Activation mechanisms of the first sphingosine‐1‐phosphate receptor

Activation of the first sphingosine‐1‐phosphate receptor (S1PR₁) promotes permeability of the blood brain barrier, astrocyte and neuronal protection, and lymphocyte egress from secondary lymphoid tissues. Although an agonist often activates the S1PR₁, the receptor exhibits high levels of basal activity. In this study, we performed long‐timescale molecular dynamics and accelerated molecular dynamics (aMD) simulations to investigate activation mechanisms of the ligand‐free (apo) S1PR₁. In the aMD enhanced sampling simulations, we observed four independent events of activation, which is characterized by close interaction between Y311^7.53 and Y221^5.58 and increased distance between the intracellular ends of transmembrane (TM) helices 3 and 6. Although TM helices TM3, TM6, TM5 and, TM7 are associated with GPCR activation, we discovered that their movements are not necessarily correlated during activation. Instead, TM5 showed a decreased correlation with each of these regions during activation. During activation of the apo receptor, Y221^5.58 and Y311^7.53 became more solvated, because a water channel formed in the intracellular pocket. Additionally, a lipid molecule repeatedly entered the receptor between the extracellular ends of TM1 and TM7, providing important insights into the pathway of ligand entry into the S1PR₁.     

Selectivity and activation of dopamine D3R from molecular dynamics

D₃ receptor, a member of dopamine (DA) D₂-like receptor family, which belongs to class A of G-protein coupled receptors (GPCRs), has been reported to play a critical role in neuropsychiatric disorders. Recently, the crystal structure of human dopamine D3 receptor was reported, which facilitates structure-based drug discovery of D3R significantly. We dock D3R-selective compounds into the crystal structure of D3R and homology structure of D2R. Then we perform 20?ns molecular dynamics (MD) of the receptor with selective compounds bound in explicit lipid and water. Our docking and MD results indicate the important residues related to the selectivity of D3R. Specifically, residue Thr^7.39 in D3R may contribute to the high selectivity of R-22 with D3R. Meanwhile, the 4-carbon linker and phenylpiperazine of R-22 improve the binding affinity and the selectivity with D3R. We also dock the agonists, including dopamine, into D3R and perform MD. Our molecular dynamics results of D3R with agonist bound show strong conformational changes from TM5, TM6, and TM7, outward movement of intracellular part of TM6, fluctuation of “ionic lock” motif and conformational change of Tyr^7.53, which is consistent with recent crystal structures of active GPCRs and illustrates the dynamical process during activation. Our results reveal the mechanism of selectivity and activation for D3R, which is important for developing high selective antagonists and agonists for D3R.     

Lipid Receptor S1P1 Activation Scheme Concluded from Microsecond All-Atom Molecular Dynamics Simulations

Sphingosine 1-phosphate (S1P) is a lysophospholipid mediator which activates G protein–coupled sphingosine 1-phosphate receptors and thus evokes a variety of cell and tissue responses including lymphocyte trafficking, endothelial development, integrity, and maturation. We performed five all-atom 700 ns molecular dynamics simulations of the sphingosine 1-phosphate receptor 1 (S1P₁) based on recently released crystal structure of that receptor with an antagonist. We found that the initial movements of amino acid residues occurred in the area of highly conserved W269^6.48 in TM6 which is close to the ligand binding location. Those residues located in the central part of the receptor and adjacent to kinks of TM helices comprise of a transmission switch. Side chains movements of those residues were coupled to the movements of water molecules inside the receptor which helped in the gradual opening of intracellular part of the receptor. The most stable parts of the protein were helices TM1 and TM2, while the largest movement was observed for TM7, possibly due to the short intracellular part starting with a helix kink at P^7.50, which might be the first helix to move at the intracellular side. We show for the first time the detailed view of the concerted action of the transmission switch and Trp (W^6.48) rotamer toggle switch leading to redirection of water molecules flow in the central part of the receptor. That event is a prerequisite for subsequent changes in intracellular part of the receptor involving water influx and opening of the receptor structure.     

Mitocryptide-2, a neutrophil-activating cryptide, is a specific endogenous agonist for formyl-peptide receptor-like 1

Peptides simultaneously produced during maturation and degradation of peptidergic hormones and functional proteins have recently become a great interest because they display unpredictably different biological roles than the parent proteins. Namely, we discovered two novel functional cryptic peptides, mitocryptide-1 (MCT-1) and mitocryptide-2 (MCT-2), hidden in mitochondrial cytochrome c oxidase and cytochrome b, that efficiently induced neutrophilic migration and activation at nanomolar concentrations. We named these functional “cryptic” peptides hidden in protein structures as “cryptides.” In this study, we investigated the receptor molecules and cellular signaling mechanisms for neutrophil-activating N-formylated cryptide MCT-2. In order to identify the receptor molecules, we established HEK-293 cells stably expressing either formyl-peptide receptor (FPR) or its homologue FPR-like 1 (FPRL1), because neutrophilic cells express these receptor molecules which recognize N-formylated peptides. We observed that MCT-2 directly bound to FPRL1 and promoted an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), and neither interacted with nor activated FPR, demonstrating that MCT-2 is a specific agonist for FPRL1. Moreover, MCT-2 induced not only [Ca²⁺]_i increase and phosphorylation of extracellular signal-regulated protein kinases 1 and 2, but also β-hexosaminidase release in neutrophilic/granulocytic cells differentiated from HL-60 cells. Such signaling events were diminished by pretreatment with pertussis toxin, indicating that MCT-2-promoted neutrophilic function is a consequence of G_i- or G_o-type G protein-dependent intracellular signaling events via FPRL1 activation. These findings suggest that MCT-2, a cryptide derived from mitochondrial cytochrome b, is a specific endogenous agonist for FPRL1 which is proposed to play key roles in inflammatory responses but whose physiological agonists are equivocal.     

Modeling of ligand binding to G protein coupled receptors: cannabinoid CB1, CB2 and adrenergic β2AR

Cannabinoid and adrenergic receptors belong to the class A (similar to rhodopsin) G protein coupled receptors. Docking of agonists and antagonists to CB₁ and CB₂ cannabinoid receptors revealed the importance of a centrally located rotamer toggle switch and its possible participation in the mechanism of agonist/antagonist recognition. The switch is composed of two residues, F3.36 and W6.48, located on opposite transmembrane helices TM3 and TM6 in the central part of the membranous domain of cannabinoid receptors. The CB₁ and CB₂ receptor models were constructed based on the adenosine A_2A receptor template. The two best scored conformations of each receptor were used for the docking procedure. In all poses (ligand-receptor conformations) characterized by the lowest ligand-receptor intermolecular energy and free energy of binding the ligand type matched the state of the rotamer toggle switch: antagonists maintained an inactive state of the switch, whereas agonists changed it. In case of agonists of β₂AR, the (R,R) and (S,S) stereoisomers of fenoterol, the molecular dynamics simulations provided evidence of different binding modes while preserving the same average position of ligands in the binding site. The (S,S) isomer was much more labile in the binding site and only one stable hydrogen bond was created. Such dynamical binding modes may also be valid for ligands of cannabinoid receptors because of the hydrophobic nature of their ligand-receptor interactions. However, only very long molecular dynamics simulations could verify the validity of such binding modes and how they affect the process of activation.     

Ligand-stabilized conformational states of human beta(2) adrenergic receptor: insight into G-protein-coupled receptor activation

G-protein-coupled receptors (GPCRs) are known to exist in dynamic equilibrium between inactive- and several active-state conformations, even in the absence of a ligand. Recent experimental studies on the β₂ adrenergic receptor (β₂AR) indicate that structurally different ligands with varying efficacies trigger distinct conformational changes and stabilize different receptor conformations. We have developed a computational method to study the ligand-induced rotational orientation changes in the transmembrane helices of GPCRs. This method involves a systematic spanning of the rotational orientation of the transmembrane helices (TMs) that are in the vicinity of the ligand for predicting the helical rotations that occur on ligand binding. The predicted ligand-stabilized receptor conformations are characterized by a simultaneous lowering of the ligand binding energy and a significant gain in interhelical and receptor-ligand hydrogen bonds. Using the β₂AR as a model, we show that the receptor conformational state depends on the structure and efficacy of the ligand for a given signaling pathway. We have studied the ligand-stabilized receptor conformations of five different ligands, a full agonist, norepinephrine; a partial agonist, salbutamol; a weak partial agonist, dopamine; a very weak agonist, catechol; and an inverse agonist, ICI-115881. The predicted ligand-stabilized receptor models correlate well with the experimentally observed conformational switches in β₂AR, namely, the breaking of the ionic lock between R131^3.50 at the intracellular end of TM3 (part of the DRY motif) and E268^6.30 on TM6, and the rotamer toggle switch on W286^6.48 on TM6. In agreement with trp-bimane quenching experiments, we found that norepinephrine and dopamine break the ionic lock and engage the rotamer toggle switch, whereas salbutamol, a noncatechol partial agonist only breaks the ionic lock, and the weak agonist catechol only engages the rotamer toggle switch. Norepinephrine and dopamine occupy the same binding region, between TM3, TM5, and TM6, whereas the binding site of salbutamol is shifted toward TM4. Catechol binds deeper into the protein cavity compared to the other ligands, making contact with TM5 and TM6. A part of the catechol binding site overlaps with those of dopamine and norepinephrine but not with that of salbutamol. Virtual ligand screening on 10,060 ligands on the norepinephrine-stabilized receptor conformation shows an enrichment of 38% compared to ligand unbound receptor conformation. These results show that ligand-induced conformational changes are important for developing functionally specific drugs that will stabilize a particular receptor conformation. These studies represent the first step toward a more universally applicable computational method for studying ligand efficacy and GPCR activation.     

In Silico Study of the Structurally Similar ORL1 Receptor Agonist and Antagonist Pairs Reveal Possible Mechanism of Receptor Activation

An opioid receptor like (ORL1) receptor is a member of a family of G-protein coupled receptors. It is a new pharmaceutical target with broad therapeutic potential in the regulation of important biological functions such as nociception, mood disorders, drug abuse, learning or cardiovascular control. The crystal structure of this receptor in complex with an antagonist was determined recently (PDB ID: 4EA3). By removing the ligand and subjecting the empty receptor to molecular dynamics simulation in a solvated lipid membrane we obtained an optimized ORL1 receptor structure which could be used in a subsequent docking study of two structurally similar agonist–antagonist ligand pairs. Ligands were docked to the empty ORL1 receptor (with and without the third intracellular loop, IC3) in different orientations, and the resulting complexes were monitored during molecular dynamics simulation in order to see how the subtle differences in structure of agonists and antagonists might affect ligand–receptor interactions and trigger receptor activation. It was established that agonists and antagonists bound to the same, relatively large, binding site in the receptor, created by residues from transmembrane helices TM2, TM3, TM5, TM6 and TM7 and close to the extra cellular end of the receptor bundle. The key difference between these two types of ligands is interaction with residue Val283^6.55 and a flexibility of ligand molecules. Ligands that cannot easily avoid this interaction will initiate movement of the intracellular end of TM6 (by a mechanism which involves Met134^3.36 and several aminoacids of TM5) and possibly activate the receptor when assisted by G-protein.     

The role of helices 5 and 6 on the human β1-adrenoceptor activation mechanism

The human β₁-adrenoceptor (β₁AR) is a G-protein-coupled receptor (GPCR) involved in sympathetic system regulation through agonist-induced activation. The conserved CWXP-motif in helix 6 (rotamer toggle switch) is one of the most important activation switches in Class A GPCRs. In order to investigate how the agonist binding disturbs this switch, we carried out molecular dynamics simulations of a hβ₁AR model in the apo and R-noradrenaline-bound forms. The results show that the agonist binding changes the β₁-angle distribution of Cys336, Trp337 and Phe341 residues and increases the helix 6 bending. Overall, we provide a functional hβ₁AR model, showing how the rotamer toggle switch mechanism works at atomic level.     

Probing the Interaction of SR141716A with the CB1 Receptor

SR141716A binds selectively to the brain cannabinoid (CB1) receptor and exhibits a potent inverse agonist/antagonist activity. Although SR141716A, also known as rimonabant, has been withdrawn from the market due to severe side effects, there remains interest in some of its many potential medical applications. Consequently, it is imperative to understand the mechanism by which SR141716A exerts its inverse agonist activity. As a result of using an approach combining mutagenesis and molecular dynamics simulations, we determined the binding mode of SR141716A. We found from the simulation of the CB1-SR141716A complex that SR141716A projects toward TM5 to interact tightly with the major binding pocket, replacing the coordinated water molecules, and secures the Trp-356^6.48 rotameric switch in the inactive state to promote the formation of an extensive water-mediated H-bonding network to the highly conserved SLAXAD and NPXXY motifs in TM2/TM7. We identify for the first time the involvement of the minor binding pocket formed by TM2/TM3/TM7 for SR141716A binding, which complements the major binding pocket formed by TM3/TM5/TM6. Simulation of the F174^2.61A mutant CB1-SR141716A complex demonstrates the perturbation of TM2 that attenuates SR141716A binding indirectly. These results suggest SR141716A exerts inverse agonist activity through the stabilization of both TM2 and TM5, securing the Trp-356^6.48 rotameric switch and restraining it from activation.     

Agonist-induced conformational changes in bovine rhodopsin: insight into activation of G-protein-coupled receptors

Activation of G-protein-coupled receptors (GPCRs) is initiated by conformational changes in the transmembrane (TM) helices and the intra- and extracellular loops induced by ligand binding. Understanding the conformational changes in GPCRs leading to activation is imperative in deciphering the role of these receptors in the pathology of diseases. Since the crystal structures of activated GPCRs are not yet available, computational methods and biophysical techniques have been used to predict the structures of GPCR active states. We have recently applied the computational method LITiCon to understand the ligand-induced conformational changes in β₂-adrenergic receptor by ligands of varied efficacies. Here we report a study of the conformational changes associated with the activation of bovine rhodopsin for which the crystal structure of the inactive state is known. Starting from the inactive (dark) state, we have predicted the TM conformational changes that are induced by the isomerization of 11-cis retinal to all-trans retinal leading to the fully activated state, metarhodopsin II. The predicted active state of rhodopsin satisfies all of the 30 known experimental distance constraints. The predicted model also correlates well with the experimentally observed conformational switches in rhodopsin and other class A GPCRs, namely, the breaking of the ionic lock between R135^3.50 at the intracellular end of TM3 (part of the DRY motif) and E247^6.30 on TM6, and the rotamer toggle switch on W265^6.48 on TM6. We observe that the toggling of the W265^6.48 rotamer modulates the bend angle of TM6 around the conserved proline. The rotamer toggling is facilitated by the formation of a water wire connecting S298^7.45, W265^6.48 and H211^5.46. As a result, the intracellular ends of TMs 5 and 6 move outward from the protein core, causing large conformational changes at the cytoplasmic interface. The predicted outward movements of TM5 and TM6 are in agreement with the recently published crystal structure of opsin, which is proposed to be close to the active-state structure. In the predicted active state, several residues in the intracellular loops, such as R69, V139^3.54, T229, Q237, Q239, S240, T243 and V250^6.33, become more water exposed compared to the inactive state. These residues may be involved in mediating the conformational signal from the receptor to the G protein. From mutagenesis studies, some of these residues, such as V139^3.54, T229 and V250^6.33, are already implicated in G-protein activation. The predicted active state also leads to the formation of new stabilizing interhelical hydrogen-bond contacts, such as those between W265^6.48 and H211^5.46 and E122^3.37 and C167^4.56. These hydrogen-bond contacts serve as potential conformational switches offering new opportunities for future experimental investigations. The calculated retinal binding energy surface shows that binding of an agonist makes the receptor dynamic and flexible and accessible to many conformations, while binding of an inverse agonist traps the receptor in the inactive state and makes the other conformations inaccessible.     

Long Range Effect of Mutations on Specific Conformational Changes in the Extracellular Loop 2 of Angiotensin II Type 1 Receptor

The topology of the second extracellular loop (ECL2) and its interaction with ligands is unique in each G protein-coupled receptor. When the orthosteric ligand pocket located in the transmembrane (TM) domain is occupied, ligand-specific conformational changes occur in the ECL2. In more than 90% of G protein-coupled receptors, ECL2 is tethered to the third TM helix via a disulfide bond. Therefore, understanding the extent to which the TM domain and ECL2 conformations are coupled is useful. To investigate this, we examined conformational changes in ECL2 of the angiotensin II type 1 receptor (AT1R) by introducing mutations in distant sites that alter the activation state equilibrium of the AT1R. Differential accessibility of reporter cysteines introduced at four conformation-sensitive sites in ECL2 of these mutants was measured. Binding of the agonist angiotensin II (AngII) and inverse agonist losartan in wild-type AT1R changed the accessibility of reporter cysteines, and the pattern was consistent with ligand-specific “lid” conformations of ECL2. Without agonist stimulation, the ECL2 in the gain of function mutant N111G assumed a lid conformation similar to AngII-bound wild-type AT1R. In the presence of inverse agonists, the conformation of ECL2 in the N111G mutant was similar to the inactive state of wild-type AT1R. In contrast, AngII did not induce a lid conformation in ECL2 in the loss of function D281A mutant, which is consistent with the reduced AngII binding affinity in this mutant. However, a lid conformation was induced by [Sar¹,Gln²,Ile⁸] AngII, a specific analog that binds to the D281A mutant with better affinity than AngII. These results provide evidence for the emerging paradigm of domain coupling facilitated by long range interactions at distant sites on the same receptor.     

Role of the extracellular amino terminus and first membrane-spanning helix of dopamine D1 and D5 receptors in shaping ligand selectivity and efficacy

Transmembrane (TM) helices of human D1-like dopaminergic receptors (hD1R and hD5R) harbor the same residues implicated in ligand binding and activation of catecholamine G protein-coupled receptors (GPCRs). Yet, hD1R and hD5R naturally display the distinct functional properties shared by wild type and constitutively active mutant GPCRs, respectively. Interestingly, we show in the present study that a class of synthetic phenylbenzazepine agonists containing a methyl on the azepine ring exhibited lower affinity for the more constitutively activated hD5R. These results cannot be explained by the “allosteric ternary complex model” postulating a higher agonist affinity for constitutively active GPCRs. We have also explored the functional role of distinct extracellular amino terminus (NT) and TM1 regions of hD1R and hD5R using a chimerical approach. Of these two regions, our studies suggest that TM1 predominantly shapes D1-like ligand affinity and selectivity. Additionally, NT and TM1 of hD1R and hD5R play no role in receptor constitutive activity but differentially modulate dopamine-mediated responsiveness. The TM1 exchange mediated drastic changes in intrinsic efficacy and activity of phenylbenzazepine drugs displaying partial agonism at hD1R and hD5R. Phenylbenzazepines were converted into strong partial agonists or full agonists in cells expressing hD1R-TM1^D5 chimera while being switched from full agonists to partial agonists and partial agonists to antagonists in cells harboring hD5R-TM1^D1 chimera. TM1 exchange had no effect on antipsychotic-mediated inverse agonism. In summary, our study shows that NT and TM1 of D1-like receptors control ligand binding and agonist-induced activation, poising these regions as important structural determinants for catecholamine GPCR function.     

His499 Regulates Dimerization and Prevents Oncogenic Activation by Asparagine Mutations of the Human Thrombopoietin Receptor

Ligand binding to the extracellular domain of the thrombopoietin receptor (TpoR) imparts a specific orientation on the transmembrane (TM) and intracellular domains of the receptors that is required for physiologic activation via receptor dimerization. To map the inactive and active dimeric orientations of the TM helices, we performed asparagine (Asn)-scanning mutagenesis of the TM domains of the murine and human TpoR. Substitution of Asn at only one position (S505N) activated the human receptor, whereas Asn substitutions at several positions activated the murine receptor. Second site mutational studies indicate that His⁴⁹⁹ near the N terminus of the TM domain is responsible for protecting the human receptor from activation by Asn mutations. Structural studies reveal that the sequence preceding His⁴⁹⁹ is helical in the murine receptor but non-helical in peptides corresponding to the TM domain of the inactive human receptor. The activating S505N mutation and the small molecule agonist eltrombopag both induce helix in this region of the TM domain and are associated with dimerization and activation of the human receptor. Thus, His⁴⁹⁹ regulates the activation of human TpoR and provides additional protection against activating mutations, such as oncogenic Asn mutations in the TM domain.     

A Pepducin Derived from the Third Intracellular Loop of FPR2 Is a Partial Agonist for Direct Activation of This Receptor in Neutrophils But a Full Agonist for Cross-Talk Triggered Reactivation of FPR2

We recently described a novel receptor cross-talk mechanism in neutrophils, unique in that the signals generated by the PAF receptor (PAFR) and the ATP receptor (P2Y₂R) transfer formyl peptide receptor 1 (FPR1) from a desensitized (non-signaling) state back to an actively signaling state (Forsman H et al., PLoS One, 8:e60169, 2013; Önnheim K, et al., Exp Cell Res, 323∶209, 2014). In addition to the G-protein coupled FPR1, neutrophils also express the closely related receptor FPR2. In this study we used an FPR2 specific pepducin, proposed to work as an allosteric modulator at the cytosolic signaling interface, to determine whether the cross-talk pathway is utilized also by FPR2. The pepducin used contains a fatty acid linked to a peptide sequence derived from the third intracellular loop of FPR2, and it activates as well as desensensitizes this receptor. We now show that neutrophils desensitized with the FPR2-specific pepducin display increased cellular responses to stimulation with PAF or ATP. The secondary PAF/ATP induced response was sensitive to FPR2-specific inhibitors, disclosing a receptor cross-talk mechanism underlying FPR2 reactivation. The pepducin induced an activity in naïve cells similar to that of a conventional FPR2 agonist, but with lower potency (partial efficacy), meaning that the pepducin is a partial agonist. The PAF- or ATP-induced reactivation was, however, much more pronounced when neutrophils had been desensitized to the pepducin as compared to cells desensitized to conventional agonists. The pepducin should thus in this respect be classified as a full agonist. In summary, we demonstrate that desensitized FPR2 can be transferred back to an actively signaling state by receptor cross-talk signals generated through PAFR and P2Y₂R, and the difference in agonist potency with respect to pepducin-induced direct receptor activation and cross-talk reactivation of FPR2 puts the concept of functional selectivity in focus.     

Rubimetide,humanin, and MMK1 exert anxiolytic-like activities via the formyl peptide receptor 2 in mice followed by the successive activation of DP1, A2A,and GABAA receptors

Rubimetide (Met-Arg-Trp), which had been isolated as an antihypertensive peptide from an enzymatic digest of spinach ribulose-bisphosphate carboxylase/oxygenase (Rubisco), showed anxiolytic-like activity prostaglandin (PG) D₂-dependent manner in the elevated plus-maze test after administration at a dose of 0.1 mg/kg (ip.) or 1 mg/kg (p.o.) in male mice of ddY strain. In this study, we found that rubimetide has weak affinities for the FPR1 and FPR2, subtypes of formyl peptide receptor (FPR). The anxiolytic-like activity of rubimetide (0.1 mg/kg, ip.) was blocked by WRW4, an antagonist of FPR2, but not by Boc-FLFLF, an antagonist of FPR1, suggesting that the anxiolytic-like activity was mediated by the FPR2. Humanin, an endogenous agonist peptide of the FPR2, exerted an anxiolytic-like activity after intracerebroventricular (icv) administration, which was also blocked by WRW4. MMK1, a synthetic agonist peptide of the FPR2, also exerted anxiolytic-like activity. Thus, FPR2 proved to mediate anxiolytic-like effect as the first example of central effect exerted by FPR agonists. As well as the anxiolytic-like activity of rubimetide, that of MMK1 was blocked by BW A868C, an antagonist of the DP₁-receptor. Furthermore, anxiolytic-like activity of rubimetide was blocked by SCH58251 and bicuculline, antagonists for adenosine A_2A and GABA_A receptors, respectively. From these results, it is concluded that the anxiolytic-like activities of rubimetide and typical agonist peptides of the FPR2 were mediated successively by the PGD₂-DP₁ receptor, adenosine-A_2A receptor, and GABA-GABA_A receptor systems downstream of the FPR2.     

The conformation of the cytoplasmic helix 8 of the CB1 cannabinoid receptor using NMR and circular dichroism

The cytoplasmic helix domain (fourth cytoplasmic loop, helix 8) of numerous GPCRs such as rhodopsin and the β-adrenergic receptor exhibits unique structural and functional characteristics. Computational models also predict the existence of such a structural motif within the CB1 cannabinoid receptor, another member of the G-protein coupled receptor superfamily. To gain insights into the conformational properties of this GPCR component, a peptide corresponding to helix 8 of the CB1 receptor with a small contiguous segment from transmembrane helix 7 (TM7) was chemically synthesized and its secondary structure determined by circular dichroism (CD) and solution NMR spectroscopy. Our studies in DPC and SDS micelles revealed significant α-helical structure while in an aqueous medium, the peptide exhibited a random coil configuration. The relative orientation of helix 8 within the CB1 receptor was obtained from intermolecular ³¹P-¹H and ¹H-¹H NOE measurements. Our results suggest that in the presence of an amphipathic membrane environment, helix 8 assumes an alpha helical structure with an orientation parallel to the phospholipid membrane surface and perpendicular to TM7. In this model, positively charged side chains interact with the lipid headgroups while the other polar side chains face the aqueous region. The above observations may be relevant to the activation/deactivation of the CB1 receptor.     

Dopamine D1 receptor and serotonin 5-HT1A receptor agonist effects of the natural product (–)-stepholidine: molecular modelling and dynamics simulations

In this study, by homology modelling and molecular dynamics (MD) simulation, models of l-stepholidine (l-SPD) activating the 5-HT_1A and D₁ receptors were constructed. In 100-ns MD simulations, the D₁ and 5-HT_1A receptors were activated by the partial agonist l-SPD, conforming with the global toggle switch activation model and the sequential activation model. The residues Y^7.53 and Y^5.58 swing significantly between different transmembrane (TM) domains after activation. Similarities between D₁ and 5-HT_1A included (1) the outward motion of TM-5; (2) the ionic lock was independent of the tilt of TM-6 and (3) there was an apparent bending of TM-6, and the ring of l-SPD formed strong π–π interactions with residue W^6.48. Differences between the two included the following: (1) in 5-HT_1A, l-SPD formed a hydrogen bond with Ala172^5.46 of TM-5, and the intracellular end of TM-5 moved outward slowly; that hydrogen bond did not form with the D₁ receptor; (2) l-SPD formed stronger interactions with D^3.32 and W^6.48 in the D₁ receptor than in the 5-HT_1A receptor and (3) the hydrogen bonding network was somewhat different in SPD-5-HT_1A and SPD-D₁ receptors. We propose the interaction between l-SPD and D^3.32 or/and W^6.48 is the original driving force during the whole activation process.     

Beta2 adrenergic receptor activation. Modulation of the proline kink in transmembrane 6 by a rotamer toggle switch

In many rhodopsin-like G-protein-coupled receptors, agonist binding to a cluster of aromatic residues in TM6 may promote receptor activation by altering the configuration of the TM6 Pro-kink and by the subsequent movement of the cytoplasmic end of TM6 away from TM3. We hypothesized that the highly conserved Cys(6.47), in the vicinity of the conserved Pro(6.50), modulates the configuration of the aromatic cluster and the TM6 Pro-kink through specific interactions in its different rotamer configurations. In the beta(2) adrenergic receptor, mutation of Cys(6.47) to Thr, which in an alpha-helix has a different rotamer distribution from Cys and Ser, produced a constitutively active receptor, whereas the Ser mutant was similar to wild-type receptor. Use of the biased Monte Carlo technique of Conformational Memories showed that the rotamer changes among Cys/Ser/Thr(6.47), Trp(6.48), and Phe(6.52) are highly correlated, representing a rotamer "toggle switch" that may modulate the TM6 Pro-kink. Differential modulation of the accessibility of Cys(6.47) and an engineered Cys(6.52) in wild type and a constitutively active background provides experimental support for the association of this rotamer switch with receptor activation.     

Study of a structurally similar kappa opioid receptor agonist and antagonist pair by molecular dynamics simulations

Among the structurally similar guanidinonaltrindole (GNTI) compounds, 5′-GNTI is an antagonist while 6′-GNTI is an agonist of the κOR opioid receptor. To explore how a subtle alteration of the ligand structure influences the receptor activity, we investigated two concurrent processes: the final steps of ligand binding at the receptor binding site and the initial steps of receptor activation. To trace these early activation steps, the membranous part of the receptor was built on an inactive receptor template while the extracellular loops were built using the ab initio CABS method. We used the simulated annealing procedure for ligand docking and all-atom molecular dynamics simulations to determine the immediate changes in the structure of the ligand–receptor complex. The binding of an agonist, in contrast to an antagonist, induced the breakage of the “3–7 lock” between helices TM3 and TM7. We also observed an action of the extended rotamer toggle switch which suggests that those two switches are interdependent.