RESUMEN
Ghrelin plays a central role in controlling major biological processes. As for other G protein-coupled receptor (GPCR) peptide agonists, the structure and dynamics of ghrelin bound to its receptor remain obscure. Using a combination of solution-state NMR and molecular modeling, we demonstrate that binding to the growth hormone secretagogue receptor is accompanied by a conformational change in ghrelin that structures its central region, involving the formation of a well-defined hydrophobic core. By comparing its acylated and nonacylated forms, we conclude that the ghrelin octanoyl chain is essential to form the hydrophobic core and promote access of ghrelin to the receptor ligand-binding pocket. The combination of coarse-grained molecular dynamics studies and NMR should prove useful in improving our mechanistic understanding of the complex conformational space explored by a natural peptide agonist when binding to its GPCR. Such information should also facilitate the design of new ghrelin receptor-selective drugs.
Asunto(s)
Ghrelina/química , Ghrelina/metabolismo , Modelos Moleculares , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Acilación , Animales , Sitios de Unión , Humanos , Espectroscopía de Resonancia Magnética , Unión Proteica , Conformación Proteica , Transducción de Señal , Relación Estructura-ActividadRESUMEN
The growth hormone secretagogue receptor (GHSR) and dopamine receptor (D2R) have been shown to oligomerize in hypothalamic neurons with a significant effect on dopamine signaling, but the molecular processes underlying this effect are still obscure. We used here the purified GHSR and D2R to establish that these two receptors assemble in a lipid environment as a tetrameric complex composed of two each of the receptors. This complex further recruits G proteins to give rise to an assembly with only two G protein trimers bound to a receptor tetramer. We further demonstrate that receptor heteromerization directly impacts on dopamine-mediated Gi protein activation by modulating the conformation of its α-subunit. Indeed, association to the purified GHSR:D2R heteromer triggers a different active conformation of Gαi that is linked to a higher rate of GTP binding and a faster dissociation from the heteromeric receptor. This is an additional mechanism to expand the repertoire of GPCR signaling modulation that could have implications for the control of dopamine signaling in normal and physiopathological conditions.
Asunto(s)
Dopamina/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Multimerización de Proteína , Receptores de Dopamina D2/química , Receptores de Ghrelina/química , Transducción de Señal , Dopamina/genética , Dopamina/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Humanos , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Receptores de Ghrelina/genética , Receptores de Ghrelina/metabolismoRESUMEN
The Catabolite Activator Protein (CAP) is a showcase example for entropic allostery. For full activation and DNA binding, the homodimeric protein requires the binding of two cyclic AMP (cAMP) molecules in an anti-cooperative manner, the source of which appears to be largely of entropic nature according to previous experimental studies. We here study at atomic detail the allosteric regulation of CAP with Molecular dynamics (MD) simulations. We recover the experimentally observed entropic penalty for the second cAMP binding event with our recently developed force covariance entropy estimator and reveal allosteric communication pathways with Force Distribution Analyses (FDA). Our observations show that CAP binding results in characteristic changes in the interaction pathways connecting the two cAMP allosteric binding sites with each other, as well as with the DNA binding domains. We identified crucial relays in the mostly symmetric allosteric activation network, and suggest point mutants to test this mechanism. Our study suggests inter-residue forces, as opposed to coordinates, as a highly sensitive measure for structural adaptations that, even though minute, can very effectively propagate allosteric signals.
Asunto(s)
Sitio Alostérico , Proteína Receptora de AMP Cíclico/química , Proteína Receptora de AMP Cíclico/metabolismo , Entropía , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Análisis de Componente Principal , Unión ProteicaRESUMEN
The dynamic character of G protein-coupled receptors is essential to their function. However, the details of how ligands stabilize a particular conformation to selectively activate a signaling pathway and how signaling proteins affect this conformational repertoire remain unclear. Using a prototypical peptide-activated class A G protein-coupled receptor (GPCR), the ghrelin receptor, reconstituted as a monomer into lipid discs and labeled with a fluorescent conformational reporter, we demonstrate that ligand efficacy and functional selectivity are directly related to different receptor conformations. Of importance, our data bring direct evidence that distinct effector proteins affect the conformational landscape of the ghrelin receptor in different ways. Whereas G proteins affect the balance between active and inactive receptor substates in favor of the active state, agonist-induced arrestin recruitment is accompanied by a marked change in the structural features of the receptor that adopt a conformation different from that observed in the absence of arrestin. In contrast to G proteins and arrestins, µ-AP2 has no significant effect on the organization of the transmembrane core of the receptor. Such a modulation of a GPCR conformational landscape by pharmacologically distinct ligands and effectors provides insights into the structural bases that decisively affect ligand efficacy and subsequent biological responses. This is also likely to have major implications for the design of drugs activating specific GPCR-associated signaling pathways.
Asunto(s)
Ghrelina/metabolismo , Receptores de Ghrelina/química , Receptores de Ghrelina/metabolismo , Transducción de Señal/fisiología , Arrestina/metabolismo , Arrestina/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Agonismo Inverso de Drogas , Fluorescencia , Ghrelina/farmacología , Humanos , Ligandos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/farmacología , Conformación Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Ghrelina/agonistas , Relación Estructura-ActividadRESUMEN
In the accepted model for human immunodeficiency virus preassembly in infected host cells, the anchoring to the intracellular leaflet of the membrane of the matrix domain (MA) that lies at the N-terminus of the viral Gag protein precursor appears to be one of the crucial steps for particle assembly. In this study, we simulated the membrane anchoring of human immunodeficiency virus-1 myristoylated MA protein using a coarse-grained representation of both the protein and the membrane. Our calculations first suggest that the myristoyl group could spontaneously release from its initial hydrophobic pocket before MA protein interacts with the lipid membrane. All-atom simulations confirmed this possibility with a related energy cost estimated to be ~5 kcal.mol(-1). The phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) head binds preferentially to the MA highly basic region as described in available NMR data, but interestingly without flipping of its 2' acyl chain into the MA protein. Moreover, MA was able to confine PI(4,5)P2 lipids all around its molecular surface after having found a stable orientation at the membrane surface. Our results suggest that this orientation is dependent on Myr anchoring and that this confinement induces a lateral segregation of PI(4,5)P2 in domains. This is consistent with a PI(4,5)P2 enrichment of the virus envelope as compared to the host cell membrane.
Asunto(s)
Antígenos VIH/metabolismo , Simulación de Dinámica Molecular , Fosfatidilinositol 4,5-Difosfato/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Aminoácidos , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/virología , Antígenos VIH/química , Humanos , Datos de Secuencia Molecular , Fosfatidilinositol 4,5-Difosfato/química , Unión Proteica , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
After extra-cellular stimulation of G-Protein Coupled Receptors (GPCRs), GDP/GTP exchange appears as the key, rate limiting step of the intracellular activation cycle of heterotrimeric G-proteins. Despite the availability of a large number of X-ray structures, the mechanism of GDP release out of heterotrimeric G-proteins still remains unknown at the molecular level. Starting from the available X-ray structure, extensive unconstrained/constrained molecular dynamics simulations were performed on the complete membrane-anchored Gi heterotrimer complexed to GDP, for a total simulation time overcoming 500 ns. By combining Targeted Molecular Dynamics (TMD) and free energy profiles reconstruction by umbrella sampling, our data suggest that the release of GDP was much more favored on its phosphate side. Interestingly, upon the forced extraction of GDP on this side, the whole protein encountered large, collective motions in perfect agreement with those we described previously including a domain to domain motion between the two ras-like and helical sub-domains of G(α).
Asunto(s)
Biología Computacional/métodos , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Proteínas de Unión al GTP Heterotriméricas/química , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Simulación de Dinámica Molecular , TermodinámicaRESUMEN
Molecular dynamics with excited normal modes (MDeNM) is an enhanced sampling method for exploring conformational changes in proteins with minimal biases. The excitation corresponds to injecting kinetic energy along normal modes describing intrinsic collective motions. Herein, we developed a new automated open-source implementation, MDexciteR (https://github.com/mcosta27/MDexciteR), enabling the integration of MDeNM with two commonly used simulation programs with GPU support. Second, we generalized the method to include the excitation of principal components calculated from experimental ensembles. Finally, we evaluated whether the use of coarse-grained normal modes calculated with elastic network representations preserved the performance and accuracy of the method. The advantages and limitations of these new approaches are discussed based on results obtained for three different protein test cases: two globular and a protein/membrane system.
RESUMEN
NDM-1 (New-Delhi-Metallo-ß-lactamase-1) is an enzyme developed by bacteria that is implicated in bacteria resistance to almost all known antibiotics. In this study, we deliver a new, curated NDM-1 bioactivities database, along with a set of unifying rules for managing different activity properties and inconsistencies. We define the activity classification problem in terms of Multiple Instance Learning, employing embeddings corresponding to molecular substructures and present an ensemble ranking and classification framework, relaying on a k-fold Cross Validation method employing a per fold hyper-parameter optimization procedure, showing promising generalization ability. The MIL paradigm displayed an improvement up to 45.7â¯%, in terms of Balanced Accuracy, in comparison to the classical Machine Learning paradigm. Moreover, we investigate different compact molecular representations, based on atomic or bi-atomic substructures. Finally, we scanned the Drugbank for strongly active compounds and we present the top-15 ranked compounds.
Asunto(s)
Antibacterianos , beta-Lactamasas , Antibacterianos/farmacología , Antibacterianos/química , beta-Lactamasas/química , BacteriasRESUMEN
The functional properties of G protein-coupled receptors (GPCRs) are intimately associated with the different components in their cellular environment. Among them, sodium ions have been proposed to play a substantial role as endogenous allosteric modulators of GPCR-mediated signaling. However, this sodium effect and the underlying mechanisms are still unclear for most GPCRs. Here, we identified sodium as a negative allosteric modulator of the ghrelin receptor GHSR (growth hormone secretagogue receptor). Combining 23Na-nuclear magnetic resonance (NMR), molecular dynamics, and mutagenesis, we provide evidence that, in GHSR, sodium binds to the allosteric site conserved in class A GPCRs. We further leveraged spectroscopic and functional assays to show that sodium binding shifts the conformational equilibrium toward the GHSR-inactive ensemble, thereby decreasing basal and agonist-induced receptor-catalyzed G protein activation. All together, these data point to sodium as an allosteric modulator of GHSR, making this ion an integral component of the ghrelin signaling machinery.
Asunto(s)
Receptores de Ghrelina , Sodio , Regulación Alostérica , Sitio Alostérico , Ghrelina/metabolismo , Iones , Receptores de Ghrelina/metabolismo , Transducción de Señal , Sodio/metabolismoRESUMEN
Heterotrimeric G-proteins' activation on the intracellular side of the cell membrane is initiated by stimulation of the G-Protein Coupled Receptors (GPCRs) extra-cellular part. This two-step activation mechanism includes (1) an exchange between GDP and GTP molecules in the G(α) subunit and (2) a dissociation of the whole G(αßγ) complex into two membrane-anchored blocks, namely the isolated G(α) and G(ßγ) subunits. Although X-ray data are available for both inactive G(αßγ):GDP and active G(α):GTP complexes, intermediate steps involved in the molecular mechanism of the dissociation have not yet been addressed at the molecular level. In this study, we first built a membrane-anchored intermediate G(iαßγ):GTP complex. This model was then equilibrated by molecular dynamics simulations before the Targeted Molecular Dynamics (TMD) technique was used to force the G(α) subunit to evolve from its inactive (GDP-bound) to its active (GTP-bound) conformations, as described by available X-ray data. The TMD constraint was applied only to the G(α) subunit so that the resulting global rearrangements acting on the whole G(αßγ):GTP heterotrimer could be analyzed. We showed how these mainly local conformational changes of G(α) could initiate large domain:domain motions of the whole complex, the G(ßγ) behaving as an almost quasi-rigid block. This separation of the two G(α):GTP and G(ßγ) subunits required the loss of several interactions at the G(α):G(ßγ) interface that were reported. This study provided an atomistic view of the crucial intermediate step of the G-proteins activation, e.g., the dissociation, that could hardly be elucidated by the experiment.
Asunto(s)
Guanosina Difosfato/química , Guanosina Trifosfato/química , Proteínas de Unión al GTP Heterotriméricas/química , Simulación de Dinámica Molecular , Fosfatidilcolinas/química , Subunidades de Proteína/química , Cristalografía por Rayos X , Cinética , Unión Proteica , TermodinámicaRESUMEN
The antidiuretic hormone arginine-vasopressin (AVP) forms a signaling complex with the V2 receptor (V2R) and the Gs protein, promoting kidney water reabsorption. Molecular mechanisms underlying activation of this critical G protein-coupled receptor (GPCR) signaling system are still unknown. To fill this gap of knowledge, we report here the cryo-electron microscopy structure of the AVP-V2R-Gs complex. Single-particle analysis revealed the presence of three different states. The two best maps were combined with computational and nuclear magnetic resonance spectroscopy constraints to reconstruct two structures of the ternary complex. These structures differ in AVP and Gs binding modes. They reveal an original receptor-Gs interface in which the Gαs subunit penetrates deep into the active V2R. The structures help to explain how V2R R137H or R137L/C variants can lead to two severe genetic diseases. Our study provides important structural insights into the function of this clinically relevant GPCR signaling complex.
RESUMEN
Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS) that causes severe motor, sensory, and cognitive impairments. Kallikrein-related peptidase (KLK)6 is the most abundant serine protease secreted in the CNS, mainly by oligodendrocytes, the myelin-producing cells of the CNS, and KLK6 is assumed to be a robust biomarker of MS, since it is highly increased in the cerebrospinal fluid (CSF) of MS patients. Here, we report the design and biological evaluation of KLK6's low-molecular-weight inhibitors, para-aminobenzyl derivatives. Interestingly, selected hit compounds were selective of the KLK6 proteolytic network encompassing KLK1 and plasmin that also participate in the development of MS physiopathology. Moreover, hits were found noncytotoxic on primary cultures of murine neurons and oligodendrocyte precursor cells (OPCs). Among them, two compounds (32 and 42) were shown to promote the differentiation of OPCs into mature oligodendrocytes in vitro constituting thus emerging leads for the development of regenerative therapies.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Calicreínas/antagonistas & inhibidores , Inhibidores de Serina Proteinasa/farmacología , Animales , Derivados del Benceno/química , Derivados del Benceno/metabolismo , Derivados del Benceno/farmacología , Sitios de Unión , Dominio Catalítico , Células Cultivadas , Diseño de Fármacos , Fibrinolisina/antagonistas & inhibidores , Fibrinolisina/metabolismo , Humanos , Calicreínas/metabolismo , Cinética , Ratones , Simulación del Acoplamiento Molecular , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Neuronas/citología , Neuronas/metabolismo , Oligodendroglía/citología , Oligodendroglía/metabolismo , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/metabolismo , Células Madre/citología , Células Madre/metabolismo , Relación Estructura-ActividadRESUMEN
The membrane is an integral component of the G protein-coupled receptor signaling machinery. Here we demonstrate that lipids regulate the signaling efficacy and selectivity of the ghrelin receptor GHSR through specific interactions and bulk effects. We find that PIP2 shifts the conformational equilibrium of GHSR away from its inactive state, favoring basal and agonist-induced G protein activation. This occurs because of a preferential binding of PIP2 to specific intracellular sites in the receptor active state. Another lipid, GM3, also binds GHSR and favors G protein activation, but mostly in a ghrelin-dependent manner. Finally, we find that not only selective interactions but also the thickness of the bilayer reshapes the conformational repertoire of GHSR, with direct consequences on G protein selectivity. Taken together, this data illuminates the multifaceted role of the membrane components as allosteric modulators of how ghrelin signal could be propagated.
Asunto(s)
Fosfatidilinositol 4,5-Difosfato/metabolismo , Receptores de Ghrelina/química , Receptores de Ghrelina/metabolismo , Regulación Alostérica , Sitios de Unión , Membrana Celular/química , Membrana Celular/metabolismo , Cisteína/genética , Transferencia Resonante de Energía de Fluorescencia , Gangliósido G(M3)/metabolismo , Humanos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Metabolismo de los Lípidos , Lípidos/química , Mutación , Fosfatidilinositol 4,5-Difosfato/química , Conformación Proteica , Receptores de Ghrelina/genética , Transducción de SeñalRESUMEN
There is increasing support for water molecules playing a role in signal propagation through G protein-coupled receptors (GPCRs). However, exploration of the hydration features of GPCRs is still in its infancy. Here, we combined site-specific labeling with unnatural amino acids to molecular dynamics to delineate how local hydration of the ghrelin receptor growth hormone secretagogue receptor (GHSR) is rearranged upon activation. We found that GHSR is characterized by a specific hydration pattern that is selectively remodeled by pharmacologically distinct ligands and by the lipid environment. This process is directly related to the concerted movements of the transmembrane domains of the receptor. These results demonstrate that the conformational dynamics of GHSR are tightly coupled to the movements of internal water molecules, further enhancing our understanding of the molecular bases of GPCR-mediated signaling.
Asunto(s)
Ghrelina , Receptores Acoplados a Proteínas G , Receptores de Ghrelina , Humanos , Ligandos , Transducción de SeñalRESUMEN
Through their coupling to G proteins, G Protein-Coupled Receptors (GPCRs) trigger cellular responses to various signals. Some recent experiments have interestingly demonstrated that the G protein can also act on the receptor by favoring a closed conformation of its orthosteric site, even in the absence of a bound agonist. In this work, we explored such an allosteric modulation by performing extensive molecular dynamics simulations on the adenosine A2 receptor (A2AR) coupled to the Mini-Gs protein. In the presence of the Mini-Gs, we confirmed a restriction of the receptor's agonist binding site that can be explained by a modulation of the intrinsic network of contacts of the receptor. Of interest, we observed similar effects with the C-terminal helix of the Mini-Gs, showing that the observed effect on the binding pocket results from direct local contacts with the bound protein partner that cause a rewiring of the whole receptor's interaction network.
Asunto(s)
Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Receptor de Adenosina A2A/química , Receptor de Adenosina A2A/metabolismo , Antagonistas del Receptor de Adenosina A2/química , Antagonistas del Receptor de Adenosina A2/farmacología , Regulación Alostérica , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Estructura Secundaria de ProteínaRESUMEN
Cytochrome P450 2C9 (CYP2C9) metabolizes about 15% of clinically administrated drugs. The allelic variant CYP2C9*30 (A477T) is associated to diminished response to the antihypertensive effects of the prodrug losartan and affected metabolism of other drugs. Here, we investigated molecular mechanisms involved in the functional consequences of this amino-acid substitution. Molecular dynamics (MD) simulations performed for the active species of the enzyme (heme in the Compound I state), in the apo or substrate-bound state, and binding energy analyses gave insights into altered protein structure and dynamics involved in the defective drug metabolism of human CYP2C9.30. Our data revealed an increased rigidity of the key Substrate Recognition Sites SRS1 and SRS5 and shifting of the ß turn 4 of SRS6 toward the helix F in CYP2C9.30. Channel and binding substrate dynamics analyses showed altered substrate channel access and active site accommodation. These conformational and dynamic changes are believed to be involved in the governing mechanism of the reduced catalytic activity. An ensemble of representative conformations of the WT and A477T mutant properly accommodating drug substrates were identified, those structures can be used for prediction of new CYP2C9 and CYP2C9.30 substrates and drug-drug interactions.
Asunto(s)
Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP2C9/metabolismo , Variantes Farmacogenómicas , Catálisis , Humanos , Errores Innatos del Metabolismo/genética , Errores Innatos del Metabolismo/metabolismo , Simulación de Dinámica Molecular , Mutación , Unión Proteica , Conformación ProteicaRESUMEN
Pharmacogenomics investigates DNA and RNA variations in the human genome related to drug responses. Cytochrome P450 (CYP) is a supergene family of drug-metabolizing enzymes responsible for the metabolism of approximately 90% of human drugs. Among the major CYP isoforms, the CYP2C subfamily is of clinical significance because it metabolizes approximately 20% of clinically administrated drugs and represents several variant alleles leading to adverse drug reactions or altering drug efficacy. Here, we review recent progress on understanding the interindividual variability of the CYP2C members and the functional and clinical impact on drug metabolism. We summarize current advances in the molecular modeling of CYP2C polymorphisms and discuss the structural bases and molecular mechanisms of amino acid variants of CYP2C members that affect drug metabolism.
Asunto(s)
Sustitución de Aminoácidos , Sistema Enzimático del Citocromo P-450 , Preparaciones Farmacéuticas/metabolismo , Aminoácidos/genética , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , HumanosRESUMEN
The human ClC-Kb channel plays a key role in exporting chloride ions from the cytosol and is known to be involved in Bartter syndrome type 3 when its permeation capacity is decreased. The ClC-Kb channel has been recently proposed as a potential therapeutic target to treat hypertension. In order to gain new insights into the sequence-structure-function relationships of this channel, to investigate possible impacts of amino-acid substitutions, and to design novel inhibitors, we first built a structural model of the human ClC-Kb channel using comparative modeling strategies. We combined in silico and in vitro techniques to analyze amino acids involved in the chloride ion pathway as well as to rationalize the possible role of several clinically observed mutations leading to the Bartter syndrome type 3. Virtual screening and drug repositioning computations were then carried out. We identified six novel molecules, including 2 approved drugs, diflusinal and loperamide, with Kd values in the low micromolar range, that block the human ClC-Kb channel and that could be used as starting point to design novel chemical probes for this potential therapeutic target.
Asunto(s)
Canales de Cloruro/química , Modelos Moleculares , Relación Estructura-Actividad Cuantitativa , Secuencia de Aminoácidos , Animales , Bovinos , Canales de Cloruro/antagonistas & inhibidores , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Cloruros/química , Cloruros/metabolismo , Susceptibilidad a Enfermedades , Evaluación Preclínica de Medicamentos , Humanos , Activación del Canal Iónico , Potenciales de la Membrana , Estructura Molecular , Mutación , Conformación ProteicaRESUMEN
GPCRs undergo large conformational changes during their activation. Starting from existing X-ray structures, we used Normal Modes Analyses to study the collective motions of the agonist-bound ß2-adrenergic receptor both in its isolated "uncoupled" and G-protein "coupled" conformations. We interestingly observed that the receptor was able to adopt only one major motion in the protein:protein complex. This motion corresponded to an anti-symmetric rotation of both its extra- and intra-cellular parts, with a key role of previously identified highly conserved proline residues. Because this motion was also retrieved when performing NMA on 7 other GPCRs which structures were available, it is strongly suspected to possess a significant biological role, possibly being the "activation mode" of a GPCR when coupled to G-proteins.
Asunto(s)
Proteínas de Unión al GTP/química , Receptores Acoplados a Proteínas G/química , Humanos , Membrana Dobles de Lípidos/química , Modelos Moleculares , Fosfolípidos/química , Unión Proteica , Conformación Proteica , Receptores Adrenérgicos beta 2/químicaRESUMEN
G-protein hetero-trimers play a fundamental role in cell function. Their dynamic behavior at the atomic level remains to be understood. We have studied the Gi hetero-trimer through a combination of molecular dynamics simulations and normal mode analyses. We showed that these big proteins could undergo large-amplitude conformational changes, without any energy penalty and with an intrinsic dynamics centered on their GDP binding pocket. Among the computed collective motions, one of the modes (mode 17) was particularly able to significantly open both the base and the phosphate sides of the GDP binding pocket. This mode describing mainly a motion between the Ras-like and the helical domains of G(α) was in close agreement with some available X-ray data and with many other biochemical/biophysical observations including the kink of helix α5. The use of a new protocol, which allows extraction of the GDP ligand along the computed normal modes, supported that the exit of GDP was largely coupled to an opening motion along mode 17. We propose for the first time a "concerted mechanism" model in which the opening of the GDP pocket and the kink of the α5 helix occur concomitantly and favor GDP release from G(αßγ) complexes. This model is discussed in the context of the G-protein-coupled receptor/G-protein interaction close to the cell membrane.