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Article Cite This: ACS Omega 2017, 2, 8557−8567

Structural Features and Ligand Selectivity for 10 Intermediates in the Activation Process of β2‑Adrenergic Receptor Tao Liang,† Yuan Yuan,§ Ran Wang,† Yanzhi Guo,† Menglong Li,† Xuemei Pu,*,† and Chuan Li*,‡ †

College of Chemistry and ‡College of Computer Science, Sichuan University, No. 29 Jiuyanqiao Wangjiang Road, Chengdu 610064, People’s Republic of China § College of Management, Southwest University for Nationalities, No. 16 South Section 4, Yihuan Road, Chengdu 610041, People’s Republic of China S Supporting Information *

ABSTRACT: It has already been suggested by researchers that there should be multiple intermediate states in the activation process for Gprotein-coupled receptors (GPCRs). However, the intermediate states are very short-lived and hardly captured by the experiments, leading to very limited understanding of their structural features and drug efficacies. In this work, a novel joint strategy of targeted molecular dynamics simulation, conventional molecular dynamics simulation, and virtual screening is developed to address the problems. The results from 10 intermediate conformations obtained from the work reveal that the ligand pocket is very unstable and fluctuates between the inactive state and the active one in the case of ligand-free, in particular for ECL2 as a gate-keeper of the ligand-binding. The ligand-binding site could be stable in the active state with a small volume and a completely closed ECL2, only when the G-protein-binding region is fully activated. In addition, the activations of the ligand-binding pocket and G-protein-binding site are relatively independent and exhibit a loose allosteric coupling, which contributes to the existence of multiple intermediate conformations. Interestingly, the screening performance of the agonists does not increase on increasing the overall activity of the intermediate state, but is dependent on the activated extent of the ligand pocket. The receptor is prone to bind the agonist when closing ECL2 and reducing the ligand-binding pocket volume, whereas it is more favorable for binding the antagonist when opening ECL2 and increasing the pocket volume. These observations added to previous studies could help us better understand the activation mechanism of GPCRs and provide valuable information for drug design.

1. INTRODUCTION β2-Adrenergic receptor (β2AR), as an important receptor of class A G-protein-coupled receptors (GPCRs), is ubiquitous in the smooth muscle throughout the body and regulates smooth muscle relaxation in the airways and vasculature.1−3 Thus, it is also an important target for cardiac and asthma drugs and has been extensively characterized by many experimental works and computational ones over several decades. In 2007, Kobilka and Stevens first resolved the inactive human β2AR crystal structure bound to a high-affinity inverse agonist carazolol.4 In the following years, more and more β2AR crystal structures were obtained, and most of them were bound to inactive ligands,5,6 thus being in inactive states. In 2011, Rasmussen resolved the first active crystal structure of β2AR,7 which is bound by a nucleotide-free Gs heterotrimer (Gα, Gβ, and Gγ) and a highaffinity agonist (BI-167107). As revealed, the activation of GPCRs from the inactive state to the active one is accompanied by a series of large conformational rearrangements. Many biochemical, biophysical, and other studies indicated that GPCR exists in a dynamic equilibrium between the inactive state and the active one,6−9 in © 2017 American Chemical Society

which there are multiple intermediate states, and different ligands would shift the equilibrium toward different directions, in turn influencing the signal transmission.10 However, the process is very fast and the intermediate states are very shortlived, which are hardly captured by the experiments. Consequently, the dynamic behavior of GPCRs in the activation process has been hardly characterized by the experiments, although its importance was well-recognized. Molecular dynamics (MD) simulation can provide a full atomic level view of protein structures over time. Thus, it has been successfully applied to study the dynamics behaviors of various proteins,11−14 including GPCRs.15−18 Previous MD studies on GPCRs mainly focused on structures of GPCRs,19−22 the interaction between various ligands and GPCRs,23−30 and internal water pathways.18,31 In addition, the activation mechanism of GPCRs was also a concern.16,32−34 However, the activation of GPCRs generally occurs on a microsecond Received: July 19, 2017 Accepted: September 14, 2017 Published: December 1, 2017 8557

DOI: 10.1021/acsomega.7b01031 ACS Omega 2017, 2, 8557−8567

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ACS Omega time scale, which significantly limits classic all-atom MD studies on the activation process started at the inactive state because of the power of the computer. Some conventional all-atom MD methods studied the activation pathway induced by different ligands and the deactivation process for two A class GPCRs within nanosecond and microsecond timescales,32,34 which provided valuable information for the dynamics change in some key residues and regions. In addition, to overcome the limitation of the simulation time and to bridge the gap between the computational timescales and the experimental ones, some biased MD methods were used to investigate the activation process of GPCRs,35−38 including targeted molecular dynamics (TMD) simulation.33,39,40 TMD can accelerate the transition process between two existing states with the aid of an external force and has been successfully applied to elucidate the large-scale conformational transition between two states of proteins.41,42 In our previous work, we also used the TMD method to overcome high barriers of the large conformational variations within the nanosecond timescale to study the activation process of β2AR from the inactive state to the active one.33 Although some structural features of the intermediate state were obtained based on one representative conformation in the activation process, the TMD conformation is not identical to the real space and hence not appropriate to further study its drug efficacies. As accepted, virtual screening based on MD conformations is an effective way to design drugs for target proteins.28,29,43 It was reported that the virtual screening based on the MD conformations of GPCRs outperforms those based on crystal structures because MD conformations incorporate the flexibility of receptor.28 However, in the previous studies, the drug selection of the intermediate states in the activation process was seldom concerned, disfavoring the designs of diverse drugs targeting GPCRs. On the whole, although GPCRs have aroused considerable interests from experiments and computations, the information concerning the activation intermediates and their drug efficacies is still very limited and highly desired for further study. Thus, in this work, we propose a facile yet efficient strategy to address the above issue for the representative β2AR through a combination of TMD, conventional MD (CMD), and virtual screening. First, we utilized TMD to overcome the high barriers of the large conformational changes in the activation process and get some initial seeds representing different intermediate stages. Then, we further performed 150 ns CMD simulations for each seed and assembled all these equilibrium trajectories to obtain reasonable intermediate conformations close to the real activation space. Finally, we studied their selectivity to ligands with the help of the virtual screening method based on the representative MD conformations, focusing on what structural features of the receptor would favor the screening toward the agonists and antagonists.

Figure 1. rmsd distributions for all conformations derived from the six 150 ns CMD trajectories.

on the TMD seeds could capture the large conformation variations in the activation process. To obtain the representative frames, we further used the k-means method to cluster all CMD conformations and obtained 80 categories based on the rmsd values of the backbone atoms of the receptor. Then, we chose representative groups to further characterize their structures. The selection is based on the populations of the groups and considered together with their rmsd values, so that the chosen groups could be representative for the activation process. Consequently, 10 groups were obtained. Figure 2

Figure 2. Popularity of conformations for the 10 representative clusters and the rmsd values (Å) of all backbone atoms to 2RH1 and 3SN6 for the 10 central conformations selected from the 10 representative clusters.

shows the populations of the 10 clusters and the rmsd values of the representative conformations from the 10 clusters. It can be seen that their rmsd values cover the range between ∼1.5 and ∼4.8 Å, thus being representative for the activation space. To characterize the conformation change of the ligandbinding pocket in the activation process, four indicators (pocket volume, distance between Ser2075.46 and Tyr3167.43 within the pocket,33 distance between Phe193ECL2 and the pocket residue Tyr3087.35, and rmsd of ECL2) were taken into account in terms of the following observations. A comparison between the inactive crystal structure (PDB ID: 2RH1)4 and the active crystal structure (PDB ID: 3SN6)7 already revealed significant differences in their structures.17,44,45 As known, the agonist is smaller than the antagonist and could form hydrogen bonding with the receptor, which necessitates a contraction of the binding pocket in the active state. Thus, the pocket volume is

2. RESULTS AND DISCUSSION 2.1. Structural Features for the Representative Conformations in the Activation Process. The rootmean-square deviation (rmsd) value between the inactive crystal structure and the active one was calculated to be 4.22 Å for all backbone atoms of the receptor. It can be seen from Figure 1 that the rmsd values of the conformations derived from the six 150 ns CMD trajectories are in the range between ∼1.5 and ∼5.0 Å, with respect to either the active crystal structure or the inactive crystal structure, exhibiting significant differences. The result implies that the CMD trajectories based 8558


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Table 1. Eight Indicators of Ten Intermediate Conformations, the Inactive Crystal Structure (2RH1), and the Active One (3SN6)a,b ligand-binding pocket rmsd_all conformation states

ina

2RH1 all-c1 all-c2 all-c3 all-c4 all-c5 all-c6 all-c7 all-c8 all-c9 all-c10 3SN6

4.52 2.03 1.88 1.82 2.89 3.87 2.71 3.34 3.04 3.60 4.22

G-protein-binding region

rmsd_ECL2

act

volume

d_207−316

ina

4.22 4.77 3.06 2.92 3.02 2.58 3.31 2.11 2.80 2.53 1.74

641 566 535 449 382 526 534 432 654 335 462 397

16.20 16.15 14.63 16.40 16.44 16.16 14.92 14.09 16.23 13.29 13.33 13.93

2.75 2.49 2.16 1.55 0.86 1.60 0.70 1.39 1.22 0.85 0.42

rmsd_NPxxY

rmsd_ICL2

act

d_193−308

d_TM3−TM6

d_ionic

ina

ina

0.42 2.80 2.42 2.13 1.57 0.91 1.57 0.77 1.41 1.29 0.76

5.86 19.15 12.33 6.27 5.46 7.47 15.56 9.05 10.70 6.69 3.94 3.84

8.37 7.12 8.48 7.65 8.43 15.91 13.99 14.60 15.42 16.94 16.20 15.41

11.15 12.78 13.38 12.22 14.65 12.21 17.05 12.34 18.92 12.01 17.96 18.97

0.61 0.73 0.91 0.56 0.93 1.12 0.94 1.19 0.86 1.35 1.22

act 1.22 1.54 1.05 1.39 1.28 1.01 1.32 1.07 0.75 1.12 0.36

1.49 1.52 1.37 1.82 2.46 3.21 3.14 3.55 2.48 3.65 3.93

act 3.93 4.26 4.62 4.55 4.14 3.73 1.89 3.05 1.40 3.48 0.90

a

The 10 intermediates are obtained by clustering based on the rmsd values of the backbone atoms of the receptor. brmsd_all, rmsd_ECL2, rmsd_NPxxY, and rmsd_ICL2 denote the rmsd values (in Å) for all backbone atoms of β2AR, ECL2 residues, NPxxY residues, and ICL2 residues, respectively; ina and act denote the rmsd (in Å) with respect to the inactive crystal structure (2RH1) and the active one (3SN6), respectively; volume (in Å3) denotes the volume of the ligand-binding pocket; d_207−316 denotes the distance (in Å) between Ser2075.46 and Tyr3167.43; d_193−308 denotes the distance (in Å) between Phe193ECL2 and Tyr3087.35; d_TM3−TM6 denotes the distance (in Å) between TM3 and TM6, measured by the distance between Arg1313.50 and Leu2726.34; and d_ionic denotes the distance (in Å) between Arg1313.50 and Glu2686.30.

Arg1313.50 and Glu2686.30 (ionic lock), rmsd values of NPxxY motif and ICL2. Herein, we referred the crystallographically observed G-protein-coupling conformational state (PDB ID: 3SN6)7 as the canonical active state and referred the crystallographically bound with the inverse agonist (PDB ID: 2RH1)4 as the standard inactive state. Figure 3 qualitatively

considered as an indicator. Compared to the inactive crystal structure, the inward bulge of TM5 centered around Ser2075.46 was found in the active state for β2AR.46 Our previous TMD observations33 indicated that Tyr3167.43 has the highest flexibility among all residues of the ligand-binding pocket in the activation process, and Ser2075.46 is second to it. Consequently, the distance between Ser2075.46 and Tyr3167.43 could act as one indicator to reflect the dynamics variation of the ligand pocket. ECL2 has been proposed to act as a “gatekeeper” for ligand binding and to associate with the specificity of ligand binding.47,48 In addition, the distance between the residue Phe193ECL2 in ECL2 and the pocket residue Tyr3087.35 was also a concern because a comparison between the fully active crystal structure and the inactive one indicated that there is a significant movement of 2−2.5 Å for the two residues, which was widely served as one main feature for the formation of the liplike structure in the ligand-binding pocket.3 Thus, the rmsd of ECL2 and the distance of Phe193ECL2···Tyr3087.35 are selected as the other two indicators. The 4 indicators characterizing the ligand-binding pocket were calculated for the 10 representative conformations from the 10 groups and are listed in Table 1. As accepted, the variation of the ligand-binding region would pass to the intracellular G-protein-binding site. The highly conserved NPxxY has been considered to be closely associated with the G-protein binding and could serve as an important activation indicator.49−51 The fully active crystal structure of β2AR-Gs displays a significant difference in the intracellular loop 2 (ICL2) conformation with respect to the inactive state because of the interaction between ICL2 and G-protein in the active state.52 When the G-protein-binding site adopts an active conformation, the ionic lock between Arg1313.50 and Glu2686.30 in the cytoplasmic end is broken and the intracellular end of TM6 obviously moves outward, leading to an opening of the Gprotein-binding region in the receptor cytoplasmic face.53 Thus, we selected the four indicators to characterize the changes of the G-protein-binding site in the activation process, such as the distance between TM3 and TM6, the distance between

Figure 3. Diagram of the activities expressed by colors for the 8 indicators in the 10 representative conformations, derived from the data in Table 1. The colors from deep to light denote the activities from high to low. Deeper the color, closer to the active state. Lighter the color, closer to the inactive state. Upper four horizontal bars correspond to the ligand-binding pocket, whereas the remaining four horizontal bars are associated with the G-protein-binding region.

shows the similarities of the intermediate conformations to the active crystal structure for the eight activation indicators above, in which the colors from light to dark denote the activity from low to high. For the all-c1 conformation, the total rmsd from the inactive state is high, up to 4.52 Å, but its rmsd from the active state is also large, up to 4.77 Å. The conformation presents large deviations from the inactive and active states. The volume of the ligand-binding pocket is 566 Å3, located between 641 Å3 of the inactive 2RH1 and 397 Å3 of the active 3SN6 but closer to that of the inactive 2RH1. The distance between the Phe193ECL2 residue and the pocket residue Tyr3087.35 is 19.15 Å, much greater than those of the inactive state and 8559


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of TM3−TM6 is almost unchanged. The two indicators display one inconsistent change. Judging from the rmsd value, the NPxxY region is also similar to the inactive state. Although ICL2 presents a significant variation with respect to the inactive state, it is still closer to the inactive state compared to the active state. The total rmsd values of all-c5 are 2.89 Å from 2RH1 and 2.58 Å from 3SN6. Its pocket volume is 526 Å3, located between the active state and the inactive one. It can be seen from Table 1 that the distance of Ser2075.46 and Tyr3167.43, the rmsd value of ECL2, and the distance of Phe193ECL2 and Tyr3087.35 are very close to those of the inactive 2RH1. For the G-protein-binding region, NPxxY does not present a significant variation relative to 2RH1, whereas ICL2 displays the feature of one intermediate state, evidenced by their rmsd values. The ionic lock is slightly increased, but still far away from 18.97 Å of the active state. By contrast, the distance of TM3−TM6 is slightly larger than the active state, displaying full activation. The activation features in the state are significantly enhanced with respect to the four states above, in particular for the Gprotein region. But, the ligand-binding region is almost in the inactive state. The total rmsd values of all-c6 are large, achieving 3.87 Å from the inactive 2RH1 and 3.31 Å from the active 3SN6. The pocket volume, the distance of Ser2075.46 and Tyr3167.43, and the rmsd of ECL2 are almost in one intermediate stage between the inactive state and the active one, as reflected by Table 1 and Figure 3. The large distance of Phe193ECL2 and Tyr3087.35 (15.56 Å) indicates that ECL2 significantly deviates from the binding pocket. The ionic lock is completely broken, similar to the active state. The distance between TM3 and TM6 also approaches the active state. ICL2 presents a more significant change than the five states above, closer to the active state. However, the NPxxY region is still closer to the inactive state. Overall, the activity of all-c6 is almost in an intermediate state. The total rmsd values of all-c7 are 2.71 Å versus 2RH1 and 2.11 Å versus 3SN6. Although the rmsd relative to the inactive state is smaller than that of all-c6, the pocket volume and the distance of Ser2075.46 and Tyr3167.43 approach the active state. The distance of Phe193ECL2 and Tyr3087.35 (9.05 Å) is also larger than those of the inactive and active states. However, the rmsd of ECL2 only presents a slight deviation from the inactive and active crystal structures. Similar to all-c5, the ionic lock does not present a significant increase, but the distance of TM3 and TM6 is close to the active state. Compared to the two crystal structures, ICL2 significantly changes, whereas the NPxxY region only exhibits a slight variation. The total rmsd of all-c8 is 3.34 Å relative to the inactive state, larger than its rmsd (2.80) from the active 3SN6. However, the ligand pocket is almost in the inactive state, as evidenced by the four pocket indicators in Table 1 and Figure 3. But, the Gprotein region almost approaches the active state. For all-c9, its total rmsd is 3.04 Å relative to 2RH1 and is 2.53 Å relative to 3SN6. Its pocket volume is 335 Å3, smaller than that of the active 3SN6. The distance of Ser2075.46 and Tyr3167.43 is close to the active site. ECL2 approaches an intermediate state, as reflected by Figure 3. Although the distance of the ionic lock only presents a slight increase with respect to the inactive state, the distance of TM3 and TM6 is significantly increased and larger than the active state. ICL2 and NPxxY regions are closer to the inactive state, judged from their rmsd values. Overall, the ligand-binding pocket of the state is almost in an active state, whereas the three indicators of the G-

the active one, indicating a significant opening of ECL2 from the pocket. As a result, the rmsd values of ECL2 is the largest (2.75 Å from 2RH1 and 2.80 Å from 3SN6) among all 10 intermediates, much greater than the rmsd value (only 0.42 Å) between the inactive 2RH1 and the active 3SN6. For the Gprotein-binding site, the distance between TM3 and TM6 is 7.12 Å, close to that of 2RH1 (8.37 Å). The distance between Arg1313.50 and Glu2686.30 (viz., ionic lock) is 12.78 Å, which is slightly larger than that of 2RH1 (11.15 Å) but much smaller than 18.97 Å of the active 3SN6. The rmsd of NPxxY is 0.61 Å from the inactive 2RH1 and 1.54 Å from the active 3SN6, much closer to the inactive state. The rmsd values of ICL2 are 1.49 Å from 2RH1 and 4.26 Å from 3SN6. As observed above, all eight indicators in the state are almost close to the inactive 2RH1, as reflected by the light colors in Figure 3. The conformation all-c2 does not present a large deviation from the inactive state, judged from its 2.03 Å value of rmsd. The rmsd is 3.06 Å from the active 3SN6 state. The value of its pocket volume (535 Å3) is located between the active state and the inactive one, displaying an intermediate feature. The distance between Ser2075.46 and Tyr3167.43 is 14.63 Å, approaching 13.93 Å of the active state. The separation between Phe193ECL2 and Tyr3087.35 is 12.33 Å, indicating that ECL2 also significantly moves away from the binding pocket. The rmsd values of ECL2 (2.49 Å from 2RH1 and 2.42 Å from 3SN6) are close to the all-c1 state. The distance of the ionic lock (8.48 Å) is larger than that of all-c1 but still closer to the inactive state with respect to the active one. Similar to all-c1, the NPxxY and ICL2 regions in the all-c2 conformation match those of the inactive state, judged from their rmsd values. Overall, most features of the state are similar to those of the allc1 state despite the small differences, both close to the inactive state. For the all-c3 state, its total rmsd values are 1.88 Å from the inactive 2RH1 and 2.92 Å from the active 3SN6. The volume of the ligand-binding pocket is 449 Å3 and close to 397 Å3 of the active 3SN6. The distance between Ser2075.46 and Tyr3167.43 within the ligand-binding pocket is 16.40 Å, almost equal to 16.20 Å of the inactive state. The rmsd values of ECL2 are 2.16 Å from the inactive 2RH1 and 2.13 Å from the active 3SN6, closer to the inactive state. The distance between Phe193ECL2 and the pocket residue Tyr3087.35 is 6.27 Å, significantly lower than those of the two conformations above (viz., all-c1 and allc2) and close to the value of the inactive 2RH1 (5.86 Å). For the G-protein-binding site, the distance between TM3 and TM6 is 7.65 Å, approaching that of 2RH1 (8.37 Å). The distance between Arg1313.50 and Glu2686.30 (ionic lock) is 12.22 Å, which is slightly higher than that of 2RH1 (11.15 Å) but much lower than 18.97 Å of the active 3SN6. The rmsd values of NPxxY are 0.91 Å versus the inactive 2RH1 and 1.39 Å versus the active 3SN6, significantly closer to the inactive state. The rmsd values of ICL2 are 1.37 Å from 2RH1 and 4.55 from 3SN6. As observed above, all parameters in the state are almost close to the inactive 2RH1 state with the exception of the pocket volume, as reflected by the light colors in Figure 3. Similar to the all-c3 state, the total rmsd of all-c4 to the inactive 2RH1 is small (1.82 Å), whereas its rmsd to the active state (3.02 Å) is relatively large. Its pocket volume (382 Å3) is very close to the active state (397 Å3). In addition, the rmsd of ECL2, the distance of Ser2075.46···Tyr3167.43, and the distance of Phe193ECL2...Tyr3087.35 are also close to the inactive state. Compared to the inactive 2RH1, the ionic lock of the all-c4 state is significantly increased to 14.65 Å, whereas the distance 8560


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ACS Omega protein region present a reverse trend with the exception of the distance of TM3 and TM6. The all-c10 state presents a large deviation from the inactive state (3.60 Å of rmsd) and a small deviation from the active state (1.74 Å of rmsd). As reflected by Table 1 and Figure 3, its ligand pocket and G-protein region almost resemble the active state, exhibiting full activation for the two regions. On the whole, all-cl, all-c2, all-c3, and all-c4 are close to the inactive state, in particular for the G-protein region. However, some structural features in these states are still present to be partly activated, for example, the binding pocket volume of allc3 and all-c4 and the ionic lock of all-c4. The G-protein region in all-c8 almost approaches the active state, but its ligandbinding pocket still matches the inactive state. By contrast, the ligand-binding pockets in all-c7 and all-c9 almost approach the active state, whereas some features in their G-protein regions are still close to the inactive state, except for the distance of TM3−TM6. Similarly, for the all-c5 and all-c6 states, some features display activation but some are in the inactive space or the intermediate state, as reflected by Figure 3. Thus, these conformations should be in different intermediate states. Only the all-c10 state is fully activated, closest to the active 3SN6. Interestingly, the observations reflect that the activations of the ligand-binding region and the G-protein-binding region are relatively independent, exhibiting a loose allosteric coupling. Some spectroscopic results suggested that the conformations of the ligand-binding pocket and the cytoplasmic domain of β2AR should not be tightly allosterically coupled.10,54 A microsecond timescale MD simulation to study the deactivation process of β2AR also showed that there is no high correlation in conformations between the G-protein couple domain and the ligand-binding region.32 Our results provide further evidences on the molecular level for the loose allosteric coupling between the two regions. The independence is mainly attributed to the instability of the ligand-binding region, which presents obvious fluctuation between the inactive and active states in the activation process. The ligand-binding pocket shows high activity only in the all-c10 state, in which the G-protein-binding region is also highly active. The observation further confirms that only when the G-protein-binding region is fully activated and the G-protein is coupled, the ligand-binding site could be stable in a closed (small volume) and active state.55 In addition, it is worth to note the ECL2 variation in the activation process. Although the rmsd of ECL2 between the active state and the inactive one is minor (only 0.42 Å), our results indicate that the distance between Phe193ECL2 and Tyr3087.35 exhibits very large fluctuations from 3.8 Å to about 20 Å among these states. Except for the fully activated all-c10 state, the distances of all other nine states are larger than that of the active crystal structure. The ECL2 in the all-c3, all-c4, all-c5, and all-c9 states is relatively close to the inactive state (vide Figure 4a), whereas in the all-c1, all-c2, all-c6, and all-c8 states, it exhibits large position deviations (vide Figure 4b), in particular for all-c1 with the largest position change (19.15 Å). ECL2 in the ligand-bound 2RH1 and 3SN6 structures presents a relatively closed state compared to the ligand-free states obtained from the work. ECL2 is completely closed only when the receptor is fully activated like all-c10 and the active 3SN6 engaging the G-protein. In general, the GPCR could bind the G-protein only when it is activated either by the agonist or by self-activation. The previous MD observation indicated that the fully activated GPCR, coupling the G-protein, would transition spontaneously

Figure 4. Superimposition of the 10 central conformations derived from the 10 representative clusters on the inactive crystal structure (2RH1) and the active crystal structure (3SN6) to display ECL2 changes. The ECL2 region and the two residues Phe193ECL2 and Tyr3087.35 are highlighted. (a) all-c3 (light orange), all-c4 (blue), all-c5 (deep pink), all-c7 (deep purple), all-c9 (limon), and all-c10 (cyan) are superimposed on the inactive 2RH1 (green) and the active 3SN6 (light pink). Displacements of ECL2 in the six intermediate conformations from the two crystal structures are relatively small. (b) all-c2 (light blue), all-c6 (yellow), all-c8 (orange), and all-c1 (purple) are superimposed on the two crystal structures. ECL2 in the four intermediate conformations presents large deviations from the two crystal structures, in particular for the all-c1 (purple). The region involved in the residues Phe193ECL2 for the four intermediate conformations are highlighted in the black circle.

from the active state to the inactive one when removing the Gprotein.32 Crystallographic GPCR structures bound by the agonist but not coupled by the G-protein still match the inactive conformation.8 On the basis of our observations above and the previous findings, it can be drawn that the discontinuous activation for the G-protein region and the ligand-binding region should contribute to the multiple intermediate states existing in the activation process. When the G-protein domain is activated and coupled by the Gprotein, the receptor is stabilized in a fully active state. 2.2. Selectivity of the Representative Conformations to Ligands. To study the selection of these intermediate states to ligands, we established a ligand validation set, which includes 40 known β2AR ligands (20 agonists and 20 antagonists) and 1440 decoys of actives. They were extracted from the DUD-E database (total of 1480 molecules). Structures of the 20 agonists and 20 antagonists are shown in Figures S1 and S2 in 8561


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pocket residues rather than the total rmsd above. On the basis of the populations of the clusters and the differences between their rmsd values, 10 representative clusters were selected. The central conformations of the 10 clusters were used to perform further ligand screening. Figure S3 and Table S1 in the Supporting Information show the four indicators of the ligand pocket for the 10 representative conformations. It can be seen that the 10 conformations exhibit different features for the 4 indicators, thus to a large extent representing the ligand-pocket variation in the activation process. The screening results of the 10 conformations are listed in Table S2. We arranged the 10 representative conformations according to their pocket sizes from small to large because of the significant effects of the pocket volume on the ligand screening observed above. It can be seen from Figure 6 that the performance to distinguish the

the Supporting Information. We docked the ligand set to the 10 representative conformations. We plot the receiver operating characteristic (ROC) curve and calculate the area under the ROC curve (AUC) to obtain the screening performance of each representative conformation, as shown in Figure 5. It can be seen that the 10 states could

Figure 5. Performances to identify the agonists and antagonists for the two crystal structures (2RH1 and 3SN6) and the 10 intermediate conformations derived from the 10 representative clusters based on the rmsd values of the backbone atoms of the receptor.

improve the screening to the agonists (AUC-agonist > 0.5) with respect to the inactive 2RH1 (AUC-agonist = 0.44). In addition, the screening to the antagonists is superior to the agonists for the nine intermediate states, except for the all-c10 state. As observed above, ECL2 is closer to the ligand-binding pocket for all-c3, all-c4, all-c5, all-c7, all-c9, and all-c10 states, whereas ECL2 of all-c1, all-c2, all-c6, and all-c8 states is far away from the pocket. The virtual screening results show that the performance to identify the agonist is higher for those states with ECL2 close to the pocket than those with ECL2 far from the pocket. In addition, it is found that the all-c3 and all-c4 conformations with a low activity have agonist affinities similar to the all-c7 and all-c9 conformations with a relatively high activity. Also, the all-c8 state with a relatively high activity presents agonist affinity similar to the all-c1 state with a low activity. As revealed above, the activity in the G-protein region is low for the all-c4 state, but its pocket is in an active state with a small volume (382 Å3). Thus, it favors the agonist binding (AUC-agonist = 0.64). For all-c8, the activity in the G-protein region is high, but its ligand-bound pocket is large (654 Å3), leading to poor affinity to the agonist (AUC-agonist = 0.50). In addition, the states like all-c3, all-c4, all-c7, all-c9, and all-c10, which have relatively high activities in the ligand-binding pocket, present to be conducive to screen the agonists. The observations indicate that the receptor’s ability to screen the agonists does not increase as the overall activity of the receptor increases, different from some previous findings that the higher the activity of the conformation, the stronger the enrichment to the agonist.56 The result provides further support to the observations from some MD and nuclear magnetic resonance experimental works that the active conformation is not necessarily the lowest energy state for agonist binding.10,55,57 Our results clearly show that the activation extent of the binding pocket is crucial to select the ligands. 2.3. Effects of the Ligand-Binding Pocket on Ligand Screening. How does the pocket affect ligand binding, and what type of pocket is suitable for agonist binding? To address the questions, we clustered all CMD trajectories into 80 categories only based on the rmsd values of the ligand-binding

Figure 6. Effects of the ligand-binding pocket volumes on the performance for screening the agonists and antagonists.

antagonist from the decoys increases with increasing pocket volume. However, the screening to the agonists is different. When the pocket volume is less than about 450 Å3, the performance for the screening agonist increases as the pocket volume increases because the large pocket volume should favor the entrance of the agonist. But, when the pocket volume is larger than about 450 Å3, the performance to identify the agonist gradually reduces with increasing pocket volume because the too large pocket disfavors the interaction between the small-size agonist and the receptor. The performance to identify the agonists is the best for the lb-c7 state with 411 Å3 of the pocket volume, in which the activity of the pocket is in an intermediate state (vide Figure S3). However, the performance to identify the antagonists is the highest for lb-c2 with 742 Å3 of the pocket volume. The result may be attributed to the fact that the antagonist of β2AR is generally bigger than the agonist. Thus, the pocket with a large volume is more favorable for binding the antagonist, whereas a relatively small volume (about 400−450 Å3) is more suitable for binding the agonist. As accepted, the distance between the Phe193ECL2 residue and the pocket residue Tyr3087.35 is associated with the opening and closing of the lid ECL2. Thus, to gain insight into its effects on ligand binding, we arranged the order of 10 representative conformations in terms of the distance from small to large, as shown in Figure 7. It can be seen that the more the opening of ECL2, the better the performance to identify the antagonists. Different from the antagonist screening, the performance for screening the agonist increases as the 8562


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distance does not play a significant role in screening the agonist and antagonist, despite the fact that the active crystal structure of β2AR shows an obvious inward bulge at Ser2075.46 compared to the inactive crystal structure.46 Previous observations derived from the interaction fingerprints (IFP) analysis of β2AR and its cocrystallized ligands indicated that full agonists possessing catechol or a catechol-mimicking moiety could provide more hydrogen bond donors to form extra hydrogen bonds with the Ser2075.46 residue.58 However, the antagonist and agonist used in this work lack catechol or the catechol-mimicking moiety. Thus, it is difficult for them to form H-bonding with the Ser2075.46 residue unlike the full agonist, leading to the result that the distance between Ser2075.46 and Tyr3167.43 does not play an observable role in influencing the receptor screening to the agonists and antagonists. In addition, although we did not calculate and analyze the effect of the G-protein coupling on the ligand binding in this work, some observations on the issue were reported by one recent experimental work.55 Their pharmacological and biochemical evidences suggested that the G-protein coupling with the active receptor would influence the passage of ligands to the orthosteric-binding site, for example, impeding disassociation of the ligand bound in the receptor or association of the ligand in the free receptor state. An early experimental work also reported that the uncoupling G-protein from β2AR would accelerate the agonist dissociation from the receptor.59 Our observations above revealed that the small volume of the ligand-binding pocket and the small distance between the gate keeper ECL2 and the pocket in some intermediate states disfavor the entrance of the large-size antagonist but favor the binding of the small-size agonists. If the ligand is already bound in the closed and active pocket, its disassociation is certainly difficult. Thus, our results are in line with the experimental findings.55,59 In addition, our observations further indicate that the ligand-binding pocket frequently fluctuates from the open and inactive conformation to the closed and active one in the absence of the G-protein coupling. Thus, it still gives a preference to the agonist in one intermediate state with the active ligand-binding pocket but the low activity of the Gprotein region, whereas the agonist binding would accelerate the activation of the intermediate state, in turn favoring the Gprotein coupling.

Figure 7. Effect of the distance between Phe193ECL2 and Tyr3087.35 (d_193−308) on the performance for screening the agonists and antagonists.

distance increases, only when the distance is in the range of about 4−9 Å. The reason should be that the increased distance would facilitate the entrance of the agonist. But, when the distance becomes too large, for example, greater than 9 Å, the performance to select the agonist reversely reduces. As reported, Phe193ECL2 and Tyr3087.35 could form a hydrophobic region to interact with the phenyl ring of the agonist, in turn capturing the agonist.46 If the distance is too large, the hydrophobic interaction would be impaired, even disappeared, thus reducing the performance for screening the agonist. For the lb-c1 and lb-c2 states, ECL2 is far away from the ligandbinding pocket, as evidenced by 16.29 and 15.59 Å distances, respectively. Consequently, their selections to the agonists are low, but the selections to the antagonists are high. The observation indicates that the receptor is not conducive to bind the agonist when ECL2 is too far away from the pocket. However, when ECL2 is gradually closed, it would disfavor the entrance of the antagonist because of the fact that the antagonist molecule is larger than the agonist. Thus, when ECL2 is in an open state, it is conducive for the entrance and binding of the antagonist. In addition, we also arranged the 10 clusters in the increasing order of the distance between Ser2075.46 and Tyr3167.43 inside the pocket (vide Figure 8) to observe the effect of the distance on ligand screening. As reflected by Figure 8, there is no obvious correlation between the screening performance and the distance, indicating that the

3. CONCLUSIONS We combined the TMD and CMD to obtain 10 representative intermediate conformations in the activation process of β2AR. The 10 intermediates exhibit different structural features in the ligand-binding region and the G-protein-bindingregion. Despite the small differences in the ligand-binding pocket between the active crystal structure and the inactive one, our observation indicates that the ligand pocket is very unstable and fluctuates between the inactive state and the active one, when the receptor is in the free-ligand state. The ligand-binding site could be stable in the active state with a small volume and a completely closed ECL2, only when the G-protein-binding region is fully activated. The observation provides a molecular evidence for some experimental observations. In addition, our results further confirm that the activations of the ligand-binding pocket and G-protein-binding site are discontinuous and relatively independent, which contribute to the existence of multiple intermediate conformations in the activation process. The virtual screening to identify the agonists and antagonists for the10 representative conformations indicates that the

Figure 8. Effect of the distance between Ser2075.46 and Tyr3167.43 (d_207−316) inside the ligand-binding pocket on the performance for screening the agonists and antagonists. 8563


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at least 9 Å from any of the box edges. Finally, there are about 30 000 water molecules in each system. The AMBER0362 force field was used for the receptor, and the GAFF force field63 was utilized for POPC. Water was represented by the TIP3P model.64 To remove bad contacts in the initial geometry of the system, a 20 000 step minimization was performed using a steepest descent method combined with the conjugate gradient algorithm. After the minimization, the system was heated from 0 to 300 K within 120 ps. The coordinates and trajectories were stored for the following TMD and CMD simulations. 4.3. TMD Simulation. To obtain the target structure from the initial structure in the TMD simulation, a restraint defined in terms of the rmsd to the target structure was applied as an extra energy term in the force field through the following timedependent energy function

screening performance to the agonist does not increase as the structure activity of the receptor increases, but is dominated by the activation of the ligand-binding pockets. The receptor is beneficial for binding the agonist when closing ECL2 (the distance of 6 Å) and increasing the pocket volume (>500 Å3). However, the distance between the residues Ser2075.46 and Tyr3167.43 in the pocket does not present correlation with the screening performance to the agonists and antagonists. The observations could provide further insights, adding into previous studies, for a better understanding of the GPCR activation mechanism and providing valuable information for designing functionally specific drugs targeting GPCRs.

4. MATERIALS AND METHODS 4.1. Workflow. In this work, a novel ensemble strategy was proposed, as shown in Figure 9. First, the TMD simulation was

UTMD =

1 NK[rmsd(t ) − rmsd 0(t )]2 2

(1)

where N is the number of atoms, K is the force constant, rmsd(t) is the rmsd of the structure by superposition to the target structure at time t, and rmsd0(t) is the prescribed rmsd relative to the target structure at time t. With the aid of the applied force, the moving structure is gradually driven toward the target structure. In this work, the inactive and active MD coordinates obtained above were used as the initial and the target structures for the TMD simulation, respectively. The NPT ensemble was utilized at a temperature of 300 K and a pressure of 1 bar. The length of all bonds involving a hydrogen atom was constrained by the SHAKE algorithm. Nonbonding interactions were handled with a 10 Å atom-based cutoff. The particle-mesh-Ewald method was applied to fix the long-range electrostatic interactions with a 10 Å nonbonded cutoff. The force constant applied to the backbone atoms of the receptor was set to be 1 kcal mol−1 Å−2, and the integration step is 2 fs.33 The simulation time is 6 ns. The trajectories were saved at an interval of 1 ps. The obtained 6000 conformations were then clustered into six clusters by the k-means algorithm based on the rmsd of the backbone atoms of the receptor, as reflected by Figure S4 in the Supporting Information. In addition, it can be seen from Figure S4 that the TMD simulation achieves an equilibrium within a time of 6 ns. The representative conformation of each cluster was selected to further conduct the CMD simulation. 4.4. CMD Simulation. To get the conformation close to the real state, a 150 ns CMD simulation was further performed with the periodic boundary condition in the NPT ensemble at 300 K for each of the six TMD representative conformations under similar simulation conditions to the TMD above. For analysis, the trajectories were saved at an interval of 2 ps in the CMD simulations. All MD simulations were performed using the sander module of AMBER 12.0 package.65 All MD results were analyzed using the analysis module of AMBER 12.0 and VMD66 as well as some other developed specific trajectory analysis softwares. 4.5. Clustering Analysis. For all six 150 ns CMD trajectories, clustering was carried out using the k-means algorithm67,68 included in the ptraj program from the AmberTools package. Clustering was based on the massweighted rmsd structural similarity matrix. In this work, two types of cluster analysis were constructed. One was based on the rmsd of the backbone atoms of all residues, and the other was based on the ligand-binding pocket residues (His93, Trp109, Thr110, Asp113, Val114, Val117, Thr118, Thr195,

Figure 9. Schematic representation of the workflow.

used to produce 6 representative initial seeds in the activation process. Then, six 150 ns CMD simulations were performed based on the six seeds. For all six 150 ns CMD trajectories, the clustering was applied to obtain the ten main intermediate states in terms of the rmsd of all backbone atoms of the receptor with respect to the inactive crystal structure. For the 10 intermediates, their structural features were analyzed, mainly focusing on the ligand-binding pocket and the G-proteincoupling region. Finally, the virtual screening was used to explore their selectivity to the ligands. 4.2. System Preparation. The inactive crystal structure (PDB ID: 2RH1)4 and the active crystal one (PDB ID: 3SN6)7 of β2AR were used as initial coordinates, in which all nonreceptor molecules were removed, but the crystal water inside the receptor was retained. For the ICL3 missed in the two crystal structures, we used MODELLER V9.1060 to rebuild it, owing to its important role in the interaction with Gprotein.49 The receptor structure was inserted into a wellprepared phospholipids bilayer, palmitoyl oleoyl phosphatidyl choline (POPC),61 and we removed the lipids whose P atoms fall within 0.5 Å of the receptor. Then, chloride ions were introduced to neutralize the receptor charge using columbic potential terms. Water molecules were added using xleap utility. The rectangle periodic box was set up so that any solute atom is 8564


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ACS Omega Tyr199, Ala200, Ser203, Ser204, Ser207, Trp286, Phe289, Phe290, Asn293, Lys305, Tyr308, Ile309, Asn312, and Tyr316).32,37 We chose several top clusters in the populations with different rmsd values for further structural analysis and virtual screening, in which the cluster center was selected as the representative structure. 4.6. Virtual Screening. A set of ligands with different efficacies was selected to explore the ligand selectivity of the intermediate states, which includes 40 known β2AR ligands (20 agonists and 20 antagonists) collected from ZINC69 database, GPCR-ligand70 database, and PubChem71 database. Their structures are shown in Figure S1 and Figure S2 in the Supporting Information. In addition, the decoys of actives were extracted from the DUD-E72 database, and the ratio of active ligands to decoys (Nactives/Ndecoys) was kept at 1:36 in terms of the rules adopted in DUD-E database. Finally, there are 1480 small molecules in the ligand set, which are docked to the representative receptor structures. All of the input files were prepared by AutoDockTools 1.5.6 package. AutoGrid 4.2 was used to create affinity grids centered on the active site. The box size is 75 Å × 75 Å × 75 Å with 0.375 Å spacing, which is enough to cover the ligand-binding site. The dockings with the rigid receptor and the flexible ligand were performed using AutoDock 4.2,73 in which 100 separate docking calculations were carried out for each ligand to ensure the accuracy of the result. Each docking calculation was composed of 1 000 000 energy evaluations using the Lamarckian genetic algorithm. The docking pose with the lowest binding energy was selected as the best binding mode for further scoring. The ROC74 plot is a common tool to evaluate the performance of a structure to discriminate the actives from the decoys, which is a curve of the true positive rate (TPR) versus the false positive rate (FPR). They are calculated in terms of the following equations TPR =

TP (TP + FN)

(2)

FPR =

FP (FP + TN)

(3)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (X.P.). *E-mail: [email protected] (C.L.). ORCID

Xuemei Pu: 0000-0002-5519-4258 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project is supported by the National Science Foundation of China (grant no. 21573151) and NSAF (grand no. U1730127).

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REFERENCES

(1) Johnson, M. Molecular mechanisms of β2-adrenergic receptor function, response, and regulation. J. Allergy Clin. Immunol. 2006, 117, 18−24. (2) Bernier, V.; Bichet, D. G.; Bouvier, M. Pharmacological chaperone action on G-protein-coupled receptors. Curr. Opin. Pharmacol. 2004, 4, 528−533. (3) Congreve, M.; Langmead, C. J.; Mason, J. S.; Marshall, F. H. Progress in Structure Based Drug Design for G Protein-Coupled Receptors. J. Med. Chem. 2011, 54, 4283−4311. (4) Cherezov, V.; Rosenbaum, D. M.; Hanson, M. A.; Rasmussen, S. G. F.; Thian, F. S.; Kobilka, T. S.; Choi, H.-J.; Kuhn, P.; Weis, W. I.; Kobilka, B. K.; Stevens, R. C. High resolution crystal structure of an engineered human β2-adrenergic G protein-coupled receptor. Science 2007, 318, 1258−1265. (5) Hanson, M. A.; Cherezov, V.; Roth, C. B.; Griffith, M. T.; Jaakola, V.-P.; Chien, E. Y. T.; Velasquez, J.; Kuhn, P.; Stevens, R. C. A specific cholesterol binding site is established by the 2.8 Å structure of the human β2-adrenergic receptor in an alternate crystal form. Structure 2008, 16, 897−905. (6) Wacker, D.; Fenalti, G.; Brown, M. A.; Katritch, V.; Abagyan, R.; Cherezov, V.; Stevens, R. C. Conserved binding mode of human β2 adrenergic receptor inverse agonists and antagonist revealed by X-ray crystallography. J. Am. Chem. Soc. 2010, 132, 11443−11445. (7) Rasmussen, S. G. F.; 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. A.; 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 β2 adrenergic receptor-Gs protein complex. Nature 2011, 477, 549−555. (8) Rosenbaum, D. M.; Zhang, C.; Lyons, J. A.; Holl, R.; Aragao, D.; Arlow, D. H.; Rasmussen, S. G. F.; Choi, H.-J.; Devree, B. T.; Sunahara, R. K. Structure and function of an irreversible agonist-β2 adrenoceptor complex. Nature 2011, 469, 236−240. (9) Munos, B. Lessons from 60 years of pharmaceutical innovation. Nat. Rev. Drug Discovery 2009, 8, 959−968. (10) Manglik, A.; Kim, T. H.; Masureel, M.; Altenbach, C.; Yang, Z.; Hilger, D.; Lerch, M. T.; Kobilka, T. S.; Thian, F. S.; Hubbell, W. L.; Prosser, R. S.; Kobilka, B. K. Structural Insights into the Dynamic

where TP (true positive) is the number of actives in the positive class, FN (false negative) is the number of actives in the negative class, FP (false positive) is the number of decoys in the positive class, and TN (true negative) is the number of decoys in the negative class. AUC is the area under the ROC curve,75 which has been widely used to quantify the screen performance.76,77 The larger the AUC value, the better the screening performance of the structure to the actives. For example, if the AUC value is equal to 0.5, it represents random screening. When it is equal to 1, the structure has the strongest screening ability for the actives in the ligand-training set. The calculation of the AUC value is involved in the ligand-binding energy but not equal to the energy. It could reflect the overall affinity of the structure for one class of actives (agonists or antagonists) from one ligand set consisting of the agonists, antagonists, and decoys, rather than the screening performance of the structure to one ligand.

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Four indicators of the ligand-binding pocket of the ten intermediate conformations derived from the clustering based on the rmsd values of the ligand-binding residues, screening performance to the ligands for the 10 intermediate conformations derived from the clustering based on the rmsd values of the ligand-binding residues, chemical structures of the 20 agonists and 20 antagonists under study, activated extent for the four indicators of the ligand-binding pocket, and projection of the six clusters on the rmsd trajectory of the 6 ns TMD simulation (PDF)

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.7b01031. 8565


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