RESUMO
G protein-coupled receptors (GPCRs) relay diverse extracellular signals into cells by catalyzing nucleotide release from heterotrimeric G proteins, but the mechanism underlying this quintessential molecular signaling event has remained unclear. Here we use atomic-level simulations to elucidate the nucleotide-release mechanism. We find that the G protein α subunit Ras and helical domains-previously observed to separate widely upon receptor binding to expose the nucleotide-binding site-separate spontaneously and frequently even in the absence of a receptor. Domain separation is necessary but not sufficient for rapid nucleotide release. Rather, receptors catalyze nucleotide release by favoring an internal structural rearrangement of the Ras domain that weakens its nucleotide affinity. We use double electron-electron resonance spectroscopy and protein engineering to confirm predictions of our computationally determined mechanism.
Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Fatores de Troca do Nucleotídeo Guanina/química , Receptores Acoplados a Proteínas G/química , Humanos , Modelos Químicos , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug-receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation-π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15 Å from the classical, 'orthosteric' ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator's allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.
Assuntos
Desenho de Fármacos , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/química , Regulação Alostérica/fisiologia , Animais , Sítios de Ligação , Células CHO , Cricetulus , Humanos , Modelos Químicos , Conformação Molecular , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Receptores Acoplados a Proteínas G/genética , Reprodutibilidade dos TestesRESUMO
It is increasingly appreciated that the rates at which drugs associate with and dissociate from receptors--the binding kinetics--directly impact drug efficacy and safety. The molecular determinants of drug-receptor binding kinetics remain poorly understood, however, especially when compared with the well-known factors that affect binding affinity. The rational modulation of kinetics during lead optimization thus remains challenging. We review some of the key factors thought to control drug-receptor binding kinetics at the molecular level--molecular size, conformational fluctuations, electrostatic interactions and hydrophobic effects--and discuss several possible approaches for the rational design of drugs with desired binding kinetics.
Assuntos
Desenho de Fármacos , Preparações Farmacêuticas/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Sítios de Ligação , Humanos , Cinética , Ligantes , Modelos Moleculares , Estrutura Molecular , Preparações Farmacêuticas/química , Ligação Proteica , Conformação Proteica , Receptores de Superfície Celular/química , Receptores Citoplasmáticos e Nucleares/química , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Structure-based drug design (SBDD) can accelerate inhibitor lead design and optimization, and efficient methods including protein purification, characterization, crystallization, and high-resolution diffraction are all needed for rapid, iterative structure determination. Janus kinases are important targets that are amenable to structure-based drug design. Here we present the first mouse Tyk2 crystal structures, which are complexed to 3-aminoindazole compounds. RESULTS: A comprehensive construct design effort included N- and C-terminal variations, kinase-inactive mutations, and multiple species orthologs. High-throughput cloning and expression methods were coupled with an abbreviated purification protocol to optimize protein solubility and stability. In total, 50 Tyk2 constructs were generated. Many displayed poor expression, inadequate solubility, or incomplete affinity tag processing. One kinase-inactive murine Tyk2 construct, complexed with an ATP-competitive 3-aminoindazole inhibitor, provided crystals that diffracted to 2.5-2.6 Å resolution. This structure revealed initial "hot-spot" regions for SBDD, and provided a robust platform for ligand soaking experiments. Compared to previously reported human Tyk2 inhibitor crystal structures (Chrencik et al. (2010) J Mol Biol 400:413), our structures revealed a key difference in the glycine-rich loop conformation that is induced by the inhibitor. Ligand binding also conferred resistance to proteolytic degradation by thermolysin. As crystals could not be obtained with the unliganded enzyme, this enhanced stability is likely important for successful crystallization and inhibitor soaking methods. CONCLUSIONS: Practical criteria for construct performance and prioritization, the optimization of purification protocols to enhance protein yields and stability, and use of high-throughput construct exploration enable structure determination methods early in the drug discovery process. Additionally, specific ligands stabilize Tyk2 protein and may thereby enable crystallization.
Assuntos
Desenho de Fármacos , Indazóis/química , Indazóis/farmacologia , TYK2 Quinase/antagonistas & inibidores , TYK2 Quinase/química , Sequência de Aminoácidos , Animais , Cristalização , Cristalografia por Raios X , Estabilidade Enzimática/efeitos dos fármacos , Humanos , Janus Quinases/antagonistas & inibidores , Janus Quinases/metabolismo , Camundongos , Dados de Sequência Molecular , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Estrutura Secundária de Proteína , Proteólise/efeitos dos fármacos , Relação Estrutura-Atividade , TYK2 Quinase/isolamento & purificaçãoRESUMO
The mechanism of ion channel voltage gating-how channels open and close in response to voltage changes-has been debated since Hodgkin and Huxley's seminal discovery that the crux of nerve conduction is ion flow across cellular membranes. Using all-atom molecular dynamics simulations, we show how a voltage-gated potassium channel (KV) switches between activated and deactivated states. On deactivation, pore hydrophobic collapse rapidly halts ion flow. Subsequent voltage-sensing domain (VSD) relaxation, including inward, 15-angstrom S4-helix motion, completes the transition. On activation, outward S4 motion tightens the VSD-pore linker, perturbing linker-S6-helix packing. Fluctuations allow water, then potassium ions, to reenter the pore; linker-S6 repacking stabilizes the open pore. We propose a mechanistic model for the sodium/potassium/calcium voltage-gated ion channel superfamily that reconciles apparently conflicting experimental data.
Assuntos
Ativação do Canal Iônico , Canal de Potássio Kv1.2/química , Canal de Potássio Kv1.2/metabolismo , Canais de Potássio Shab/química , Canais de Potássio Shab/metabolismo , Animais , Interações Hidrofóbicas e Hidrofílicas , Potenciais da Membrana , Modelos Biológicos , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Molecular dynamics simulations can now track rapid processes--those occurring in less than about a millisecond--at atomic resolution for many biologically relevant systems. These simulations appear poised to exert a significant impact on how new drugs are found, perhaps even transforming the very process of drug discovery. We predict here future results we can expect from, and enhancements we need to make in, molecular dynamics simulations over the coming 25 years, and in so doing set out several Grand Challenges for the field. In the context of the problems now facing the pharmaceutical industry, we ask how we can best address drug discovery needs of the next quarter century using molecular dynamics simulations, and we suggest some possible approaches.
Assuntos
Descoberta de Drogas/tendências , Simulação de Dinâmica Molecular/tendências , Proteínas/química , Indústria Farmacêutica/tendências , Humanos , Ligantes , Ligação ProteicaRESUMO
A third of marketed drugs act by binding to a G-protein-coupled receptor (GPCR) and either triggering or preventing receptor activation. Although recent crystal structures have provided snapshots of both active and inactive functional states of GPCRs, these structures do not reveal the mechanism by which GPCRs transition between these states. Here we propose an activation mechanism for the ß(2)-adrenergic receptor, a prototypical GPCR, based on atomic-level simulations in which an agonist-bound receptor transitions spontaneously from the active to the inactive crystallographically observed conformation. A loosely coupled allosteric network, comprising three regions that can each switch individually between multiple distinct conformations, links small perturbations at the extracellular drug-binding site to large conformational changes at the intracellular G-protein-binding site. Our simulations also exhibit an intermediate that may represent a receptor conformation to which a G protein binds during activation, and suggest that the first structural changes during receptor activation often take place on the intracellular side of the receptor, far from the drug-binding site. By capturing this fundamental signaling process in atomic detail, our results may provide a foundation for the design of drugs that control receptor signaling more precisely by stabilizing specific receptor conformations.
Assuntos
Receptores Adrenérgicos beta 2/metabolismo , Sítio Alostérico , Motivos de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Simulação por Computador , Cristalografia por Raios X/métodos , Proteínas de Ligação ao GTP/química , Humanos , Ligantes , Modelos Biológicos , Conformação Molecular , Conformação Proteica , Prótons , Transdução de Sinais , Tirosina/químicaRESUMO
How drugs bind to their receptors--from initial association, through drug entry into the binding pocket, to adoption of the final bound conformation, or "pose"--has remained unknown, even for G-protein-coupled receptor modulators, which constitute one-third of all marketed drugs. We captured this pharmaceutically critical process in atomic detail using the first unbiased molecular dynamics simulations in which drug molecules spontaneously associate with G-protein-coupled receptors to achieve final poses matching those determined crystallographically. We found that several beta blockers and a beta agonist all traverse the same well-defined, dominant pathway as they bind to the ß(1)- and ß(2)-adrenergic receptors, initially making contact with a vestibule on each receptor's extracellular surface. Surprisingly, association with this vestibule, at a distance of 15 Å from the binding pocket, often presents the largest energetic barrier to binding, despite the fact that subsequent entry into the binding pocket requires the receptor to deform and the drug to squeeze through a narrow passage. The early barrier appears to reflect the substantial dehydration that takes place as the drug associates with the vestibule. Our atomic-level description of the binding process suggests opportunities for allosteric modulation and provides a structural foundation for future optimization of drug-receptor binding and unbinding rates.
Assuntos
Preparações Farmacêuticas , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais , Alprenolol/química , Alprenolol/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Dessecação , Espaço Extracelular/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Receptores Adrenérgicos beta 1/química , Receptores Adrenérgicos beta 2/química , TermodinâmicaRESUMO
Wolfe-Simon et al. (Research Articles, 3 June 2011, p. 1163; published online 2 December 2010) reported the discovery of an unusual bacterium, strain GFAJ-1, that can grow in the presence of high concentrations of arsenate. The authors' contention, however, that this microbe can appreciably vary the elemental composition of its fundamental biomolecules by substituting arsenic for phosphorus appears premature based on the data presented.
Assuntos
Arsênio/análise , Arsênio/metabolismo , DNA Bacteriano/química , Halomonadaceae/metabolismo , Fósforo/análise , Fósforo/metabolismo , Arseniatos/metabolismo , Meios de Cultura/química , DNA Bacteriano/metabolismo , Halomonadaceae/crescimento & desenvolvimento , Halomonadaceae/isolamento & purificação , Fosfatos/metabolismoRESUMO
Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38α (involved in the formation of TNFα and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional (1)H/(13)C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38α both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in similar fashion to Jnk-1 siRNA and to rosiglitazone treatment. Together, the data suggest that these new ligand series bind to a novel, allosteric, and physiologically relevant site and therefore represent a unique approach to identify kinase inhibitors.
Assuntos
Descoberta de Drogas , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Ensaios de Triagem em Larga Escala , Humanos , Proteína Quinase 8 Ativada por Mitógeno/química , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Bibliotecas de Moléculas Pequenas , Estereoisomerismo , Relação Estrutura-Atividade , Proteínas Quinases p38 Ativadas por Mitógeno/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
We present the first atomic-resolution observations of permeation and gating in a K(+) channel, based on molecular dynamics simulations of the Kv1.2 pore domain. Analysis of hundreds of simulated permeation events revealed a detailed conduction mechanism, resembling the Hodgkin-Keynes "knock-on" model, in which translocation of two selectivity filter-bound ions is driven by a third ion; formation of this knock-on intermediate is rate determining. In addition, at reverse or zero voltages, we observed pore closure by a novel "hydrophobic gating" mechanism: A dewetting transition of the hydrophobic pore cavity-fastest when K(+) was not bound in selectivity filter sites nearest the cavity-caused the open, conducting pore to collapse into a closed, nonconducting conformation. Such pore closure corroborates the idea that voltage sensors can act to prevent pore collapse into the intrinsically more stable, closed conformation, and it further suggests that molecular-scale dewetting facilitates a specific biological function: K(+) channel gating. Existing experimental data support our hypothesis that hydrophobic gating may be a fundamental principle underlying the gating of voltage-sensitive K(+) channels. We suggest that hydrophobic gating explains, in part, why diverse ion channels conserve hydrophobic pore cavities, and we speculate that modulation of cavity hydration could enable structural determination of both open and closed channels.
Assuntos
Ativação do Canal Iônico , Canal de Potássio Kv1.2/química , Canal de Potássio Kv1.2/metabolismo , Animais , Fenômenos Biofísicos , Condutividade Elétrica , Interações Hidrofóbicas e Hidrofílicas , Técnicas In Vitro , Cinética , Modelos Biológicos , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica , Estrutura Terciária de Proteína , RatosRESUMO
MK2 is a Ser/Thr kinase of significant interest as an anti-inflammatory drug discovery target. Here we describe the development of in vitro tools for the identification and characterization of MK2 inhibitors, including validation of inhibitor interactions with the crystallography construct and determination of the unique binding mode of 2,4-diaminopyrimidine inhibitors in the MK2 active site. Use of these tools in the optimization of a potent and selective inhibitor lead series is described in the accompanying Letter.
Assuntos
Anti-Inflamatórios/química , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/química , Trifosfato de Adenosina/química , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/farmacologia , Sítios de Ligação , Ligação Competitiva , Simulação por Computador , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Pirimidinas/síntese química , Pirimidinas/farmacologia , Relação Estrutura-AtividadeRESUMO
We describe structure-based optimization of a series of novel 2,4-diaminopyrimidine MK2 inhibitors. Co-crystal structures (see accompanying Letter) demonstrated a unique inhibitor binding mode. Resulting inhibitors had IC(50) values as low as 19nM and moderate selectivity against a kinase panel. Compounds 15, 31a, and 31b inhibit TNFalpha production in peripheral human monocytes.
Assuntos
Anti-Inflamatórios/química , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/química , Administração Oral , Animais , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/farmacocinética , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Serina-Treonina Quinases/metabolismo , Pirimidinas/síntese química , Pirimidinas/farmacocinética , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The unique cytokine interleukin-18 (IL-18) acts synergistically with IL-12 to regulate T-helper 1 and 2 lymphocytes and, as such, seems to underlie the pathogenesis of various autoimmune and allergic diseases. Several anti-IL-18 agents are in clinical development, including the recombinant human antibody ABT-325, which is entering trials for autoimmune diseases. Given competing cytokine/receptor and cytokine/receptor decoy interactions, understanding the structural basis for recognition is critical for effective development of anti-cytokine therapies. Here we report three crystal structures: the murine antibody 125-2H Fab fragment bound to human IL-18, at 1.5 A resolution; the 125-2H Fab (2.3 A); and the ABT-325 Fab (1.5 A). These structures, along with human/mouse IL-18 chimera binding data, allow us to make three key observations relevant to the biology and antigenic recognition of IL-18 and related cytokines. First, several IL-18 residues shift dramatically (> 10 A) upon binding 125-2H, compared with unbound IL-18 (Kato, Z., Jee, J., Shikano, H., Mishima, M., Ohki, I., Ohnishi, H., Li, A., Hashimoto, K., Matsukuma, E., Omoya, K., Yamamoto, Y., Yoneda, T., Hara, T., Kondo, N., and Shirakawa, M. (2003) Nat. Struct. Biol. 10, 966-971). IL-18 thus exhibits plasticity that may be common to its interactions with other receptors. Related cytokines may exhibit similar plasticity. Second, ABT-325 and 125-2H differ significantly in combining site character and architecture, thus explaining their ability to bind IL-18 simultaneously at distinct epitopes. These data allow us to define the likely ABT-325 epitope and thereby explain the distinct neutralizing mechanisms of both antibodies. Third, given the high 125-2H potency, 10 well ordered water molecules are trapped upon complex formation in a cavity between two IL-18 loops and all six 125-2H complementarity-determining regions. Thus, counterintuitively, tight and specific antibody binding may in some cases be water-mediated.
Assuntos
Anticorpos Monoclonais/química , Antígenos/química , Epitopos/química , Fragmentos Fab das Imunoglobulinas/química , Interleucina-18/química , Água/química , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Antígenos/imunologia , Antígenos/metabolismo , Cristalografia por Raios X , Epitopos/imunologia , Epitopos/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Interleucina-18/imunologia , Interleucina-18/metabolismo , Camundongos , Ligação Proteica/imunologia , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: Structure-based drug design (SBDD) can provide valuable guidance to drug discovery programs. Robust construct design and expression, protein purification and characterization, protein crystallization, and high-resolution diffraction are all needed for rapid, iterative inhibitor design. We describe here robust methods to support SBDD on an oral anti-cytokine drug target, human MAPKAP kinase 2 (MK2). Our goal was to obtain useful diffraction data with a large number of chemically diverse lead compounds. Although MK2 structures and structural methods have been reported previously, reproducibility was low and improved methods were needed. RESULTS: Our construct design strategy had four tactics: N- and C-terminal variations; entropy-reducing surface mutations; activation loop deletions; and pseudoactivation mutations. Generic, high-throughput methods for cloning and expression were coupled with automated liquid dispensing for the rapid testing of crystallization conditions with minimal sample requirements. Initial results led to development of a novel, customized robotic crystallization screen that yielded MK2/inhibitor complex crystals under many conditions in seven crystal forms. In all, 44 MK2 constructs were generated, ~500 crystals were tested for diffraction, and ~30 structures were determined, delivering high-impact structural data to support our MK2 drug design effort. CONCLUSION: Key lessons included setting reasonable criteria for construct performance and prioritization, a willingness to design and use customized crystallization screens, and, crucially, initiation of high-throughput construct exploration very early in the drug discovery process.
Assuntos
Desenho de Fármacos , Peptídeos e Proteínas de Sinalização Intracelular/química , Mutagênese Sítio-Dirigida , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/química , Substituição de Aminoácidos , Simulação por Computador , Cristalização , Cristalografia por Raios X , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Isoenzimas/biossíntese , Isoenzimas/química , Isoenzimas/isolamento & purificação , Conformação Proteica , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Fully understanding the mechanisms of signaling proteins such as G protein-coupled receptors (GPCRs) will require the characterization of their conformational states and the pathways connecting those states. The recent crystal structures of the beta(2)- and beta(1)-adrenergic receptors in a nominally inactive state constituted a major advance toward this goal, but also raised new questions. Although earlier biochemical observations had suggested that these receptors possessed a set of contacts between helices 3 and 6, known as the ionic lock, which was believed to form a molecular switch for receptor activation, the crystal structures lacked these contacts. The unexpectedly broken ionic lock has raised questions about the true conformation(s) of the inactive state and the role of the ionic lock in receptor activation and signaling. To address these questions, we performed microsecond-timescale molecular dynamics simulations of the beta(2)-adrenergic receptor (beta(2)AR) in multiple wild-type and mutant forms. In wild-type simulations, the ionic lock formed reproducibly, bringing the intracellular ends of helices 3 and 6 together to adopt a conformation similar to that found in inactive rhodopsin. Our results suggest that inactive beta(2)AR exists in equilibrium between conformations with the lock formed and the lock broken, whether or not the cocrystallized ligand is present. These findings, along with the formation of several secondary structural elements in the beta(2)AR loops during our simulations, may provide a more comprehensive picture of the inactive state of the beta-adrenergic receptors, reconciling the crystal structures with biochemical studies.