipping (Fig. S15). In one of the two runs, the receptor reaches the active-like state during the prolonged equilibration phase and stays trapped in this state (Fig. S7–8). Indeed, we experienced similar sampling issues with brute-force MD in a recent work on the β2-adrenergic receptor and an odorant receptor 33. Therefore, the REST2 MD protocol proves to be an effective approach to studying GPCR activation and could also find wide applications in GPCR ligand screening and design. It represents a major step forward from existing approaches, which are specifically focused on the affinity between the ligand and the receptor

34,35

. The contraction-

oscillation of the orthosteric pocket and the cleft opening upon activation could be used as structural properties to guide agonist design and might also find wide applications in other GPCRs. METHODS The initial receptor models were built based on an inactive-state crystal structure of rat NTSR1 (PDB ID 4BUO) 20. Each receptor was then embedded in explicit POPC membrane and aqueous solvent neutralized with 0.15 M Na+ and Cl- ions. All-atom MD simulations were carried out with Amber 16 36, using the Amber 99SB-ildn

37

and lipid 14 38 force fields. We performed two


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independent runs (with different initial velocities) of 1.7–2.8 µs long production brute-force MD for each system. The REST2 MD were realized with 32 replicas. The effective temperatures used for generating the REST2 scaling factors ranged from 310 K to 560 K, following a distribution calculated with the Patriksson-van der Spoel approach

39

. This choice resulted in an average exchange

probability of 23%. We first carried out 65 ns × 32 replicas MD for each of the four systems starting from the inactive state. Another 125 ns × 32 replicas were subsequently performed for apo-wt starting from an active-like state. Discarding the first 5 ns for equilibration, trajectories of the original unscaled replica (at 310 K effective temperature) were collected for analysis. Further details are provided in SI-Methods. ASSOCIATED CONTENT Supporting Information. A single PDF file containing detailed methods; information on Xray structures of NTSR1 and other selected class A GPCRs; RMSD and RMSF plots; details on the PCA analysis and PCA-based clusters; results of the brute-force MD, as well as time series plot of the properties discussed in the main text. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *To

whom

correspondence

may

be

addressed:

[email protected]

or

[email protected] Author Contributions


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X.C. and J.G. designed research; X.C. performed research; X.C., S.F. and J.G. analyzed data; and X.C., S.F. and J.G. wrote the paper. All authors have given approval to the final version of the manuscript. Funding Sources This

work

is

supported

by

the

German

Research

Foundation

(Deutsche

Forschungsgemeinschaft, DFG, grant number CO 1715/1-1 to X.C.), the French National Research Agency (Agence Nationale de la Recherche, ANR) and the US National Science Foundation (NSF) as part of the Collaborative Research in Computational Neuroscience to J.G., and by the French government, through the UCAJEDI Investments in the Future project managed by the National Research Agency (ANR) with the reference number ANR-15-IDEX01, to X.C. and J.G.. Notes The authors declare no competing financial interests. ACKNOWLEDGMENT We acknowledge PRACE for awarding us access to MareNostrum at Barcelona Supercomputing Center (BSC), Spain. ABBREVIATIONS GPCR, G protein-coupled receptor; TM, transmembrane helix; NTSR1, neurotensin receptor type 1; NTS, neurotensin; wt, wild-type; cam, constitutively active mutation; wt-nts, wild-type neurotensin receptor type 1 bound with NTS; mut-nts, thermostabilized NTSR1 mutant bound with neurotensin; REST2, replica exchange with solute scaling.


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REFERENCES (1) Santos, R.; Ursu, O.; Gaulton, A.; Bento, A. P.; Donadi, R. S.; Bologa, C. G.; Karlsson, A.; Al-Lazikani, B.; Hersey, A.; Oprea, T. I.; Overington, J. P. A comprehensive map of molecular drug targets. Nat Rev Drug Discov 2017, 16, 19. (2) Venkatakrishnan, A. J.; Deupi, X.; Lebon, G.; Heydenreich, F. M.; Flock, T.; Miljus, T.; Balaji, S.; Bouvier, M.; Veprintsev, D. B.; Tate, C. G.; Schertler, G. F.; Babu, M. M. Diverse activation pathways in class A GPCRs converge near the G-protein-coupling region. Nature 2016, 536, 484. (3) Isberg, V.; Mordalski, S.; Munk, C.; Rataj, K.; Harpsoe, K.; Hauser, A. S.; Vroling, B.; Bojarski, A. J.; Vriend, G.; Gloriam, D. E. GPCRdb: an information system for G proteincoupled receptors. Nucleic Acids Res 2016, 44, D356. (4) Tehan, B. G.; Bortolato, A.; Blaney, F. E.; Weir, M. P.; Mason, J. S. Unifying family A GPCR theories of activation. Pharmacol Ther 2014, 143, 51. (5) Miao, Y.; Nichols, S. E.; Gasper, P. M.; Metzger, V. T.; McCammon, J. A. Activation and dynamic network of the M2 muscarinic receptor. Proc Natl Acad Sci U S A 2013, 110, 10982. (6) Kohlhoff, K. J.; Shukla, D.; Lawrenz, M.; Bowman, G. R.; Konerding, D. E.; Belov, D.; Altman, R. B.; Pande, V. S. Cloud-based simulations on Google Exacycle reveal ligand modulation of GPCR activation pathways. Nat Chem 2014, 6, 15. (7) De Sena Jr, D. M.; Cong, X.; Giorgetti, A.; Kless, A.; Carloni, P. Structural heterogeneity of the µ-opioid receptor’s conformational ensemble in the apo state. Sci Rep 2017, 7, 45761.


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(8) Vilardaga, J. P.; Bunemann, M.; Krasel, C.; Castro, M.; Lohse, M. J. Measurement of the millisecond activation switch of G protein-coupled receptors in living cells. Nat Biotechnol 2003, 21, 807. (9) Li, J.; Jonsson, A. L.; Beuming, T.; Shelley, J. C.; Voth, G. A. Ligand-dependent activation and deactivation of the human adenosine A(2A) receptor. J Am Chem Soc 2013, 135, 8749. (10) Yuan, S.; Hu, Z.; Filipek, S.; Vogel, H. W246(6.48) opens a gate for a continuous intrinsic water pathway during activation of the adenosine A2A receptor. Angew Chem Int Ed Engl 2015, 54, 556. (11) Yuan, S.; Palczewski, K.; Peng, Q.; Kolinski, M.; Vogel, H.; Filipek, S. The mechanism of ligand-induced activation or inhibition of mu- and kappa-opioid receptors. Angew Chem Int Ed Engl 2015, 54, 7560. (12) de March, C. A.; Yu, Y.; Ni, M. J.; Adipietro, K. A.; Matsunami, H.; Ma, M.; Golebiowski, J. Conserved Residues Control Activation of Mammalian G Protein-Coupled Odorant Receptors. J Am Chem Soc 2015, 137, 8611. (13) Shan, J.; Khelashvili, G.; Mondal, S.; Mehler, E. L.; Weinstein, H. Ligand-dependent conformations and dynamics of the serotonin 5-HT(2A) receptor determine its activation and membrane-driven oligomerization properties. PLoS Comput Biol 2012, 8, e1002473. (14) Carraway, R.; Leeman, S. E. The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami. J Biol Chem 1973, 248, 6854.


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Page 20 of 29

(15) Morgat, C.; Mishra, A. K.; Varshney, R.; Allard, M.; Fernandez, P.; Hindie, E. Targeting neuropeptide receptors for cancer imaging and therapy: perspectives with bombesin, neurotensin, and neuropeptide-Y receptors. J Nucl Med 2014, 55, 1650. (16) Griebel, G.; Holsboer, F. Neuropeptide receptor ligands as drugs for psychiatric diseases: the end of the beginning? Nat Rev Drug Discov 2012, 11, 462. (17) Schimpff, R. M.; Avard, C.; Fenelon, G.; Lhiaubet, A. M.; Tenneze, L.; Vidailhet, M.; Rostene, W. Increased plasma neurotensin concentrations in patients with Parkinson's disease. J Neurol Neurosurg Psychiatry 2001, 70, 784. (18) Ballesteros, J. A.; Weinstein, H. Integrated methods for the construction of threedimensional models and computational probing of structure-function relations in G proteincoupled receptors. Methods Neurosci 1995, 25, 366. (19) Krumm, B. E.; Lee, S.; Bhattacharya, S.; Botos, I.; White, C. F.; Du, H.; Vaidehi, N.; Grisshammer, R. Structure and dynamics of a constitutively active neurotensin receptor. Sci Rep 2016, 6, 38564. (20) Egloff, P.; Hillenbrand, M.; Klenk, C.; Batyuk, A.; Heine, P.; Balada, S.; Schlinkmann, K. M.; Scott, D. J.; Schutz, M.; Pluckthun, A. Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli. Proc Natl Acad Sci U S A 2014, 111, E655. (21) Wang, L.; Friesner, R. A.; Berne, B. J. Replica Exchange with Solute Scaling: A More Efficient Version of Replica Exchange with Solute Tempering (REST2). J Phys Chem B 2011, 115, 9431.


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Page 21 of 29 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


(22) Rasmussen, S. G.; DeVree, B. T.; Zou, Y.; Kruse, A. C.; Chung, K. Y.; Kobilka, T. S.; Thian, F. S.; Chae, P. S.; Pardon, E.; Calinski, D.; Mathiesen, J. M.; Shah, S. T.; Lyons, J. A.; Caffrey, M.; Gellman, S. H.; Steyaert, J.; Skiniotis, G.; Weis, W. I.; Sunahara, R. K.; Kobilka, B. K. Crystal structure of the beta2 adrenergic receptor-Gs protein complex. Nature 2011, 477, 549. (23) Sounier, R.; Mas, C.; Steyaert, J.; Laeremans, T.; Manglik, A.; Huang, W.; Kobilka, B. K.; Demene, H.; Granier, S. Propagation of conformational changes during mu-opioid receptor activation. Nature 2015, 524, 375. (24) West, G. M.; Chien, E. Y.; Katritch, V.; Gatchalian, J.; Chalmers, M. J.; Stevens, R. C.; Griffin, P. R. Ligand-dependent perturbation of the conformational ensemble for the GPCR beta2 adrenergic receptor revealed by HDX. Structure 2011, 19, 1424. (25) Sungkaworn, T.; Jobin, M. L.; Burnecki, K.; Weron, A.; Lohse, M. J.; Calebiro, D. Single-molecule imaging reveals receptor-G protein interactions at cell surface hot spots. Nature 2017, 550, 543. (26) Okude, J.; Ueda, T.; Kofuku, Y.; Sato, M.; Nobuyama, N.; Kondo, K.; Shiraishi, Y.; Mizumura, T.; Onishi, K.; Natsume, M.; Maeda, M.; Tsujishita, H.; Kuranaga, T.; Inoue, M.; Shimada, I. Identification of a Conformational Equilibrium That Determines the Efficacy and Functional Selectivity of the µ-Opioid Receptor. Angew Chem Int Ed 2015, 54, 15771. (27) Yu, Y.; de March, C. A.; Ni, M. J.; Adipietro, K. A.; Golebiowski, J.; Matsunami, H.; Ma, M. Responsiveness of G protein-coupled odorant receptors is partially attributed to the activation mechanism. Proc Natl Acad Sci U S A 2015, 112, 14966.


21


Page 22 of 29

(28) Dror, R. O.; Arlow, D. H.; Maragakis, P.; Mildorf, T. J.; Pan, A. C.; Xu, H.; Borhani, D. W.; Shaw, D. E. Activation mechanism of the beta2-adrenergic receptor. Proc Natl Acad Sci U S A 2011, 108, 18684. (29) Hua, T.; Vemuri, K.; Nikas, S. P.; Laprairie, R. B.; Wu, Y.; Qu, L.; Pu, M.; Korde, A.; Jiang, S.; Ho, J. H.; Han, G. W.; Ding, K.; Li, X.; Liu, H.; Hanson, M. A.; Zhao, S.; Bohn, L. M.; Makriyannis, A.; Stevens, R. C.; Liu, Z. J. Crystal structures of agonist-bound human cannabinoid receptor CB1. Nature 2017, 547, 468. (30) Katritch, V.; Abagyan, R. GPCR agonist binding revealed by modeling and crystallography. Trends Pharmacol Sci 2011, 32, 637. (31) Warne, T.; Moukhametzianov, R.; Baker, J. G.; Nehme, R.; Edwards, P. C.; Leslie, A. G.; Schertler, G. F.; Tate, C. G. The structural basis for agonist and partial agonist action on a beta(1)-adrenergic receptor. Nature 2011, 469, 241. (32) Han, S. J.; Hamdan, F. F.; Kim, S. K.; Jacobson, K. A.; Bloodworth, L. M.; Li, B.; Wess, J. Identification of an agonist-induced conformational change occurring adjacent to the ligandbinding pocket of the M(3) muscarinic acetylcholine receptor. J Biol Chem 2005, 280, 34849. (33) de March, C. A.; Topin, J.; Bruguera, E.; Novikov, G.; Ikegami, K.; Matsunami, H.; Golebiowski, J. Odorant Receptor 7D4 Activation Dynamics. Angew Chem Int Ed Engl 2018, 57, 4554. (34) Shoichet, B. K.; Kobilka, B. K. Structure-based drug screening for G-protein-coupled receptors. Trends Pharmacol Sci 2012, 33, 268.


22

Page 23 of 29 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


(35) Andrews, S. P.; Brown, G. A.; Christopher, J. A. Structure-based and fragment-based GPCR drug discovery. ChemMedChem 2014, 9, 256. (36) D.A. Case; D.S. Cerutti; T.E. Cheatham, I.; T.A. Darden; R.E. Duke; T.J. Giese; H. Gohlke; A.W. Goetz; D. Greene; N. Homeyer; S. Izadi; A. Kovalenko; T.S. Lee; S. LeGrand; P. Li, C.; Lin, J. L., T. Luchko, R. Luo, D. Mermelstein, K.M. Merz, G. Monard, H. Nguyen, I. Omelyan, A. Onufriev, F. Pan, R. Qi, D.R. Roe, A. Roitberg, C. Sagui, C.L. Simmerling, W.M. Botello-Smith, J. Swails, R.C. Walker, J. Wang, R.M. Wolf, X. Wu, L. Xiao, D.M. York, P.A. Kollman; University of California: San Francisco, USA, 2017. (37) Lindorff-Larsen, K.; Piana, S.; Palmo, K.; Maragakis, P.; Klepeis, J. L.; Dror, R. O.; Shaw, D. E. Improved side-chain torsion potentials for the Amber ff99SB protein force field. Proteins 2010, 78, 1950. (38) Dickson, C. J.; Madej, B. D.; Skjevik, A. A.; Betz, R. M.; Teigen, K.; Gould, I. R.; Walker, R. C. Lipid14: The Amber Lipid Force Field. J Chem Theory Comput 2014, 10, 865. (39) Patriksson, A.; van der Spoel, D. A temperature predictor for parallel tempering simulations. Phys Chem Chem Phys 2008, 10, 2073.


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For Table of Contents use only

Activation dynamics of the neurotensin G proteincoupled receptor 1 Xiaojing Cong,* Sébastien Fiorucci, Jérôme Golebiowski*


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Figure 1. Prominent differential dynamics among the four systems that display intracellular cleft opening (pink arrows) or binding pocket widening (green arrows). 490x237mm (96 x 96 DPI)



Figure 2. NTSR1 apo-cam and wt-nts show activation features while apo-wt and mut-nts do not. 412x265mm (96 x 96 DPI)


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Figure 3. In silico reversal mutation of apo-cam to apo-wt recovers structural characteristics of the apo-wt and inactive-state features. 343x297mm (96 x 96 DPI)



Figure 4. Communication between NTS and the transmission switch is impaired by mutations in mut-nts compared to wt-nts. 359x225mm (96 x 96 DPI)


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Figure 5. Schematic view of the activation mechanism: conformational rearrangements at the orthosteric pocket propagate toward the intracellular side via the transmission switch residues, which act as connectors. 308x314mm (96 x 96 DPI)


Activation Dynamics of the Neurotensin G Protein-Coupled Receptor 1

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