RESUMO
Despite the important role of the carboxyl-terminus (Ct) of the activated brain cannabinoid receptor one (CB1) in the regulation of G protein signaling, a structural understanding of interactions with G proteins is lacking. This is largely due to the highly flexible nature of the CB1 Ct that dynamically adapts its conformation to the presence of G proteins. In the present study, we explored how the CB1 Ct can interact with the G protein by building on our prior modeling of the CB1-Gi complex (Shim, Ahn, and Kendall, The Journal of Biological Chemistry 2013;288:32449-32465) to incorporate a complete CB1 Ct (Glu416(Ct)-Leu472(Ct)). Based on the structural constraints from NMR studies, we employed ROSETTA to predict tertiary folds, ZDOCK to predict docking orientation, and molecular dynamics (MD) simulations to obtain two distinct plausible models of CB1 Ct in the CB1-Gi complex. The resulting models were consistent with the NMR-determined helical structure (H9) in the middle region of the CB1 Ct. The CB1 Ct directly interacted with both Gα and Gß and stabilized the receptor at the Gi interface. The results of site-directed mutagenesis studies of Glu416(Ct), Asp423(Ct), Asp428(Ct), and Arg444(Ct) of CB1 Ct suggested that the CB1 Ct can influence receptor-G protein coupling by stabilizing the receptor at the Gi interface. This research provided, for the first time, models of the CB1 Ct in contact with the G protein.
Assuntos
Proteínas de Ligação ao GTP/química , Subunidades Proteicas/química , Receptor CB1 de Canabinoide/química , Sequência de Aminoácidos , Analgésicos/química , Sítios de Ligação , Membrana Celular/química , Membrana Celular/metabolismo , Cicloexanóis/química , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica , Guanosina 5'-O-(3-Tiotrifosfato)/química , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The cannabinoid (CB1) receptor is a member of the rhodopsin-like G protein-coupled receptor superfamily. The human CB1 receptor, which is among the most expressed receptors in the brain, has been implicated in several disease states, including drug addiction, anxiety, depression, obesity, and chronic pain. Different classes of CB1 agonists evoke signaling pathways through the activation of specific subtypes of G proteins. The molecular basis of CB1 receptor coupling to its cognate G protein is unknown. As a first step toward understanding CB1 receptor-mediated G protein signaling, we have constructed a ternary complex structural model of the CB1 receptor and Gi heterotrimer (CB1-Gi), guided by the x-ray structure of ß2-adrenergic receptor (ß2AR) in complex with Gs (ß2AR-Gs), through 824-ns duration molecular dynamics simulations in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer environment. We identified a group of residues at the juxtamembrane regions of the intracellular loops 2 and 3 (IC2 and IC3) of the CB1 receptor, including Ile-218(3.54), Tyr-224(IC2), Asp-338(6.30), Arg-340(6.32), Leu-341(6.33), and Thr-344(6.36), as potential key contacts with the extreme C-terminal helix α5 of Gαi. Ala mutations of these residues at the receptor-Gi interface resulted in little G protein coupling activity, consistent with the present model of the CB1-Gi complex, which suggests tight interactions between CB1 and the extreme C-terminal helix α5 of Gαi. The model also suggests that unique conformational changes in the extreme C-terminal helix α5 of Gα play a crucial role in the receptor-mediated G protein activation.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/química , Modelos Moleculares , Receptor CB1 de Canabinoide/química , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismoRESUMO
The cannabinoid receptor 1 (CB1) is a G protein-coupled receptor primarily expressed in brain tissue that has been implicated in several disease states. CB1 allosteric compounds, such as ORG27569, offer enormous potential as drugs over orthosteric ligands, but their mechanistic, structural, and downstream effects upon receptor binding have not been established. Previously, we showed that ORG27569 enhances agonist binding affinity to CB1 but inhibits G protein-dependent agonist signaling efficacy in HEK293 cells and rat brain expressing the CB1 receptor (Ahn, K. H., Mahmoud, M. M., and Kendall, D. A. (2012) J. Biol. Chem. 287, 12070-12082). Here, we identify the mediators of CB1 receptor internalization and ORG27569-induced G protein-independent signaling. Using siRNA technology, we elucidate an ORG27569-induced signaling mechanism for CB1 wherein ß-arrestin 1 mediates short term signaling to ERK1/2 with a peak at 5 min and other upstream kinase components including MEK1/2 and c-Src. Consistent with these findings, we demonstrate co-localization of CB1-GFP with red fluorescent protein-ß-arrestin 1 upon ORG27569 treatment using confocal microscopy. In contrast, we show the critical role of ß-arrestin 2 in CB1 receptor internalization upon treatment with CP55940 (agonist) or treatment with ORG27569. These results demonstrate for the first time the involvement of ß-arrestin in CB1-biased signaling by a CB1 allosteric modulator and also define the differential role of the two ß-arrestin isoforms in CB1 signaling and internalization.
Assuntos
Arrestinas/metabolismo , Indóis/farmacologia , Piperidinas/farmacologia , Receptor CB1 de Canabinoide/metabolismo , Sítio Alostérico , Animais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/metabolismo , Microscopia Confocal/métodos , Conformação Proteica , Isoformas de Proteínas , Estrutura Secundária de Proteína , RNA Interferente Pequeno/metabolismo , Ratos , Transdução de Sinais , beta-Arrestina 1 , beta-Arrestina 2 , beta-Arrestinas , Proteína Vermelha FluorescenteRESUMO
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(2.61)A 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.
Assuntos
Piperidinas/química , Pirazóis/química , Receptor CB1 de Canabinoide/química , Sequência de Aminoácidos , Sítios de Ligação , Antagonistas de Receptores de Canabinoides/química , Membrana Celular/metabolismo , Colesterol/química , Humanos , Ligação de Hidrogênio , Ligantes , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutação , Ligação Proteica , Receptores de Canabinoides/química , Receptores Acoplados a Proteínas G , Rimonabanto , Transdução de SinaisRESUMO
The classical cannabinoid agonist HU210, a structural analog of (-)-Δ(9)-tetrahydrocannabinol, binds to brain cannabinoid (CB1) receptors and activates signal transduction pathways. To date, an exact molecular description of the CB1 receptor is not yet available. Utilizing the minor binding pocket of the CB1 receptor as the primary ligand interaction site, we explored HU210 binding using lipid bilayer molecular dynamics (MD) simulations. Among the potential ligand contact residues, we identified residues Phe-174(2.61), Phe-177(2.64), Leu-193(3.29), and Met-363(6.55) as being critical for HU210 binding by mutational analysis. Using these residues to guide the simulations, we determined essential cannabinoid-binding domains in the CB1 receptor, including the highly sought after hydrophobic pocket important for the binding of the C3 alkyl chain of classical and nonclassical cannabinoids. Analyzing the simulations of the HU210-CB1 receptor complex, the CP55940-CB1 receptor complex, and the (-)-Δ(9)-tetrahydrocannabinol-CB1 receptor complex, we found that the positioning of the C3 alkyl chain and the aromatic stacking between Trp-356(6.48) and Trp-279(5.43) is crucial for the Trp-356(6.48) rotamer change toward receptor activation through the rigid-body movement of H6. The functional data for the mutant receptors demonstrated reductions in potency for G protein activation similar to the reductions seen in ligand binding affinity for HU210.
Assuntos
Canabinoides/metabolismo , Receptor CB1 de Canabinoide/química , Receptor CB1 de Canabinoide/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Sítios de Ligação , Canabinoides/química , Cicloexanóis/química , Cicloexanóis/metabolismo , Dronabinol/análogos & derivados , Dronabinol/química , Dronabinol/metabolismo , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
Our previous studies have shown that aberrant arachidonic acid metabolism, especially the 5-lipoxygenase (5-Lox) pathway, is involved in oral carcinogenesis and can be targeted for cancer prevention. To develop potent topical agents for oral cancer chemoprevention, 5 known 5-Lox inhibitors from dietary and synthetic sources (Zileuton, ABT-761, licofelone, curcumin, and garcinol) were evaluated in silico for their potential efficacy. Garcinol, a polyisoprenylated benzophenone from the fruit rind of Garcinia spp., was found to be a promising agent based on the calculation of a theoretical activity index. Computer modeling showed that garcinol well fit the active site of 5-Lox, and potentially inhibited enzyme activity through interactions between the phenolic hydroxyl groups and the non-heme catalytic iron. In a short-term study on 7,12-dimethylbenz[a]anthracene (DMBA)-treated hamster cheek pouch, topical garcinol suppressed leukotriene B4 (LTB4) biosynthesis and inhibited inflammation and cell proliferation in the oral epithelium. In a long-term carcinogenesis study, topical garcinol significantly reduced the size of visible tumors, the number of cancer lesions, cell proliferation, and LTB4 biosynthesis. These results demonstrated that topical application of a 5-Lox inhibitor, garcinol, had chemopreventive effect on DMBA-induced hamster cheek pouch carcinogenesis.
Assuntos
9,10-Dimetil-1,2-benzantraceno , Anticarcinógenos/administração & dosagem , Carcinógenos , Inibidores de Lipoxigenase/administração & dosagem , Neoplasias Bucais/prevenção & controle , Terpenos/administração & dosagem , Animais , Proliferação de Células/efeitos dos fármacos , Bochecha , Cricetinae , Leucotrieno B4/antagonistas & inibidores , Leucotrieno B4/biossíntese , Masculino , Mesocricetus , Neoplasias Bucais/induzido quimicamente , Neoplasias Bucais/patologia , Extratos VegetaisRESUMO
The G-protein-coupled receptor (GPCR) second extracellular loop (E2) is known to play an important role in receptor structure and function. The brain cannabinoid (CB(1)) receptor is unique in that it lacks the interloop E2 disulfide linkage to the transmembrane (TM) helical bundle, a characteristic of many GPCRs. Recent mutation studies of the CB(1) receptor, however, suggest the presence of an alternative intraloop disulfide bond between two E2 Cys residues. Considering the oxidation state of these Cys residues, we determine the molecular structures of the 17-residue E2 in the dithiol form (E2(dithiol)) and in the disulfide form (E2(disulfide)) of the CB(1) receptor in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer, using a combination of simulated annealing and molecular dynamics simulation approaches. We characterize the CB(1) receptor models with these two E2 forms, CB(1)(E2(dithiol)) and CB(1)(E2(disulfide)), by analyzing interaction energy, contact number, core crevice, and cross correlation. The results show that the distinct E2 structures interact differently with the TM helical bundle and uniquely modify the TM helical topology, suggesting that E2 of the CB(1) receptor plays a critical role in stabilizing receptor structure, regulating ligand binding, and ultimately modulating receptor activation. Further studies on the role of E2 of the CB(1) receptor are warranted, particularly comparisons of the ligand-bound form with the present ligand-free form.
Assuntos
Domínios e Motivos de Interação entre Proteínas , Receptor CB1 de Canabinoide/química , Motivos de Aminoácidos , Sítios de Ligação , Dicroísmo Circular , Cistina/química , Ditiotreitol/química , Humanos , Simulação de Dinâmica Molecular , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptor CB1 de Canabinoide/metabolismo , Triptofano/químicaRESUMO
Δ9-tetrahydrocannabinol (Δ9-THC), the main active ingredient of Cannabis sativa (marijuana), interacts with the human brain cannabinoid (CB1) receptor and mimics pharmacological effects of endocannabinoids (eCBs) like N-arachidonylethanolamide (AEA). Due to its flexible nature of AEA structure with more than 15 rotatable bonds, establishing its binding mode to the CB1 receptor is elusive. The aim of the present study was to explore possible binding conformations of AEA within the binding pocket of the CB1 receptor confirmed in the recently available X-ray crystal structures of the CB1 receptor and predict essential AEA binding domains. We performed long time molecular dynamics (MD) simulations of plausible AEA docking poses until its receptor binding interactions became optimally established. Our simulation results revealed that AEA favors to bind to the hydrophobic channel (HC) of the CB1 receptor, suggesting that HC holds essential significance in AEA binding to the CB1 receptor. Our results also suggest that the Helix 2 (H2)/H3 region of the CB1 receptor is an AEA binding subsite privileged over the H7 region.
Assuntos
Ácidos Araquidônicos/química , Endocanabinoides/química , Alcamidas Poli-Insaturadas/química , Receptor CB1 de Canabinoide/ultraestrutura , Animais , Ácidos Araquidônicos/metabolismo , Encéfalo/metabolismo , Moduladores de Receptores de Canabinoides/química , Canabinoides/farmacologia , Endocanabinoides/metabolismo , Endocanabinoides/farmacologia , Humanos , Simulação de Acoplamento Molecular/métodos , Simulação de Dinâmica Molecular , Alcamidas Poli-Insaturadas/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas/fisiologia , Receptor CB1 de Canabinoide/química , Receptor CB1 de Canabinoide/metabolismoRESUMO
Brain cannabinoid (CB(1)) receptors are G-protein coupled receptors and belong to the rhodopsin-like subfamily. A homology model of the inactive state of the CB(1) receptor was constructed using the x-ray structure of beta(2)-adrenergic receptor (beta(2)AR) as the template. We used 105 ns duration molecular-dynamics simulations of the CB(1) receptor embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer to gain some insight into the structure and function of the CB(1) receptor. As judged from the root mean-square deviations combined with the detailed structural analyses, the helical bundle of the CB(1) receptor appears to be fully converged in 50 ns of the simulation. The results reveal that the helical bundle structure of the CB(1) receptor maintains a topology quite similar to the x-ray structures of G-protein coupled receptors overall. It is also revealed that the CB(1) receptor is stabilized by the formation of extensive, water-mediated H-bond networks, aromatic stacking interactions, and receptor-lipid interactions within the helical core region. It is likely that these interactions, which are often specific to functional motifs, including the S(N)LAxAD, D(E)RY, CWxP, and NPxxY motifs, are the molecular constraints imposed on the inactive state of the CB(1) receptor. It appears that disruption of these specific interactions is necessary to release the molecular constraints to achieve a conformational change of the receptor suitable for G-protein activation.
Assuntos
Modelos Moleculares , Conformação Proteica , Receptor CB1 de Canabinoide/química , Receptor CB1 de Canabinoide/metabolismo , Sítios de Ligação , Simulação por Computador , Sequência Conservada , Cristalografia por Raios X , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/metabolismo , Fosfatidilcolinas/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores Adrenérgicos beta 2/química , Software , Água/metabolismoRESUMO
As a cancer chemotherapeutic drug, arsenic acts on numerous intracellular signal transduction pathways in cancer cells. However, its mechanism of actions is still not fully understood. Previous studies suggest that arsenic reacts with closely spaced cysteine (Cys) residues of proteins with high Cys content and accessible sulfhydryl (SH) groups. In this study, human breast cancer cell line MCF-7 was examined as a cellular model to explore arsenic-binding proteins and the mechanism of binding. An arsenic-biotin conjugate was synthesized by coupling the pentafluorophenol ester of biotin with p-aminophenylarsenoxide. Arsenic-binding proteins were eluted with streptavidin resin from arsenic-biotin treated MCF-7 cells, separated by polyacrylamide gel electrophoresis, and identified by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). Arsenic-binding properties of two of these proteins, beta-tubulin and pyruvate kinase M2 (PKM2), were studied further in vitro and the biological consequences of this binding was evaluated. Binding assay with Western blotting confirmed binding of beta-tubulin and PKM2 by arsenic in a concentration-dependent manner. Arsenic binding inhibited tubulin polymerization, but surprisingly had no effect on PKM2 activity. Molecular modeling showed that binding of Cys(12) alone or vicinal Cys residues (Cys(12) and Cys(213)) of beta-tubulin by arsenic blocked the active site for access of GTP, which is necessary for tubulin polymerization. On the contrary, all Cys residues of PKM2 were far away from the active site of the enzyme. In summary, this study confirmed beta-tubulin and PKM2 as arsenic-binding proteins in MCF-7 cells. Functional consequence of such binding may depend on whether arsenic binding causes conformational changes or blocks active sites of target proteins.
Assuntos
Arsênio/química , Neoplasias da Mama/metabolismo , Proteínas de Transporte/metabolismo , Antineoplásicos/farmacologia , Biotina/química , Linhagem Celular Tumoral , Cisteína/química , Humanos , Modelos Químicos , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Piruvato Quinase/metabolismo , Tubulina (Proteína)/químicaRESUMO
In the present study, we compared the estrogenic activity of zearalenone (ZEN) and zeranol (ZOL) by determining their relative receptor binding affinities for human ERalpha and ERbeta and also by determining their uterotropic activity in ovariectomized female mice. ZOL displayed a much higher binding affinity for human ERalpha and ERbeta than ZEN did. The IC(50) values of ZEN and ZOL for binding to human ERalpha were 240.4 and 21.79nM, respectively, and the IC(50) values for binding to ERbeta were 165.7 and 42.76nM, respectively. In ovariectomized female ICR mice, s.c. administration of ZEN at doses >or=2mg/kg/day for 3 consecutive days significantly increased uterine wet weight compared with the control group, and administration of ZOL increased the uterine wet weight at lower doses (>or=0.5mg/kg/day for 3 days). Based on available X-ray crystal structures of human ERalpha and ERbeta, we have also conducted molecular modeling studies to probe the binding characteristics of ZEN and ZOL for human ERalpha and ERbeta. Our data revealed that ZEN and ZOL were able to occupy the active site of the human ERalpha and ERbeta in a strikingly similar manner as 17beta-estradiol, such that the phenolic rings of ZEN and ZOL occupied the same receptor region as occupied by the A-ring of 17beta-estradiol. The primary reason that ZOL and ZEN is less potent than 17beta-estradiol is likely because 17beta-estradiol could bind to the receptor pocket without significantly changing its conformation, while ZOL or ZEN would require considerable conformational alterations upon binding to the estrogen receptors (ERs).
Assuntos
Estrogênios não Esteroides/farmacologia , Zearalenona/farmacologia , Zeranol/farmacologia , Animais , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos ICR , Modelos Biológicos , Modelos Moleculares , Ovariectomia , Ligação ProteicaRESUMO
To search for endogenous estrogens that may have preferential binding affinity for human estrogen receptor (ER) alpha or beta subtype and also to gain insights into the structural determinants favoring differential subtype binding, we studied the binding affinities of 74 natural or synthetic estrogens, including more than 50 steroidal analogs of estradiol-17beta (E2) and estrone (E1) for human ER alpha and ER beta. Many of the endogenous estrogen metabolites retained varying degrees of similar binding affinity for ER alpha and ER beta, but some of them retained differential binding affinity for the two subtypes. For instance, several of the D-ring metabolites, such as 16 alpha-hydroxyestradiol (estriol), 16 beta-hydroxyestradiol-17 alpha, and 16-ketoestrone, had distinct preferential binding affinity for human ER beta over ER alpha (difference up to 18-fold). Notably, although E2 has nearly the highest and equal binding affinity for ER alpha and ER beta, E1 and 2-hydroxyestrone (two quantitatively predominant endogenous estrogens in nonpregnant woman) have preferential binding affinity for ER alpha over ER beta, whereas 16 alpha-hydroxyestradiol (estriol) and other D-ring metabolites (quantitatively predominant endogenous estrogens formed during pregnancy) have preferential binding affinity for ER beta over ER alpha. Hence, facile metabolic conversion of parent hormone E2 to various metabolites under different physiological conditions may serve unique functions by providing differential activation of the ER alpha or ER beta signaling system. Lastly, our computational three-dimensional quantitative structure-activity relationship/comparative molecular field analysis of 47 steroidal estrogen analogs for human ER alpha and ER beta yielded useful information on the structural features that determine the preferential activation of the ER alpha and ER beta subtypes, which may aid in the rational design of selective ligands for each human ER subtype.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Estrogênios/química , Estrogênios/metabolismo , Relação Quantitativa Estrutura-Atividade , Dietilestilbestrol/química , Dietilestilbestrol/metabolismo , Estradiol/química , Estradiol/metabolismo , Estriol/metabolismo , Antagonistas de Estrogênios/metabolismo , Estrona/metabolismo , Humanos , Hidroxiestronas/metabolismo , Modelos Moleculares , Estrutura Molecular , Fitoestrógenos/química , Fitoestrógenos/metabolismo , Transdução de Sinais , Eletricidade EstáticaRESUMO
It has been earlier established (Pozzi et al. Biochemistry 50 (2011) 10195-10202) that prethrombin-2 crystallizes into two similar but distinct forms: a collapsed form and an alternative form. We employed long molecular dynamics (MD) simulations for these two forms to obtain solvent-equilibrated forms. We find that, at 200ns, the simulated solution collapsed form is quite similar to the X-ray crystal collapsed form, while the simulated solution alternative form deviates from the X-ray crystal alternative form as well as from the solution collapsed form. A detailed structural analysis suggests that the fluctuation of the 140s-loop, in cross-talk with the 220s-loop, may alter the conformation of the W215-E217 segment near the nascent thrombin active site. A rationale is provided for the manner in which interactions of prethrombin-2 with FVa may affect the equilibrium between the two forms of prethrombin-2.
Assuntos
Protrombina/química , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , SoluçõesRESUMO
An all-atom human ternary model for the prothrombinase-prothrombin complex, including metal ions and post-translationally modified residues, was constructed from existing X-ray crystal structures. The factor Xa-prothrombin interface was taken from an existing ternary model, which locates the active site of factor Xa in the vicinity of prothrombin cleavage positions. The three sulfotyrosine residues at the C-terminal sequence of factor Va A2 domain are accommodated by modelling rational interactions with positively charged patches on the surface of prothrombin. The entire model is then solvent-equilibrated with molecular dynamics. This ternary model for the thrombin-generating complex provides an estimate as to the role of the C-terminus of the factor Va A2 domain: to establish an interface between FXa and prothrombin and to stabilize the orientation of this interface.
Assuntos
Fator Va/química , Modelos Biológicos , Trombina/química , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Processamento de Proteína Pós-Traducional/genética , Estrutura Terciária de Proteína/genéticaRESUMO
N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716; 1) is a potent and selective antagonist for the CB1 cannabinoid receptor. Using the AM1 molecular orbital method, conformational analysis of 1 around the pyrazole C3 substituent identified four distinct conformations designated Tg, Ts, Cg, and Cs. The energetic stability of these conformers followed the order Tg > Cg > Ts > Cs for the neutral (unprotonated) form of 1 and Ts > Tg > Cs > Cg for its piperidine N-protonated form. Unified pharmacophore models for the CB1 receptor ligands were developed by incorporating the protonated form of 1 into the superimposition model for the cannabinoid agonists 4-[4-(1,1-dimethylheptyl)-2-hydroxyphenyl]perhydro-2alpha,6beta-dihydroxynaphthalene (CP55244; 2) and the protonated form of (R)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN55212-2; 3) reported previously (Shim et al. In Rational Drug Design Symposium Series; Parrill, A. L., Reddy, M. R., Eds.; American Chemical Society: Washington, DC, 1999; pp 165-184). Values of K(i) for 1 and a series of 31 structural analogues were determined from radioligand binding analyses by competitive displacement of [3H]CP55940 from cannabinoid receptors in a rat brain membrane preparation. Comparative molecular field analysis (CoMFA) was employed to construct three-dimensional (3D)-quantitative structure-activity relationship (QSAR) models for this data set as unprotonated species assuming the Tg, Cg, and Ts conformers and for the protonated species assuming the Ts, Tg, and Cs conformers. Values of the conventional r2 and cross-validated r2 (r(cv)2) associated with these CoMFA models exceeded the threshold for statistical robustness (r2 > or = 0.90) and internal predictive ability (r(cv)2 > or = 0.50) in each of these six cases except for the protonated species assuming the Tg conformer (i.e., r2 = 0.97; r(cv)2 = 0.36). Results from conformational analyses, superimposition models, and 3D-QSAR models suggest that the N1 aromatic ring moiety of 1 dominates the steric binding interaction with the receptor in much the same way as does the C3 alkyl side chain of cannabinoid agonists and the C3 aroyl ring of the aminoalkylindole agonists. We also determined that several of the conformers considered in this study possess the proper spatial orientation and distinct electrostatic character to bind to the CB1 receptor. We propose that the unique region in space occupied by the C5 aromatic ring of 1 might contribute to conferring antagonist activity. We further propose that the pyrazole C3 substituent of 1 might contribute to conferring either neutral antagonist or inverse agonist activity, depending upon the interaction with the receptor.
Assuntos
Piperidinas/química , Piperidinas/farmacologia , Pirazóis/química , Pirazóis/farmacologia , Receptores de Droga/química , Algoritmos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Cinética , Ligantes , Modelos Químicos , Modelos Moleculares , Naftalenos/farmacologia , Ligação Proteica , Conformação Proteica , Prótons , Ratos , Receptores de Canabinoides , Receptores de Droga/metabolismo , Rimonabanto , Software , Relação Estrutura-AtividadeRESUMO
Effects of cannabinoid compounds on neurons are predominantly mediated by the CB(1) cannabinoid receptor. Onset of signaling cascades in response to cannabimimetic drugs is triggered by the interaction of the cannabinoid receptor with G(i/o) proteins. Much work has been done to delineate the cannabinoid agonist-induced downstream signaling events; however, it remains to define the molecular basis of cannabinoid receptor-G protein interactions that stimulate these signaling pathways. In this review, we discuss several signal transduction pathways, focusing on studies that demonstrate the efficacy of CB(1) receptor agonists through G protein mediated pathways.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores de Droga/metabolismo , Adenilil Ciclases/metabolismo , Sequência de Aminoácidos , Animais , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Canabinoides/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Óxido Nítrico/biossíntese , Receptores de Canabinoides , Receptores de Droga/agonistas , Transdução de Sinais/fisiologiaRESUMO
Recently developed X-ray crystal structures of active state G-protein-coupled receptors have opened the way for detailed examination of the movement of the transmembrane (TM) helices and the specific residues involved in the receptor activation upon ligand binding. Previous modeling studies have indicated that the brain cannabinoid (CB1) receptor binds with a ligand at least in part through a hydrophobic tail on the ligand. This interaction is believed to be similar to the rotameric toggle switch proposed for the ß2 adrenergic receptor (ß2AR). In the present study, an active state model for the CB1 receptor, guided by the X-ray structure of the active state for ß2AR, was constructed with HU210 bound as a ligand. Molecular dynamics (MD) simulations were employed to provide a smooth progression between inactive and active states of the receptor. This model was compared with our previously published CB1 receptor model to identify the functional residues that play key roles in triggering receptor conformational changes upon agonist binding. Movements in TM helices and functional residues are discussed. W279(5.43), contributing to an inward movement of the fifth TM helix (TM5) to the helical core, could serve as another rotameric switch for receptor activation. V282(5.46), interacting with the ligand's hydrophobic C3 alkyl chain, appears to play a key role in TM5 inward movement centered at L286(5.50) and subsequent coupling to V204(3.40). V204(3.40), closely interacting with the TM5 and TM6 hydrophobic patch residues in the middle of the receptor, particularly I290(5.54) and L352(6.44), appears to facilitate helical rearrangements, leading to the breakage of the ionic lock and the rotameric change of Y397(7.53), which are key features of the active state.
Assuntos
Receptor CB1 de Canabinoide/química , Receptor CB1 de Canabinoide/metabolismo , Animais , Humanos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
Mounting evidence suggests that imbalances in immune regulation contribute to cell transformation. Women of African descent are an understudied group at high risk for developing aggressive breast cancer (BrCa). Therefore, we examined the role of 16 innate immune single nucleotide polymorphisms (SNPs) in relation to BrCa susceptibility among 174 African-American women in Atlanta, GA, USA. SNPs were examined in germ-line DNA collected from 102 BrCa patients and 72 women with benign nodules using SNPstream methodology. Inheritance of the TLR3 rs10025405 GG genotype was associated with an 82% decrease in BrCa risk. In contrast, individuals who possessed at least one IRAK4 rs4251545 T allele had a 1.68- to 4.99-fold increase in the risk of developing BrCa relative to those with the referent genotype (OR = 4.99; 95% CI = 1.00, 25.00; p = 0.0605). However, the IRAK4 rs4251545 locus was only significant under the additive genetic model (p trend = 0.0406). In silico predictions suggest IRAK4 rs4251545 SNP falls within a transcription enhancer/silencer region of the gene and codes for an Ala428Thr amino acid change. This missense mutation introduces a potential phosphorylation site in the extreme carboxy terminus (XCT) of the IRAK4 kinase domain. Preliminary molecular modeling predicts that this SNP stabilizes two alpha helices within the XCT on the surface of the IRAK4 kinase domain and increases the size of the groove between them. Our in silico results, combined with previous reports noting the presence of IRAK4 and XCT fragments in mouse and human serum, suggest the possibility that the XCT subdomain of IRAK4 possesses biological function. These findings require further evaluation and validation in larger populations, additional molecular modeling as well as functional studies to explore the role of IRAK4 and its XCT in cell transformation and innate immunity.
RESUMO
The brain cannabinoid (CB(1)) receptor that mediates numerous physiological processes in response to marijuana and other psychoactive compounds is a G protein coupled receptor (GPCR) and shares common structural features with many rhodopsin class GPCRs. For the rational development of therapeutic agents targeting the CB(1) receptor, understanding of the ligand-specific CB(1) receptor interactions responsible for unique G protein signals is crucial. For a more than a decade, a combination of mutagenesis and computational modeling approaches has been successfully employed to study the ligand-specific CB(1) receptor interactions. In this review, after a brief discussion about recent advances in understanding of some structural and functional features of GPCRs commonly applicable to the CB(1) receptor, the CB(1) receptor functional residues reported from mutational studies are divided into three different types, ligand binding (B), receptor stabilization (S) and receptor activation (A) residues, to delineate the nature of the binding pockets of anandamide, CP55940, WIN55212-2 and SR141716A and to describe the molecular events of the ligand-specific CB(1) receptor activation from ligand binding to G protein signaling. Taken these CB(1) receptor functional residues, some of which are unique to the CB(1) receptor, together with the biophysical knowledge accumulated for the GPCR active state, it is possible to propose the early stages of the CB(1) receptor activation process that not only provide some insights into understanding molecular mechanisms of receptor activation but also are applicable for identifying new therapeutic agents by applying the validated structure-based approaches, such as virtual high throughput screening (HTS) and fragment-based approach (FBA).
Assuntos
Canabinoides/química , Canabinoides/farmacologia , Receptor CB1 de Canabinoide/química , Receptor CB1 de Canabinoide/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Canabinoides/uso terapêutico , Domínio Catalítico , Ensaios de Triagem em Larga Escala , Humanos , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
BACKGROUND: CB1 cannabinoid receptors are G-protein coupled receptors for endocannabinoids including anandamide and 2-arachidonoylglycerol. Because these arachidonic acid metabolites possess a 20-carbon polyene chain as the alkyl terminal moiety, they are highly flexible with the potential to adopt multiple biologically relevant conformations, particularly those in a bent form. To better understand the molecular interactions associated with binding and steric trigger mechanisms of receptor activation, a series of conformationally-restricted anandamide analogs having a wide range of affinity and efficacy were evaluated. RESULTS: A CB1 receptor model was constructed to include the extracellular loops, particularly extracellular loop 2 which possesses an internal disulfide linkage. Using both Glide (Schrödinger) and Affinity (Accelrys) docking programs, binding conformations of six anandamide analogs were identified that conform to rules applicable to the potent, efficacious and stereoselective non-classical cannabinoid CP55244. Calculated binding energies of the optimum structures from both procedures correlated well with the reported binding affinity values. The most potent and efficacious of the ligands adopted conformations characterized by interactions with both the helix-3 lysine and hydrophobic residues that interact with CP55244. The other five compounds formed fewer or less energetically favorable interactions with these critical residues. The flexibility of the tested anandamide analogs, measured by torsion angles around the benzene as well as the stretch between side chain moieties, could contribute to the differences in ability to interact with the CB1 receptor. CONCLUSION: Analyses of multiple poses of conformationally-restricted anandamide analogs permitted identification of favored amino acid interactions within the CB1 receptor binding pocket. A ligand possessing both high affinity and cannabinoid agonist efficacy was able to interact with both polar and hydrophobic interaction sites utilized by the potent and efficacious non-classical cannabinoid CP55940. In contrast, other analogs characterized by reduced affinity or efficacy exhibited less favorable interactions with those key residues.