Allosteric modulation mechanism of the mGluR5 transmembrane domain

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Computational Biochemistry 5

Allosteric modulation mechanism of the mGluR transmembrane domain Xiaojing Cong, Jean-Baptiste Chéron, Jérôme Golebiowski, Serge Antonczak, and Sebastien Fiorucci J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.9b00045 • Publication Date (Web): 26 Apr 2019 Downloaded from http://pubs.acs.org on April 27, 2019

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Journal of Chemical Information and Modeling

Allosteric modulation mechanism of the mGluR5 transmembrane domain Xiaojing Cong,1* Jean-Baptiste Chéron,1 Jérôme Golebiowski,1,2 Serge Antonczak1 and Sébastien Fiorucci1*

1

Université Côte d’Azur, CNRS, Institut de Chimie de Nice UMR7272, Nice 06108, France

2 Department

of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology, Daegu 711-873, South Korea

ABSTRACT

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor type 5 (mGluR5), a prototypical class C G protein-coupled receptor (GPCR), have shown therapeutic potentials for various neurological disorders. Understanding the allosteric

1 ACS Paragon Plus Environment

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activation mechanism is essential for the rational design of mGluR5 PAMs. We studied the actions of positive and negative allosteric modulators within the transmembrane domain of mGluR5, using enhance-sampling all-atom molecular dynamics simulations. We found dual binding modes of the PAM, associated with distinct shapes of the allosteric pocket. The negative allosteric modulators, in contrast, showed only one binding mode. The simulations revealed the mechanism by which the PAM activated the receptor, in the absence of orthosteric agonist (the so-called allosteric agonism). The mechanism relied on dynamic communications between amino-acid motifs that are highly conserved across class C GPCRs. The findings may guide structure-based design and virtual screening of allosteric modulators for mGluR5 as well as for other class C GPCRs.

INTRODUCTION

The metabotropic glutamate receptors (mGluRs) regulate synaptic activities and are promising therapeutic targets for various neurological and psychiatric disorders 1. This family of eight receptors (mGluR1mGluR8) are class C G protein-coupled receptors 2 ACS Paragon Plus Environment

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(GPCRs) that commonly consist of an extracellular ligand-binding domain (ECD) and a heptahelical transmembrane domain (7TM) 2. The ECD houses the orthosteric site for natural ligands, whereas the 7TM contains a binding pocket for allosteric modulators (AM) and is responsible for G protein activation 2. Because allosteric sites are less conserved than orthosteric sites within the same receptor family, AMs often offer better subtype selectivity and functional fine tuning than orthosteric ligands. Indeed, allosteric modulation of mGluRs has emerged as a very promising field for drug development 3. In this context, the 7TM is key to the rational design of AMs for therapeutic purposes. mGluRs are strict constitutive dimers where agonist binding to the orthosteric site triggers conformational rearrangements of the dimeric ECD, which induce activation of the 7TM (Fig. 1A)

4.

The ECD-7TM cooperativity requires dimers since mGluR

monomers are not activated by orthosteric agonists 4. However, monomeric mGluRs are also found in human brain samples and the monomer-dimer ratio changes under certain pathologic conditions 5. Monomers can be activated directly by positive AMs (PAMs) binding within the 7TM (allosteric agonism) 4. The allosteric agonism does not require the ECD because truncated mGluR 7TM monomers can activate G proteins upon PAM 3 ACS Paragon Plus Environment


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binding 4, 6. They also exhibit constitutive activities that can be inhibited by negative AMs (NAMs)

4, 6.

Therefore, truncated mGluR 7TMs behave like class A GPCRs and may

serve as excellent models for investigating the agonistic part of the allosteric modulation mechanism 4, 6.

Figure 1. Structure of mGluR5. (A) Schematic view of inactive mGluR5 dimer. The ECD contains the orthosteric site (green) and the 7TM houses an AM-binding pocket (red). (B) X-ray crystal structure of the 7TM of a thermostabilized mGluR5 bound with a NAM (m-MPEP, in ball-and-stick) (PDB ID: 6FFI 7). For clarity, the N-terminal fragment and the T4-lysozyme are not shown. At the conserved triad (Y6593.44, T7816.46 and S8097.39), a water molecule mediates a H-bond network (blue dashed lines) with the


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NAM. On the cytoplasmic side, K6653.50, E7706.35 and K8217.51 form ionic locks between TM3, TM6 and TM7.

The full-length dimeric mGluR5 structures have been resolved by high-resolution cryoEM in both inactive and active states 8. However, no AMs are present within the 7TM. Xray crystal structures are available for the 7TM of mGluR5

7, 9, 10

and mGluR1

11

bound

with NAMs. The NAM-binding site comprises a shallower sub-pocket that correspond to the orthosteric pocket in class A GPCRs and a deeper sub-pocket that overlaps with the allosteric Na+-binding site in the class A. At the 7TM core underneath the NAM, three residues (Y3.44, T6.46 and S7.39, superscripts indicating the nomenclature proposed by Pin

et al.

12)

coordinate a water molecule via a hydrogen-bond (H-bond) network (Fig. 1B).

The triad is conserved in the mGluR family and may play similar roles to the so-called “transmission switch” in class A GPCRs, which connects the ligand-binding pocket to the receptor intracellular half

13.

On the intracellular side, two ionic locks between

residues K3.50, E6.35 and K7.51 are conserved in mGluRs as well as the homologous GABAB receptors within the class C GPCR family (Fig. 1B)

14.

The K3.50-E6.35 ionic lock,



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in particular, is suggested to stabilize the 7TM in inactive state, like the TM3-TM6 ionic lock conserved in many class A GPCRs

14, 15.

How PAMs bind the 7TM and promote

activation is still unknown. Site-directed mutagenesis data suggest two possible binding modes for mGluR4 PAMs, corresponding to the two overlapping sub-pockets occupied by the NAMs

16.

The shallower pocket is likely responsible for allosteric agonism (the

direct agonist activity of PAMs), whereas the deeper one is possibly involved in the cooperativity with the ECD 16. To gain better understanding of the binding modes and the actions of PAMs, we performed molecular dynamics (MD) simulations of the truncated mGluR5 7TM in apo form, in complex with 3,3′-difluorobenzaldazine (DFB, PAM) and 2-methyl-6-(3methoxyphenyl)-ethynyl pyridine (m-MPEP, the co-crystalized NAM in PDB ID: 6FFI 7). In addition, we studied the role of the F7886.53A mutation in the DFB-bound case, because the PAM becomes a NAM for the mutated receptor in vitro

17.

We studied the

four cases (Table 1) with an enhanced-sampling MD protocol (see Methods), which had proven to efficiently sample the activation dynamics of class A GPCRs

18-20.

The

simulations revealed class A-like activation mechanism of the PAM-bound 7TM and 6 ACS Paragon Plus Environment

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dual binding modes of the PAM. This study provided a dynamic insightful view of the action of DFB as a PAM for the 7TM wild type (wt) and as a NAM for the F7886.53A mutant (mut). Table 1. Simulation systems and chemical structures of the ligands.

Denotatio n

Receptor

Ligand type Ligand structure

apo-wt

wt-m-MPEP

wt-DFB

wt

wt 7TM bound with

wt 7TM bound with

7TM

m-MPEP

DFB

NA a

NAM

PAM

mut-DFB F7886.53A mutant of 7TM bound with DFB NAM b

NA

a

NA: not applicable

b

DFB is a PAM for the wt and a NAM for mut

17.

See Fig. 1B for the location of

F7886.53.

RESULTS AND DISCUSSION

PAM induced class A-like activation features



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The PAM-bound receptor (wt-DFB) exhibited conformational changes that resembled typical features of class A GPCR activation

21.

Namely, at the intracellular G protein-

coupling site the TM3-TM6 ionic lock was destabilized (Table 2) and TM6 moved outward from the 7TM bundle (Fig. 2). These clearly differentiated the PAM-bound receptor from the other three systems, which remained in the inactive state during the same simulation course. Destabilization of the TM3-TM6 ionic lock was also seen in a previous study of PAM-bound mGluR4

15.

In class A GPCRs, agonist binding is known

to generate an active-like state featuring TM6 outward movements on the intracellular side. This leads to the coupling of G proteins that induce and stabilize more drastic TM6 outward displacements

21-24.

The TM6 outward movement and interruption of the TM3-

TM6 ionic lock have been the principal benchmarks for monitoring class A GPCR activation 18-20, 25, 26. The class A-like features observed here in the PAM-bound receptor point to intrinsic efficacy of the PAM to activate the receptor independently of orthosteric agonists. This phenomenon, known as allosteric agonism, has been observed in both full-length and truncated mGluRs

6, 16.

During the preparation of this manuscript, high-

resolution cryo-EM structures of full-length mGluR5 dimers were published, in apo state 8 ACS Paragon Plus Environment

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and in an agonist-bound state (PDB IDs 6N52 and 6N51, respectively) 8. However, the 7TM domain of both states turned out nearly identical to the NAM-bound crystal structures of truncated mGluR5 7TM

7, 9, 10.

The authors suggested that the orthosteric

agonist was unable to stabilize an active conformation within the 7TM, and the structure of an active 7TM domain remains unknown so far 8.

Figure 2. PAM destabilized the TM3-TM6 ionic lock and induced TM6 outward movements on the intracellular side, in contrast to the other three systems. (Top)



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Superimposition of representative conformations of wt-DFB (red) and wt-m-MPEP (gray), illustrating the outward TM6 movement of the former. For clarity, only the TM region (in ribbons) and the ionic-lock residues (in sticks) are shown. (Bottom) Probability density maps of the Cα distances between the ionic-lock residues sampled during the simulations of each system. We consider the K6653.50-E7706.35 Cα distance > 12 Å (red dashed lines) as “active-like”. Magenta triangle indicates the values in the cryo-EM structures of mGluR5 dimers (PDB IDs 6N51 and 6N52) 8.

Table 2. Lifetimea of the ionic locks in the four simulation systems. PAM destabilized the TM3-TM6 ionic lock but not the TM6-TM7 one.

wt-m-MPEP apo-wt wt-DFB mut-DFB K6653.50-E7706.35 51.4%

62.6%

28.1%

63.6%

E7706.35-K8217.51 99.5%

88.8%

94.7%

96.6%

a

Percentage of the total simulation time, calculated with a distance cutoff of 5

Å between N-O atom pairs of the charged functional groups.

PAM exhibits two binding modes


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The initial binding mode of DFB obtained from docking was very similar to that of mMPEP in the crystal structure (Fig. S1). During the simulations, wt-DFB exhibited two binding modes of the PAM, each associated with a distinct cluster of the AM-binding pocket conformation (Fig. 3, S2, Movie S1 and S2). In wt-m-MPEP and mut-DFB, however, only one binding mode was observed for the NAMs (Fig. 3A and B). Correspondingly, the NAMs restricted the pocket conformation near the initial crystal structure (Fig. 3C). In contrast, apo-wt without any ligand displayed remarkable changes of the pocket shape during the same simulation course (Fig. 3C). The changes were mostly due to concerted movements of TM6 and TM7, as revealed by principal component analysis (PCA) (Fig. 3D). Comparing the pocket conformations in apo-wt,

wt-DFB and wt-m-MPEP (Fig. 3C), it is plausible that binding of the ligands involves both conformational selection and induced fit. This is because the conformations in the presence of the ligands overlapped significantly with pre-existing conformations in the ligand-free receptor. Indeed, it has been suggested for other GPCRs that ligand binding and receptor activation may require a dynamic equilibrium of both conformational selection and induced fit 27, 28. 11 ACS Paragon Plus Environment


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The deeper binding mode of the PAM in wt-DFB resembled that of m-MPEP, in which the upper ring of the ligands formed π-π stacking and edge-to-face interactions with W7856.50 and F7886.53, respectively (Fig. 3A). However, the PAM was positioned closer to TM5 than the NAM, which altered remarkably the pocket conformation: TM6 tilted outward and TM7 moved inward (Fig. 3). The water mediated H-bond network at the conserved triad was weakened as the lower ring of DFB inserted between Y6593.44 and S8097.39. The shallower binding mode of DFB in wt-DFB was up shifted by about 3 Å (Fig. 3B) and was more populated during the simulation (Fig. 3C). It exhibited several distinct features: i) F7886.53 inserted between DFB and TM7, forming π-π stacking with the former while the latter moved outward; ii) W7856.50 adopted a “swung-out” rotameric state, letting in water molecules that H-bonded with W7856.50 and DFB; and iii) the Hbond network of the triad was altered as T7816.46 moved away from S8097.39 to form a new H-bond with Y6593.44 via its backbone (Fig. 3A).


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Figure 3. Ligand-binding modes and pocket conformations sampled in the simulations. (A) Binding modes of the ligands (ball-and-stick) and key interacting residues (sticks). Blue dashed lines indicate hydrogen bonds. (B) Probability density distributions of the depth of the ligands inside the pocket sampled in the three systems. The depth was



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measured by the center-of-mass distance between the ligand and the Cα atoms of the triad residues. Magenta dashed line indicates the value in the initial crystal structure. (C) Probability density maps of the pocket geometry sampled in the four simulation systems. The geometry was represented by the Cα center-of-mass distances between TM3 (residues I6423.27 I6493.36) and TM6 (residues W7856.50 G7946.59), and between TM5 (residues N7375.42

L7445.49) and TM7 (residues I7997.27

V8067.36). Magenta

triangle indicates the values in the initial crystal structure bound with m-MPEP (PDB ID: 6FFI 7). (D) Pocket movements of apo-wt from the extracellular viewpoint, illustrated as projection of the simulation trajectory along the first eigenvector from PCA.

Mechanism of the allosteric agonism The alternation of the PAM binding mode is consistent with the notion that mGluR PAMs may bind in two distinct modes within the 7TM: while the deeper one is possibly involved in the cooperativity with the ECD, the shallower one is likely responsible for the intrinsic efficacy (or allosteric agonism)

16.

To investigate the mechanism of allosteric

agonism, we analyzed the differential conformational dynamics of wt-DFB versus wt-m-


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MPEP and mut-DFB. We found that in the deeper binding mode, F7886.53 and M8027.32 formed a “lid” above the PAM. As the PAM shifted up to the shallower mode, F7886.53 rotated aside to a unique position between TM6 and TM7, underneath M8027.32 (Fig. 3A). The F7886.53 rotation prompted notable TM6 displacements with respect to TM7 (Movies S1 and S2), which altered the conformation and the H-bond network of the triad (Fig. 3A). Reconfiguration of the triad induced outward displacements of TM6 on the intracellular side. The rotation of F7886.53 was strongly correlated with the movements of the PAM, TM6 and the triad (Table 3 and Fig. S2). These were loosely coupled with the TM6 outward displacements on the intracellular side (Table 3 and Fig. S2). The results suggest a key role of F7886.53 in switching the PAM binding modes and in the allosteric agonism. The action of F7886.53 required M8027.32, consistent with the in vitro finding that point mutations F7886.53A and M8027.32T both abolished DFB’s PAM effect and turned the ligand into a NAM

17.

The simulations of mut-DFB verified the above

mechanism: as the F7886.53A mutation removed the “lid”, only one binding mode was observed (Fig. 3 A and B). The binding mode resembled the shallower one in wt-DFB, in terms of the ligand position, the orientation of W7856.50, A7886.53 and M8027.32, as 15 ACS Paragon Plus Environment


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well as the H-bond pattern (Fig. 3A). However, the A7886.53 side chain in mut-DFB could not adequately displace TM6 to reconfigure the triad like in wt-DFB (Fig. 3A), and the TM3-TM6 ionic lock remained intact (Table 2).

Table 3. Spearman’s correlation testa on the distinct conformational changes in wt-DFB. Listed here are the r values, while all the P-values are < 2.2e-16 unless otherwise noted in parenthesis.

r

Pocket

Pocket

width:

width:

TM3-TM6

TM5-TM7

DFB

F7886.53

binding orientation modeb

c

W7856.5 0

Triad

dihedral

configurationd

1

Pocket width: TM5- -0.69 TM7 DFB binding mode F7886.53 orientation W7856.50 dihedral 1

-0.32

0.51

0.64

-0.75

-0.72

0.56

-0.44

-0.33

0.52


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Triad configuratio

0.64

-0.71

-0.30

0.31

-0.72

0.81

0.51

n Intracellular side

TM6

movements

0.03 (0.463)

-0.22

-0.006

-0.20

(0.851)

(2.31e-9)

e

a Tested b

with a sample size of 850 simulation frames.

Measured by the center-of-mass distance between the ligand and the triad Cα

atoms. c

Distance between F7886.53 side chain and V8067.36 Cα as a measure of F7886.53

movements upon alternation of the PAM binding mode. d

Upon interruption of the H-bond network at the triad, T7816.46 moved toward Y6593.44

to form a new H-bond with the latter, here measured by the T7816.46- Y6593.44 Cα distance. e

Measured by the E7706.35-K6653.50 Cα distance.

Overall, the findings attribute the shallower binding mode to the allosteric agonism. Based on these, we propose an activation mechanism similar to class A GPCRs: the extracellular and the intracellular portions of the 7TM are loosely coupled through a conserved “connector” region in the 7TM core as a pivot (Fig. 4)

18-20, 25, 26.

F7886.53 is

conserved in all the eight mGluRs while M8027.32 is not (T in mGluR1 and L in mGluR4



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and mGluR6-8). Therefore, the role of F7886.53 is likely conserved within the mGluR family whereas the position 7.32 may be explored for subtype selectivity in mGluR PAM design.

Figure 4. Proposed scheme of allosteric agonism of mGluR5 7TM.

AMs differentially alter correlated motions within the 7TM The above analyses revealed the roles of conserved residues in the allosteric agonism. Nevertheless, GPCR activation is known to involve large-scale, long-range collective motions. To shed light on these motions and the effects of the AMs therein, we performed network analysis of the dynamic correlations during the simulations. The


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four systems exhibited different subnetworks (or clusters of residues), in which the nodes coupled more strongly with each other than with those in the other clusters (Fig. 5). The results, consistent with the correlated motions discussed above (Table 3), provide a more comprehensive view of the allosteric modulation pathways from the extracellular to the intracellular side (Fig. 5).

Figure 5. Cartoon presentation of the four simulation systems colored by subnetworks. The AMs are shown in balls. Compared to apo-wt, the AMs generally enhanced the TM6-TM7 coupling above the AMs (blue) and decoupled the lower part of TM6 from the upper end. In wt-DFB, the PAM showed strong coupling to the TM6-TM7 upper ends, reflecting its concerted motions with F7886.53 and M8027.32. In mut-DFB, however, DFB belonged to the subnetwork of TM3, TM4 and TM5 (orange). In wt-m-MPEP, the NAM



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connected TM2, TM3 and TM7 in the middle (magenta), while the lower part of TM6 was strongly coupled to TM3 and TM5 (cyan), reflecting restricted TM6 movements.

The extra- and intra-cellular loops (ECL1

ECL3 and ICL1

ICL3) also displayed

differential dynamics in the four systems. Namely, in wt-DFB and mut-DFB, the loops were highly flexible; whereas in wt-m-MPEP, they displayed stronger coupling to the TMs (Fig. 5), likely owing to the overall receptor stability (Fig. S4). Particularly in wt-

DFB, the TM6 movements reduced the overall ICL1-ICL3 interactions (Table S1). However, it is difficult to identify specific contributions of the loops to the 7TM activation. W7856.50 as a gatekeeper of water flux Water has been shown to mediate the interactions of NAMs with the mGluR5 7TM in crystal structures

7, 9, 10.

In particular, these structures conserve the water-binding site in

the center of the triad residues. In class A GPCRs, water networks display conserved patterns and are generally believed to contribute to the receptors’ structural stability and activation

29.

Therefore, we examined the water dynamics within the 7TM during the

simulations. We found that while the upper part of the AM-pocket (above the ligand)


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was highly hydrated in all the four systems, hydration of the lower part till the triad was associated with the rotameric state of W7856.50 (Fig. S3). In wt-m-MPEP, W7856.50 largely maintained the initial π-π stacking with the NAM, preserving the crystal water at the triad (46% water occupancy during the simulation). In mut-DFB, the swung-out position of W7856.50 hydrated the DFB-occupied pocket and enabled rapid water exchange with the triad site (22% water occupancy at the triad). In wt-DFB, W7856.50 swung in-and-out and the triad site collapsed in the shallower PAM-binding mode, resulting in partial AM-pocket hydration and only 16% of water occupancy at the triad. In

apo-wt, W7856.50 was highly dynamic and the AM-pocket was constantly hydrated till the triad site. The water-mediated AM-7TM interactions can be appreciated in the simulation snapshots included in Supplementary Information (bulk water, ions and membrane lipids are removed for clarity). We did not observe water flux through the 7TM bundle. However, in wt-DFB, the intracellular half was slightly more hydrated than in the other systems as TM6 moved outward (Fig. S3). The location of the water-binding site at the triad is comparable to the conserved allosteric Na+-binding site in non-olfactory class A GPCRs. We speculate that water 21 ACS Paragon Plus Environment


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occupancy at this site may stabilize inactive states of the 7TM, similar to the effect of Na+ ions on class A GPCRs. W7856.50 is highly conserve in class C GPCRs and it corresponds to W6.48 (superscript refers to the Ballesteros-Weinstein nomenclature 30) in the class A. W6.48 is conserved in 78% of the non-olfactory class A receptors and acts as a water gatekeeper

31.

Therefore, W6.50 may have a similar role in class C GPCRs in

modulating the 7TM stability and activation.

CONCLUSIONS

This study revealed dual binding modes of an mGluR5 PAM. In its shallower binding mode, the PAM could activate the receptor through a mechanism similar to class A GPCRs. The mechanism might be conserved in other class C GPCRs, as it involves several highly conserved amino-acid motifs. The deeper PAM binding mode might be involved in cooperativity with the ECD or dimer conformations, the mechanism of which is however beyond the scope of the current study. While DFB has exhibited agonist activity for the truncated mGluR5 7TM in vitro 6, our findings suggest that it may be a partial agonist for 7TM since only one of its two binding modes could trigger activation. 22 ACS Paragon Plus Environment

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The F7886.53A mutation in the 7TM resulted in only one binding mode of DFB in the mutant, which stabilized the inactive state. This explains why DFB acts as a NAM for the mutant receptor in vitro. Similarly, m-MPEP also showed one binding mode that stabilized the inactive state. The findings may guide rational design of selective mGluR5 AMs as new therapeutic agents. The REST2 MD unambiguously differentiated the PAM- and NAM-bound receptors. Using the activation features as benchmark, the REST2 MD protocol could be used to classify mGluR PAMs and NAMs from the output of standard virtual screening for binders. Moreover, it could provide insights into the actions of the identified PAMs versus NAMs, to guide the optimization of the hits.

METHODS

The initial receptor 7TM models were constructed based on the X-ray crystal structure of a thermostabilized human mGluR5 7TM in complex with m-MPEP (PDB ID 6FFI) 7. The protonation state of titrable residues were predicted at pH 6.5 using the H++ server (Gordon et al., 2005). DFB was docked to the m-MPEP-binding pocket in wt 7TM using Autodock Vina 32. An 18×18×22 Å3 grid box was centered at the center-of-mass position 23 ACS Paragon Plus Environment


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of m-MPEP, encompassing the residues within 6 Å of m-MPEP. Six binding-site residues (S6583.43, L7445.44, T7816.46, F7886.53, W7856.50 and M8027.32) were set flexible, which had been identified previously by site-directed mutagenesis to contribute to DFB binding

17.

Other

parameters were kept at their default values. The top-ranked cluster (vina score -9.2 kcal•mol-1, 1.8 kcal•mol-1 lower than the 2nd cluster) turned out to be very similar to the binding mode proposed in Ref.

17.

Therefore, it was selected as the initial binding mode for both wt-DFB and

mut-DFB. The receptor systems were then embedded in explicit POPC membrane and aqueous solvent neutralized with Cl- ions. Effective point charges of the ligands were obtained by RESP fitting

33

of the electrostatic potentials calculated with the HF/6-31G*

basis set using Gaussian 09

34.

The Amber 99SB-ildn

35,

lipid 14

36

and GAFF

37

force

fields were used for the proteins, the lipids and the ligands, respectively. The TIP3P

38

and the Joung-Cheatham 39 models were used for the water and the ions, respectively. After energy minimization, all-atom MD simulations were carried out using Gromacs 5.1 patched with the PLUMED 2.3 plugin

40.

Each system was gradually heated to 310

K and pre-equilibrated during 10 ns of brute-force MD in the NPT-ensemble (see SIMethods for details). The replica exchange with solute scaling (REST2)

41

technique

was then employed to enhance the sampling with 48 replicas in the NVT ensemble. The 24 ACS Paragon Plus Environment

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protein and the ligands were considered as “solute” in the REST2 scheme–force constants of their van der Waals, electrostatic and dihedral terms were subject to scaling. The effective temperatures used for generating the REST2 scaling factors ranged from 310 K to 700 K, following a distribution calculated with the Patriksson-van der Spoel approach

42.

Exchange between replicas was attempted every 1000

simulation steps. This setup resulted in an average exchange probability of ~40%. The original unscaled replica (at 310 K effective temperature) was collected and analyzed. The first 10 ns were discarded for equilibration, as the RMSD of the ligands and the TM domain Cα atoms stabilized (Fig. S5). The production-phase trajectory was saved every 20 ps. To assess the convergence, we plotted the cumulative averages of the above RMSDs along the simulation time (Fig. S5). The cumulative average is defined as ∑Ni=1

Xi / N, where N is the number of frames sampled till the current simulation time, i is the current frame number, and Xi is the observation of interest (here the RMSD) for frame i. In total, 170 ns (× 48 replicas) of the production-phase simulations were collected for each system. PCA was performed with Bio3d after aligning each trajectory to the core residues (Cα fluctuations within a volume of 2 Å3)

43.

Network analysis was carried out 25

ACS Paragon Plus Environment


Page 26 of 46

using the NetworkView plugin 44 for VMD. Further details are provided in Supplementary

Methods.

ASSOCIATED CONTENT

Supporting Information. The following files are available free of charge via the Internet at http://pubs.acs.org. Supporting Information file including Figs. S1S5, Table S1 and Supplementary Methods (file type PDF). Movie S1 (file type AVI).

Movie S2 (file type AVI).

Simulation snapshots (file type ZIP).

AUTHOR INFORMATION

Corresponding Author


Page 27 of 46 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


*

To

whom

correspondence

may

be

addressed:

[email protected]

or

[email protected]

Author Contributions

X.C, J.G, S.A and S.F designed research; X.C, J.B.C and S.F performed research; X.C, J.B.C, S.A, J.G and S.F analyzed data; and X.C, J.B.C and S.F wrote the paper. All authors have given approval to the final version of the manuscript. 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) through grant CO 1715/1-1 to X.C, by GIRACT (Geneva, Switzerland) and the Gen Foundation (Registered UK Charity No. 1071026) through fellowships to J.B.C, 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-IDEX-01, to X.C, J.G and S.F.



Page 28 of 46

ACKNOWLEDGMENT

We acknowledge PRACE for awarding us access to MareNostrum at Barcelona Supercomputing Center (BSC), Spain. Part of the work was performed using HPC resources from GENCI-CINES, France (grant A0040710477). The authors thank Dr. Jérémie Topin for critical reading of the manuscript.

ABBREVIATIONS AM, allosteric modulator; PAM, Positive allosteric modulator; NAM, negative allosteric modulator; mGluR5, metabotropic glutamate receptor type 5; GPCR, G protein-coupled receptor; ECD, extracellular ligand-binding domain; 7TM, heptahelical transmembrane domain;

H-bond,

hydrogen

bond;

MD,

molecular

dynamics;

DFB,

3,3′-

difluorobenzaldazine; m-MPEP, 2-methyl-6-(3-methoxyphenyl)-ethynyl pyridine; wt, wild type; mut, the F7886.53A mutant; ECL, extracellular loop; ICL, intracellular loop; PCA, principal component analysis; REST2, replica exchange with solute scaling.

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Table of Contents graphic


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Figure 1. Structure of mGluR5. 119x61mm (300 x 300 DPI)



Figure 2. PAM destabilized the TM3-TM6 ionic lock and induced TM6 outward movements on the intracellular side, in contrast to the other three systems. 87x121mm (300 x 300 DPI)


Page 42 of 46

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Figure 3. Ligand-binding modes and pocket conformations sampled in the simulations. 138x185mm (300 x 300 DPI)



Figure 4. Proposed scheme of allosteric agonism of mGluR5 7TM. 138x131mm (300 x 300 DPI)


Page 44 of 46

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Figure 5. Cartoon presentation of the four simulation systems colored by subnetworks. 198x79mm (300 x 300 DPI)



Table of Contents graphic 87x106mm (300 x 300 DPI)


Page 46 of 46

Allosteric modulation mechanism of the mGluR5 transmembrane domain

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