Investigation of allosteric coupling in human β2-adrenergic receptor in the presence of intracellular loop 3
© The Author(s). 2016
Received: 23 March 2016
Accepted: 24 June 2016
Published: 2 July 2016
This study investigates the allosteric coupling that exists between the intra- and extracellular parts of human β2-adrenergic receptor (β2-AR), in the presence of the intracellular loop 3 (ICL3), which is missing in all crystallographic experiments and most of the simulation studies reported so far. Our recent 1 μs long MD run has revealed a transition to the so-called very inactive state of the receptor, in which ICL3 packed under the G protein’s binding cavity and completely blocked its accessibility to G protein. Simultaneously, an outward tilt of transmembrane helix 5 (TM5) caused an expansion of the extracellular ligand-binding site. In the current study, we performed independent runs with a total duration of 4 μs to further investigate the very inactive state with packed ICL3 and the allosteric coupling event (three unrestrained runs and five runs with bond restraints at the ligand-binding site).
In all three independent unrestrained runs (each 500 ns long), ICL3 preserved its initially packed/closed conformation within the studied time frame, suggesting an inhibition of the receptor’s activity. Specific bond restraints were later imposed between some key residues at the ligand-binding site, which have been experimentally determined to interact with the ligand. Restraining the binding site region to an open state facilitated ICL3 closure, whereas a relatively constrained/closed binding site hindered ICL3 packing. However, the reverse operation, i.e. opening of the packed ICL3, could not be realized by restraining the binding site region to a closed state. Thus, any attempt failed to free the ICL3 from its locked state due to the presence of persistent hydrogen bonds.
Overall, our simulations indicated that starting with very inactive states, the receptor stayed almost irreversibly inhibited, which in turn decreased the overall mobility of the receptor. Bond restraints which represented the geometric restrictions caused by ligands of various sizes when bound at the ligand-binding site, induced the expected conformational changes in TM5, TM6 and consequently, ICL3. Still, once ICL3 was packed, the allosteric coupling became ineffective due to strong hydrogen bonds connecting ICL3 to the core of the receptor.
Keywordsβ2-adrenergic receptor Intracellular loop 3 (ICL3) G protein-coupled receptor Allosteric coupling Transmembrane helix
Human β2-adrenergic receptor (β2-AR) is a member of the G-protein coupled receptor (GPCR) superfamily that is responsible in the eukaryotic signal transduction, responding to hormones adrenaline and noradrenaline to mainly induce the smooth muscle relaxation in the lung tissue. As all members of GPCRs, β2-AR shares the 7TM structural motif, which consists of seven transmembrane-spanning alpha helices connected by loop regions at the intra- and extracellular sides of the membrane.
As the first hormone-activated GPCR structure to be reported by X-ray crystallography [1, 2], the high resolution structural information was obtained through the elimination of the third intracellular loop (ICL3) replaced with the protein T4 lysozyme (T4L) and also the C-terminal tail in order to increase both the proteolytic stability and crystallizability. ICL3 links the cytoplasmic ends of transmembrane helices V and VI (TM5 and TM6) and has a functional role for both the recognition and the activation of G proteins [3, 4]. Unlike other intracellular loop regions, ICL3 has a highly variable length and sequence among the members of the GPCR superfamily, even the closely related subtypes. It is believed that a GPCR’s selectivity to different G proteins originated in the structural uniqueness of ICL3.
One experimental study conducted by West et al.  investigated the ligand-dependent perturbation of the conformational ensemble for β2-AR, which incorporated the ICL3 region through hydrogen/deuterium exchange (HDX) coupled with mass spectroscopy. HDX data suggested ICL3’s role as a molecular switch, where antagonist and inverse agonist binding shifted the equilibrium toward inactive states, which is characterized by protection to exchange (i.e., stabilization) in the ICL3 loop and flanking regions of TM helices V and VI. In contrast, binding of a full agonist shifted equilibrium toward higher accessibility and/or destabilization of ICL3 region. The lack of mobility or the stabilization of ICL3 was also observed in our previous work by Ozcan et al., where the presence of ICL3 had a significant impact on the overall dynamics of the receptor, especially the arrangements of some key residues at both the ligand-binding site and the G-protein binding site. In that study, in addition to the loop model where the missing ICL3 region was generated in fully atomistic detail, a second model called clipped model was created with the two open ends of TM5 and TM6 covalently bonded to each other. During the 1 μs long MD run, the loop model found a very inactive conformation towards 600 ns, when TM6 moved towards the receptor’s core region with ICL3 packing underneath the membrane and blocking the G-protein’s binding site. ICL3 preserved its closed conformation and consequently, the receptor’s overall dynamics has decreased significantly with only minor fluctuations for the remaining 400 ns, and became similar to the clipped model’s dynamics, which showed no major variations.
Early experimental peptide studies showed that the peptides synthesized with the primary sequence 259–273 corresponding to ICL3 region of Gs-coupled β2-AR, selectively bind with G proteins, stimulate their functional activity, trigger signaling cascade in the absence of hormonal stimulus, and decrease the regulatory effects of β2-AR agonists [7–9]. Another peptide study revealed that the ICL3-derived peptides can form helical structures and contain clusters of positively charged residues exposed to one helix side which is crucial for interacting with the negatively charged receptor binding site of the Gαs subunit (Shinagawa et al. , Okuda et al, ). The last result is in good agreement with the simulation study by Ozcan et al.  during which the unstructured loop region generated through modeling techniques adopted a few turns of helices before blocking the G-protein binding cavity.
Yet, despite its functional significance, many experimental and simulation studies conducted so far have neglected its presence [12–15]. The present work focuses on the effect of the intracellular loop region ICL3 on the intrinsic dynamics of the receptor. The intrinsic dynamics is the key determinant of the receptor’s function, whereas the tertiary structure encodes the dynamic behavior. Thus, it is important to have a complete 3D structure of the receptor in order to understand the system’s function to a greater degree.
The study presented here adopted the same atomistic model of the receptor that incorporates the ICL3 region generated in our previous study . First, we start by investigating the stability of the alternative inactive state of the receptor, which was observed during the last half of the 1 μs long MD simulation. The most distinguishing features of the alternative state was the closure of ICL3 that completely blocked the G protein binding site at the intracellular region, and the simultaneous enlargement of the ligand-binding site at the extracellular part. Both regions of the receptor changed their conformation almost simultaneously which suggested a strong allosteric coupling. In two independent continuation MD runs (500 ns long each), the receptor preserved its stable ICL3 conformation as well as the extracellular part of the receptor.
In addition to the presence of ICL3, the allosteric effect was further investigated through specific bond restraints between key residues at the ligand-binding site, which led to an alternative closed state. Also, the probability of open-to-closed or closed-to-open transition in ICL3 conformation was revealed for the first time. Addition of restraints mimics the presence/interaction of the ligand and possibly accelarates the closure event. So, the conformational shift that would take place in the presence of the ligand would require much longer simulations, whereas the restraints have provided an enhanced event sampling by providing an exaggerated driving force or perturbation at the binding site.
Even though the allosteric coupling that exists between the intracellular and the extracellular regions of the receptor is a well-known feature of β2-AR and many other GPCRs, the presence of ICL3, which directly influences the overall dynamics of the receptor, was not taken into consideration when the coupling behavior was investigated. Therefore, the present study will be the first in providing this correlated motion that exists between the extracellular ligand-binding site and the intracellular G-protein binding site that incorporates the ICL3 region. Through imposing distance restraints between some key residues at the ligand-binding site, we were able to trigger a series of conformational changes in the transmembrane helices that led to the close packing of ICL3.
Protein-membrane system preparation
The system under study was adopted from Ozcan’s work  where the initial conformation was the x-ray crystallographic structure of human β2AR in complex with T4 lysozyme and the partial inverse agonist carazolol (PDB id: 2RH1) at 2.4 Å resolution . The anchor protein T4L was covalently attached to two ends of helices 5 and 6 in order to facilitate crystallization. After removal of T4L, the missing intracellular loop region ICL3 was added between residues 230 and 262 after being modeled as an unstructured loop of 32 residues long via homology modeling tool, MODWEB . Also, the bound ligand at the ligand-binding site was removed and the apo form of the receptor was used as an initial conformation.
The receptor was then embedded in the double-layered 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) phospholipid cell membrane generated with VMD’s Plug-in tool . The receptor was positioned with an oblique angle of 8° between its main principal component along the membrane and the z-axis . After solvating the protein-lipid system with VMD’s solvate module, Na+ and Cl- ions were added to neutralize the total charge of the system, which is necessary for Particle-Mesh Ewald summation method used in electrostatic energy calculations . The resulting periodic box dimensions were (86x86x100) in Angstrom. The total number of atoms in the system was 68.001, of which 5.055 belong to protein, 20.770 to lipids, 42.135 to water molecules, and 41 to ions.
The system’s equilibration consisted of several stages such as melting of lipid tails, minimization and equilibration with protein restrained, equilibration with protein released and lastly the production runs . Nanoscale Molecular Dynamics (NAMD) v2.8 software tool was used for all our MD runs . The force fields used were CHARMM27 [21, 22] for lipids, CHARMM22 [23, 24] for proteins and TIP3P model for water in the system . In this work, the isothermal-isobaric (NPT) ensemble was employed using Langevin dynamics in order to keep the temperature constant with a Langevin damping coefficient (gamma) of 5/ps for all non-hydrogen atoms. The pressure was kept constant at 1 atm using a Nose-Hoover Langevin piston with 100 fs period and 50 ps damping timescale . Long-range electrostatic interactions were treated by particle mesh Ewald (PME) method, with a grid point density of over 1 Å. A cutoff of 12 Å was used for van der Waals and short-range electrostatics interactions with a switching function. Time step was set to 2 fs by using SHAKE algorithm for bonds involving hydrogens  and the data was recorded at every 200 ps. The number of time steps between each full electrostatics evaluation was set to 2. Short-range non-bonded interactions were calculated at every time step.
Independent MD runs with and without restraints
Details of several MD runs with and without restraints
Total time (ns)
inactive crystal structure, 2RH1
last frame of Run #1
last frame of Run #1
initial frame of Run #1
initial frame of Run #1
frame @ 470th ns of Run #1
frame @ 470th ns of Run #1
last frame of Run #7 (rstr4)
last frame of Run #1
Restraint distances in all seven MD runs and their corresponding values in experimentally reported active and inactive states
Distances in crystallographic structures (Å)
Bond Restraints (Å)
Inactive (PDB id: 2RH1)
Active (PDB id: 3SN6)
Table 2 also lists the same distances in both active and inactive x-ray structures (PDB ids: 3SN6 and 2RH1). For the inactive state, all distances are higher than the experimentally observed ranges, as expected. Surprisingly, for the active state, almost all distance values are slightly out of range. Yet, most of them are smaller than those in the inactive state, especially those between three serine residues on TM5 and Asp113 on TM3. Therefore, we will focus on the first three distances (rows in Table 2), which clearly distinguish the active states from the inactive ones.
In the first restrained MD run (rstr1), Ser203-Asp113 distance was set to 16 Å for the first 300 ns and then increased to 17 Å for the remaining 200 ns. Other distances were set to values closer to those observed in the inactive crystal structure. Here, the value of 17 Å was explicitly selected as it was recorded in the last frame of the original MD run (MD1μs), when ICL3 closed upon G-protein binding site.
In the second restrained MD run (rstr2), the same three critical distances between serine residues and Asp113 were restrained to 8 Å, 10 Å and 8 Å, which all fall within the experimental range of the active state. Similarly, the other distances were set closer to those in the crystal structures. The third run (rstr3) almost share the same set of restraints as in rstr1 and rstr2, except that it starts with a different initial conformation of the receptor (See Table 1). In the fourth run (rstr4), the same initial conformation was used as in rstr3, with an additional restraint distance set between two backbone alpha-carbon atoms in Ser207 and Asp113. Finally, in the last run (rstr5), the final frame of the original MD run (MD1μs) was used as a starting conformation and the ligand-binding site was severely constricted.
Results and discussion
Analysis of two continuation runs indicates stability of packed ICL3
During the first continuation run, ICL3 stayed in its close form with only minor fluctuations (~2 Å) as shown in the RMSD profiles illustrated in Fig. 2a. All the RMSD calculations were carried out after the alignment of each MD snapshot in the trajectory to the initial snapshot based on the transmembrane region, as it is the least mobile part of the receptor. The RMSD profiles labeled as All, Core, and Tmemb represent the RMSD of the whole receptor, the core which is the receptor without ICL3 and the transmembrane region which consists of seven alpha helices located inside the membrane, respectively (See Additional file 1: Figure S1). This complete blockage of the G protein’s binding site suggests an inhibition of the receptor’s activity.
In the second continuation run using a different velocity distribution, a temporary increase up to 5 Å in the RMSD value was observed halfway through the trajectory, which led to a slight opening of ICL3 as reflected by the white colored snapshot in Fig. 2b. However, this opening was only temporary and lasted for about 20 ns. This was followed by a sharp decrease in RMSD to 4 Å caused by the closure of ICL3 to a position slightly different than the initial state. ICL3 stayed there for the rest of the simulation.
In both continuation runs, the closed state of ICL3 was preserved, representing an extreme inactive state, where the G-protein binding site was completely blocked. Experimental studies revealed both active and inactive states of the receptor, but none of these structures incorporated the ICL3 region. Here, it is the presence of ICL3 that caused the receptor to adopt such a novel inactive state, which was found to be noticeably stable.
The fluctuation of each residue averaged over the whole trajectory was determined for each simulation and illustrated in Fig. 2d. As expected, in both continuation runs, the mobility of ICL3 stayed at a much lower level than in the original MD simulation. Moreover, a slight decrease of mobility was observed in every part of the receptor, especially on two important loop regions, ICL2 (intracellular) and ECL2 (extracellular), in conjunction with the decrease in ICL3’s mobility.
In our simulation studies, the closure of ICL3 was strongly coupled with the lower part of TM6, which exhibited an inward movement of 7.5 Å, in the opposite direction of the outward movement of 14 Å observed during activation (experimentally measured at the Cα carbon of Glu 268 ). The RMSD profiles of the intracellular part of TM6 with respect to the active state (PDB id:3SN6) illustrated at the top section of Additional file 2: Figure S2 show that as ICL3 started to change its conformation to a closed state, TM6’s intracellular part shifted to the opposite direction of activation and stayed there for both continuation runs. On the other hand, the intracellular part of TM5 attached to ICL3 at the other end, seemed unaffected by these conformational variations. As illustrated at the lower section of Additional file 1: Figure S1, TM5 stabilized at around 2 Å during the original MD as well as both continuation runs.
Two of the key residues at the binding site are Asp113 on TM3 and Ser207 on TM5, which are known to interact both with agonists and antagonists, via hydrogen bonds or close contacts. They are situated at the two distant corners of the binding site and when the ligand is favorably bound, Ser207 is near the ligand’s aromatic moiety, while Asp113 usually makes multiple hydrogen bonds with the ligand’s polar end group (See Fig. 1b). Therefore, the distance between these two residues directly controls the binding capability of the ligand. Experimental measurements already determined an approximate distance range of [8 Å -10 Å] between the two side chain atoms, Oγ of Ser207 and Cγ of Asp113, when the receptor was found in its active state [31, 32]. As the receptor passes from an active state to an inactive one, the same distance also increases and stabilizes roughly at around 11 Å -12 Å. Thus, as an indicator of activation/inactivation, the same distance was monitored for both continuation runs.
Rapid closure of ICL3 is observed as restraints expand the ligand-binding site
The goal here was to investigate the allosteric coupling between the intra- and extracellular parts of the receptor, by applying some distance restraints to several key residues at the extracellular ligand-binding site region (See Fig. 1). As listed in Table 1 (See Methods section), there exist seven distances between side chain atoms that were experimentally observed within a certain range when the receptor adopted an active state [28–33]. In our first constrained simulation (rstr1), one of those distances which exists between S203Oγ and D113Cγ, was restrained to 16 Å for 300 ns and later increased to 17 Å for another 200 ns, while the remaining six were restrained to those observed in the inactive crystal structure (PDB id: 2RH1) for the whole 500 ns long simulation (See Table 2).
The high value of 17 Å was especially selected for S203Oγ-D113Cγ distance in order to reveal the same allosteric response of the intracellular part of TM6 and ICL3 observed previously in the original MD run. As expected, a close correspondence was observed between the extracellular and intracellular parts of the receptor, as ICL3 packed towards the core of the receptor by the end of 200 ns, which is about 400 ns earlier than in the original MD1μs.
ICL3 preserves its open conformation as restraints narrow the ligand-binding site
ICL3 closure necessitates the outward tilt of TM5
One important finding about the closure of ICL3 in the first restrained run and also the original run was the simultaneous outward tilt of TM5 towards the lipid bilayer, which is crucial in initiating the conformational changes along TM5 and TM6 and consequently on ICL3 (See Fig. 4c). In both runs, the distance restraints applied to residues on TM3 and TM5 shifted TM5 but not the more stationary TM3. Consequently, this desired outward tilt in the extracellular part of TM5 was followed by the inward tilt of the intracellular part of TM5 and also of TM6, which induced the expected ICL3 closure (See Fig. 4d).
The necessity of TM5’s outward tilt was demonstrated in another 500 ns long restrained run (rstr3 in Table 2) that used similar restraints as in the first restrained run, but an alternative initial conformation, in which ICL3 was in an extended form but slightly packed and oriented towards the core of the receptor (See Additional file 3: Figure S3). The applied restraints simply necessitated an expanded ligand-binding site, which was expected to induce the closure of ICL3. However, no closure was observed in ICL3, which covered a wide range of alternative states nearby G-protein binding site and towards the end of 500 ns, ended up close to its initial position (See Additional file 4: Figure S4). When the conformational change in TM5 was observed, it was clear that as a result of the distance restraint, the outward tilt in TM5 was not notable since both TM3 and TM5 moved apart at the extracellular side (See Additional file 5: Figure S5). Furthermore, no major conformational change in the intracellular part of TM5 was observed. Consequently, ICL3’s motion stayed random between open and close states, and no closure was observed. This result shows that the inward tilt of TM6 at the intracellular side was not enough to induce the closure of ICL3, which necessitates the inward tilt in both TM5 and TM6.
Packed ICL3 could not be opened by constricting the ligand-binding site
One activation mechanism proposed by Dror et al.  also supports this finding. They have shown that in its basal form, the receptor’s intracellular part of TM6 fluctuated between open or half open (intermediate) states, and adopted a fully open active state only when a G protein was bound from the intracellular region and pushed the binding site to an open form. If an agonist was bound at the extracellular binding site, then this active state was stabilized. On the other hand, when G protein was released from this agonist-bound state, it was observed that the receptor’s intracellular part of TM6 quickly returned to its inactive state obstructing the G-protein binding site. This finding indicated that the active state cannot be induced by some agonists alone and can only be reached by some exterior forces.
The very inactive state of the receptor , was further investigated by two 500 ns long MD runs, which presented a highly stable packed state of ICL3. Although a slight tendency for its opening was observed in one of these simulations (Fig. 2), the closed state was adopted shortly afterwards. The hydrogen bond network analysis revealed several hydrogen bonds connecting ICL3 with adjacent TM5 and TM6 regions, thus stabilizing this novel packed state.
Several distance restraints were applied to key residues at the extracellular ligand-binding site in order to investigate their effect on the intracellular G-protein binding site including ICL3. Bond restraints caused either an expansion or constriction of the ligand-binding site. Key distances that majorly control the size of the cavity were between Asp113 on TM3 and three serine residues (S203, S204 and S207) on TM5. When the ligand-binding site was forced to an expanded/open state via these restraints (rstr1), ICL3 closure took place following a straight pathway towards the G-protein binding site, as in the original MD1μs run. On the other hand, when the same ligand-binding site was forced to a constricted state (rstr2), no change at the intracellular part was observed as ICL3 preserved its initial open conformation. These two observations were both in agreement with the ‘pincer-like’ behavior of the receptor, where the intracellular part becomes wider as the extracellular part becomes narrower, and vice versa .
In both runs MD1μs and rstr1, closure of ICL3 was observed to closely couple with the inward tilt of both TM5’s and TM6’s intracellular parts and also the outward tilt in TM5’s extracellular part, where the signal propagation starts. In our third restrained run (rstr3) which started with an alternative state of the receptor, directed closure of ICL3 was not observed. When initial and final snapshots were aligned, it was clear that the distance restraints forced TM3 rather than TM5 to be displaced and consequently, only a minor outward tilt in TM5’s upper half was observed with no significant displacement at its intracellular region. Addition of an extra restraint between backbone atoms of the same residue pair (S203-D113) produced the desired outward tilt of TM5 at the upper half in rstr4 run. Consequently both TM5 and TM6 were displaced at the intracellular part and ICL3 closed instantaneously on G-protein binding site within 100 ns, which was the most rapid closure to be observed so far. Here, ICL3 adopted an alternative packed state, which was further observed to be stable for another 500 ns when the restraints were removed.
Another set of restraints that constrict the ligand-binding site was applied on the closed ICL3 state (rstr5) in order to free the loop from its interactions with the receptor. However, our attempt to open up ICL3 failed during the time scale of our runs. To summarize, our current study revealed alternative packed states of ICL3, which are stabilized by several hydrogen bonds between ICL3 and the rest of the receptor. Furthermore, in contrast to the persistence of ICL3 in its locked position, it was almost always straightforward to bring ICL3 from a loose, free state to a locked one by simply applying a few distance restraints that expand the extracellular ligand-binding site.
Starting with such very inactive states, the receptor stayed almost irreversibly inhibited during our runs, which in turn decreased the overall mobility of the receptor. Experimental support is currently lacking for the locked, inactive state of β2AR, due to the fact that ICL3 has been missing in most studies including the crystal structures.
The bond restraints imposed in our study simply represent the restrictions caused by ligands of various sizes bound at the ligand-binding site. Small agonist molecules tend to fit to a narrow region, whereas the antagonists and inverse agonists of larger size induce a more expanded binding site. As a result of allosteric coupling between intra- and extracellular regions, which is mediated through the transmembrane helices, particularly TM5 and TM6, the population of conformational states of ICL3 between unpacked and packed positions and thereby the binding of G-protein were modulated.
ANM, anisotropic network model; ECL, extracellular loop; GPCRs, G protein coupled receptors; ICL, intracellular loop; MD, molecular dynamics; PCA, principal component analysis; POPC, palmitoyloleoyl-phosphatidylcholine; RMSD, root mean square deviation; RMSF, root mean square fluctuation; T4L, T4-lysozyme; TM, transmembrane helix; TMEMB, transmembrane region; β2AR, β2-adrenergic receptor.
This work has been fully supported by The Scientific and Technological Research Council of Turkey (TÜBİTAK, Project #213 M544).
Availability of data and materials
All results are presented in the main text and the following additional supporting files provided in.pdf format.
CO has carried out the molecular dynamics simulations. All authors have participated in the analysis and interpretation of MD trajectory, and writing of the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Rasmussen SG, Choi HJ, Rosenbaum DM, Kobilka TS, Thian FS, Edwards PC, Burghammer M, Ratnala VR, Sanishvili R, Fischetti RF, Schertler GF, Weis WI, Kobilka BK. Crystal structure of the human beta2 adrenergic G-protein-coupled receptor. Nature. 2007;450:383–7.View ArticlePubMedGoogle Scholar
- Cherezov V, Rosenbaum DM, Hanson MA, Rasmussen SG, Thian FS, Kobilka TS, Choi HJ, Kuhn P, Weis WI, Kobilka BK, Steven RC. High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science. 2007;318:1258–65.View ArticlePubMedPubMed CentralGoogle Scholar
- Kobilka BK, Deupi X. Conformational complexity of G-protein-coupled receptors. Trends Pharmacol Sci. 2007;28:397–406.View ArticlePubMedGoogle Scholar
- Rasmussen SG, DeVree BT, Zou Y, Kruse AC, Chung KY, Kobilka TS, Thian FS, Chae PS, Pardon E, Calinski D, Mathiesen JM, Shah ST, Lyons JA, Caffrey M, Gellman SH, Steyaert J, Skiniotis G, Weis WI, Sunahara RK, Kobilka BK. Crystal structure of the beta2 adrenergic receptor-Gs protein complex. Nature. 2011;477:549–55.View ArticlePubMedPubMed CentralGoogle Scholar
- West GM, Chien EY, Katritch V, Gatchalian J, Chalmers MJ, Stevens RC, Griffin PR. Ligand-dependent perturbation of the confor- mational ensemble for the GPCR β2 adrenergic receptor revealed by HDX. Structure. 2011;19(10):1424–32.View ArticlePubMedPubMed CentralGoogle Scholar
- Ozcan O, Uyar A, Doruker P, Akten ED. Effect of intracellular loop 3 on intrinsic dynamics of human β2-adrenergic receptor. BMC Struct Biol. 2013;13:29.View ArticlePubMedPubMed CentralGoogle Scholar
- Palm D, Munch G, Dees C, Hekman M. Mapping of β-adrenoceptor coupling domains to Gs-protein by site- specific synthetic peptides. FEBS Lett. 1989;254:89–93.View ArticlePubMedGoogle Scholar
- Munch G, Dees C, Hekman M, Palm D. Multisite contacts involved in coupling of the β-adrenergic receptor with the stimulatory guanine-nucleotide-binding regulatory protein. Structural and functional studies by β-receptor-site- specific synthetic peptides. Eur J Biochem. 1991;198(2):357–64.View ArticlePubMedGoogle Scholar
- Okamoto T, Murayama Y, Hayashi Y, Inagaki M, Ogata E, Nishimoto I. Identification of a Gs activator region of the β2-adrenergic receptor that is autoregulated via protein kinase A-dependent phosphorylation. Cell. 1991;67(4):723–30.View ArticlePubMedGoogle Scholar
- Shinagawa K, Ohya M, Higashijima T, Wakamatsu K. Circular dichroism studies of the interaction between synthetic peptides corresponding to intracellular loops of ß-adrenergic receptors and phospholipid vesicles. J Biochem. 1994;115:463–68.PubMedGoogle Scholar
- Okuda A, Matsumoto O, Akaji M, Taga T, Ohkudo T, Kobayashi Y. Solution structure of intracellular signal-transducing peptide derived from human ß -adrenergic receptor. Biochem Biophys Res Commun. 2002;291:1297–1301.View ArticlePubMedGoogle Scholar
- Kim TH, Chung KY, Manglik A, Hansen AL, Dror RO, Mildorf TJ, Shaw DE, Kobilka BK, Prosser RS. The Role of Ligands on the Equilibria Between Functional States of a G Protein-Coupled Receptor. J Am Chem Soc. 2013;135:9465–74.View ArticlePubMedPubMed CentralGoogle Scholar
- Bhattacharya S, Vaidehi N. Differences in Allosteric Communication Pipelines in the Inactive and Active States of a GPCR. Biophys J. 2014;107:422–34.View ArticlePubMedPubMed CentralGoogle Scholar
- Niesen MJM, Bhattacharya S, Vaidehi N. The Role of Conformational Ensembles in Ligand Recognition in G-Protein Coupled Receptors. J Am Chem Soc. 2011;133:13197–204.View ArticlePubMedGoogle Scholar
- Liu JJ, Horst R, Katritch V, Stevens RC, Wüthrich K. Biased Signaling Pathways in β2-adrenergic Receptor Characterized by 19F-NMR. Science. 2012;335:1106–10.View ArticlePubMedPubMed CentralGoogle Scholar
- Pieper U, Eswar N, Webb BM, Eramian D, Kelly L, Barkan DT, Carter H, Mankoo P, Karchin R, Marti-Renom MA, Davis FP, Sali A. MODBASE, a Database of Annotated Comparative Protein Structure Models and Associated Resources. Nucleic Acids Res. 2009;37(Database issue):D347–54.View ArticlePubMedGoogle Scholar
- Humphrey W, Dalke A, Schulten K. VMD - Visual Molecular Dynamics. J Mol Graph. 1996;14:33–8.View ArticlePubMedGoogle Scholar
- Lomize MA, Lomize AL, Pogozheva ID, Mosberg HI. OPM: orientations of proteins in membranes database. Bioinformatics. 2006;22:623–5.View ArticlePubMedGoogle Scholar
- Essmann U, Perera L, Berkowitz ML, Darden T, Lee H, Pedersen LG. A Smooth Particle Mesh Ewald Method. J Chem Phys. 1995;103(19):8576–93.View ArticleGoogle Scholar
- Phillips JC, Braun R, Wang W, Gumbart J, Tajkhorshid E, Villa E, Chipot C, Skeel RD, Kale L, Schulten K. Scalable Molecular Dynamics with NAMD. J Comput Chem. 2005;26(16):1781–802.View ArticlePubMedPubMed CentralGoogle Scholar
- Schlenkrich M, Brickmann J, MacKerell AD Jr, Karplus M: An empirical potential energy function for phospholipids: criteria for parameter optimization and applications. In Biological Membranes: A Molecular Perspective from Computation and Experiment. 1st edition. Edited by Merz KM Jr, Roux B. Birkhauser, Boston; 1996.Google Scholar
- Feller SE, Yin D, Pastor RW, MacKerell Jr AD. Molecular dynamics simulation of unsaturated lipids at Low hydration: parametrization and comparison with diffraction studies. Biophys J. 1997;73:2269–79.View ArticlePubMedPubMed CentralGoogle Scholar
- Mackerell AD, Bashford D, Bellott M, Dunbrack RL, Evanseck JD, Field MJ, Fischer S, Gao J, Guo H, Ha S, Joseph D, Kuchnir L, Kuczera K, Lau FTK, Mattos C, Michnick S, Ngo T, Nguyen DT, Prodhom B, Roux B, Schlenkrich M, Smith J, Stote R, Straub J, Watanabe M, Wiorkiewicz-Kuczera J, Yin D, Karplus M. Self-consistent parameterization of biomolecules for molecular modeling and condensed phase simulations. FASEB J. 1992;6:A143–A143.Google Scholar
- Mackerell AD, Bashford D, Bellott M, Dunbrack RL, Evanseck JD, Field MJ, Fischer S, Gao J, Guo H, Ha S, Joseph D, Kuchnir L, Kuczera K, Lau FTK, Mattos C, Michnick S, Ngo T, Nguyen DT, Prodhom B, Roux B, Schlenkrich M, Smith J, Stote R, Straub J, Watanabe M, Wiorkiewicz-Kuczera J, Yin D, Karplus M. All-atom empirical potential for molecular modeling and dynamics studies of proteins. J Phys Chem B. 1998;102:3586–616.View ArticlePubMedGoogle Scholar
- Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML. Comparison of simple potential functions for simulating liquid water. J Chem Phys. 1983;79:926–35.View ArticleGoogle Scholar
- Feller SE, Zhang Y, Pastor RW. Computer-Simulation of Liquid/Liquid Interfaces II. Surface-Tension Area Dependence of a Bilayer and Monolayer. J Chem Phys. 1995;103:10267–76.View ArticleGoogle Scholar
- Ryckaert JP, Ciccotti G, Berendsen HJC. Numerical integration of Cartesian equations of motion of a system with constraints – molecular dynamics of N-alkanes. J Comput Phys. 1977;23:327–41.View ArticleGoogle Scholar
- Elling CE, Frimurer TM, Gerlach LO, Jorgensen R, Holst B, Schwartz TW. Metal ion site engineering indicates a global toggle switch model for seven-transmembrane receptor activation. J Biol Chem. 2006;281:17337–46.View ArticlePubMedGoogle Scholar
- Elling CE, Thirstrup K, Holst B, Schwartz TW. Conversion of agonist site to metal-ion chelator site in the beta(2)-adrenergic receptor. Proc Natl Acad Sci U S A. 1999;96:12322–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Liapakis G, Ballesteros JA, Papachristou S, Chan WC, Chen X, Jav- itch JA. The forgotten serine. A critical role for Ser-2035.42 in ligand binding to and activation of the beta 2-adrenergic receptor. J Biol Chem. 2000;275:37779–88.View ArticlePubMedGoogle Scholar
- Sato T, Kobayashi H, Nagao T, Kurose H. Ser203 as well as Ser204 and Ser207 in fifth transmembrane domain of the human beta2-adrenoceptor contributes to agonist binding and receptor activation. Br J Pharmacol. 1999;128:272–4.View ArticlePubMedPubMed CentralGoogle Scholar
- Gouldson PR, Winn PJ, Reynolds CA. A molecular dynamics approach to receptor mapping: application to the 5HT3 and beta 2-adrenergic receptors. J Med Chem. 1995;38:4080–6.View ArticlePubMedGoogle Scholar
- Wieland K, Zuurmond HM, Krasel C, IJzerman AP, Lohse MJ. Involvement of Asn-293 in stereospecific agonist recognition and in activation of the beta 2-adrenergic receptor. Proc Natl Acad Sci U S A. 1996;93:9276–81.View ArticlePubMedPubMed CentralGoogle Scholar
- Dror RO, Arlow DH, Maragakis P, Mildorf TJ, Pan AC, Xu H, Borhani DW, Shaw DE. Activation mechanism of the β2-adrenergic receptor. PNAS. 2011;108:18684–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Bokoch MP, Zou Y, Rasmussen SG, Liu CW, Nygaard R, Rosenbaum DM, Fung JJ, Choi H, Thian FS, Kobilka TS, Puglisi JD, Weis WI, Pardo L, Prosser RS, Mueller L, Kobilka BK. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor. Nature. 2010;463:108–12.View ArticlePubMedPubMed CentralGoogle Scholar