Free energy landscape of G-protein coupled receptors, explored by accelerated molecular dynamics

Miao, Yinglong; Nichols, Sara E.; McCammon, J. Andrew

doi:10.1039/c3cp53962h

Cited by 78 publications

(105 citation statements)

References 66 publications

(146 reference statements)

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“…Moreover, long-timescale MD simulations captured ligand binding to GPCRs (29), in particular for several prototypical negative allosteric modulators (NAMs) of the M 2 mAChR (30). In recent studies, we have also successfully depicted the activation of both the M 2 and M 3 mAChRs in accelerated MD (aMD) simulations (31)(32)(33).…”

Section: Significancementioning

confidence: 94%

“…Although these crystal structures provided enormously important insights into different specific conformational states, as well as atomistic protein-ligand interactions of GPCRs, they nonetheless represent snapshots of the highly dynamic nature of GPCRs (20). To address the above issue, extensive computational simulations have been performed to characterize the structural dynamics of GPCRs (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33). All-atom molecular dynamics (MD) simulations are capable of exploring large-scale conformational changes during both receptor activation (23) and deactivation (20) by using fast supercomputers.…”

Section: Significancementioning

confidence: 99%

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Accelerated structure-based design of chemically diverse allosteric modulators of a muscarinic G protein-coupled receptor

Miao

Goldfeld

Moo

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Design of ligands that provide receptor selectivity has emerged as a new paradigm for drug discovery of G protein-coupled receptors, and may, for certain families of receptors, only be achieved via identification of chemically diverse allosteric modulators. Here, the extracellular vestibule of the M 2 muscarinic acetylcholine receptor (mAChR) is targeted for structure-based design of allosteric modulators. Accelerated molecular dynamics (aMD) simulations were performed to construct structural ensembles that account for the receptor flexibility. Compounds obtained from the National Cancer Institute (NCI) were docked to the receptor ensembles. Retrospective docking of known ligands showed that combining aMD simulations with Glide induced fit docking (IFD) provided much-improved enrichment factors, compared with the Glide virtual screening workflow. Glide IFD was thus applied in receptor ensemble docking, and 38 top-ranked NCI compounds were selected for experimental testing. In [ 3 H]Nmethylscopolamine radioligand dissociation assays, approximately half of the 38 lead compounds altered the radioligand dissociation rate, a hallmark of allosteric behavior. In further competition binding experiments, we identified 12 compounds with affinity of ≤30 μM. With final functional experiments on six selected compounds, we confirmed four of them as new negative allosteric modulators (NAMs) and one as positive allosteric modulator of agonist-mediated response at the M 2 mAChR. Two of the NAMs showed subtype selectivity without significant effect at the M 1 and M 3 mAChRs. This study demonstrates an unprecedented successful structure-based approach to identify chemically diverse and selective GPCR allosteric modulators with outstanding potential for further structure-activity relationship studies.GPCR | allosteric modulators | ensemble docking | affinity | cooperativity

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Section: Significancementioning

confidence: 94%

Section: Significancementioning

confidence: 99%

Accelerated structure-based design of chemically diverse allosteric modulators of a muscarinic G protein-coupled receptor

Miao

Goldfeld

Moo

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

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“…The last 50 ns of each run was used for analysis on conformational flexibility, positional correlation, and community. In addition, we ran accelerated molecular dynamics (aMD) simulations, which have been shown to significantly broaden conformational sampling (26,(29)(30)(31). For each system, six replicate aMD runs were performed; each run was 20 ns long, and the last 10 ns was used for calculating chemical shifts and analyzing conformational changes.…”

Section: Resultsmentioning

confidence: 99%

Unidirectional allostery in the regulatory subunit RIα facilitates efficient deactivation of protein kinase A

Guo

Zhou

2016

Proc. Natl. Acad. Sci. U.S.A.

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The holoenzyme complex of protein kinase A is in an inactive state; activation involves ordered cAMP binding to two tandem domains of the regulatory subunit and release of the catalytic subunit. Deactivation has been less studied, during which the two cAMPs unbind from the regulatory subunit to allow association of the catalytic subunit to reform the holoenzyme complex. Unbinding of the cAMPs appears ordered as indicated by a large difference in unbinding rates from the two sites, but the cause has remained elusive given the structural similarity of the two tandem domains. Even more intriguingly, NMR data show that allosteric communication between the two domains is unidirectional. Here, we present a mechanism for the unidirectionality, developed from extensive molecular dynamics simulations of the tandem domains in different cAMP-bound forms. Disparate responses to cAMP releases from the two sites (A and B) in conformational flexibility and chemical shift perturbation confirmed unidirectional allosteric communication. Community analysis revealed that the A-site cAMP, by forming across-domain interactions, bridges an essential pathway for interdomain communication. The pathway is impaired when this cAMP is removed but remains intact when only the B-site cAMP is removed. Specifically, removal of the A-site cAMP leads to the separation of the two domains, creating room for binding the catalytic subunit. Moreover, the A-site cAMP, by maintaining interdomain coupling, retards the unbinding of the B-site cAMP and stalls an unproductive pathway of cAMP release. Our work expands the perspective on allostery and implicates functional importance for the directionality of allostery.unidirectional allostery | cAMP | molecular dynamics | community analysis | NMR spectroscopy P rotein kinase A (PKA), also known as cAMP-dependent protein kinase, plays an important role in multiple cellular processes by catalyzing protein phosphorylation (1). Its dysregulation is involved in many diseases including cancer and inflammatory disorders (2). The holoenzyme complex, formed by two catalytic (C) subunits bound to a homodimer of regulatory (R) subunits, is inactive. Each regulatory subunit contains two tandem cAMP-binding domains (referred to as CBD-A and CBD-B) (3); in the holoenzyme complex, the A site is masked by the C subunit (4). Activation occurs when two cAMP molecules bind sequentially and cooperatively to the two sites on each R subunit (5). Possible structural changes in the activation process have been constructed from crystallographic studies (4). The first cAMP molecule binds to the B site and makes the A site accessible. Binding of the second cAMP molecule then leads to the release of the C subunit for catalyzing substrate phosphorylation. Deactivation, involving the unbinding of the two cAMP molecules from an R subunit and association of the R and C subunits to reform the holoenzyme complex, has been less studied, and a number of important questions remain open. In particular, kinetic studies have shown that the cAMP un...

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“…Accelerated molecular dynamics (aMD) is particularly advantageous for this purpose because in combination with the recent advances in graphical processing unit-enabled hardware, it can provide access to ligand-binding events that occur on the millisecond time scale without requisite knowledge of the binding pathway. (16) Hundredsof-nanosecond aMD simulations have captured the activation of the M2 and M3 muscarinic Gprotein coupled receptors (17)(18)(19) and ligand binding to the M3 receptor. (20) In this work, aMD is extended to CYP3A4 to simulate TST binding to the enzyme free of a predefined reaction coordinate.…”

mentioning

confidence: 99%

Membrane-embedded substrate recognition by cytochrome P450 3A4

Hackett

2018

Journal of Biological Chemistry

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Cytochrome P450 3A4 (CYP3A4) is the dominant xenobiotic-metabolizing enzyme in the liver and intestine and is involved in the disposition of more than 50% of drugs. Owing to its ability to bind multiple substrates, its reaction kinetics are complex, and its association with the microsomal membrane confounds our understanding of how this enzyme recognizes and recruits diverse substrates. Testosterone (TST) hydroxylation is the prototypical CYP3A4 reaction, displaying positive homotropic cooperativity with three binding sites. Here, exploiting the capability of accelerated molecular dynamics (aMD) to sample events in the millisecond regime, I performed > 25-µs aMD simulations in the presence of three TST molecules. These simulations identified high-occupancy surfacebinding sites as well as a pathway for TST ingress into the CYP3A4 active site originating in the membrane. Adaptive biasing force analysis of the latter pathway revealed a metastable intermediate that could constitute a third binding site at high TST concentrations. Prompted by the observation that interactions between TST and the G'-helix mobilize the ligand into the active site, a free-energy analysis of TST distribution in the membrane was conducted and revealed that the depth of the G'-helix is optimal for extracting TST. In summary, these simulations confirm separate, but adjacent substrate-binding sites within the enzyme and the existence of an auxiliary TSTbinding site. The broader impact of these simulations is that they support a mechanism in which cytochromes P450 directly recruit membrane-solubilized substrates. ________________The cytochromes P450s metabolize nearly all drugs and environmental xenobiotics to which humans are exposed. Of the 57 P450s encoded by the human genome, cytochrome P450 3A4 (CYP3A4) is involved in the clearance of more than half of approved drugs and presents s i g n i f i c a n t c h a l l e n g e t o o p t i m i z i n g pharmacokinetics in drug development and avoiding adverse effects in the clinic.(1,2) CYP3A4 enzyme kinetics can demonstrate marked cooperativity.(3) Positive cooperativity is characterized by a continuous, positive increase in the rate of catalysis with increasing substrate concentration; whereas positive heterotropic cooperativity occurs when the rate of substrate turnover is enhanced by a second distinct effector molecule. Cooperativity in CYP catalysis was first recognized in microsome preparations(4-6) and later confirmed using purified CYP3A4 with several substrates including testosterone (TST), 17β-estradiol, amitriptyline, and aflatoxin B1. (7) A shift in the regioselectivity of midazolam (MDZ) oxidation in patients by concurrent fluconazole administration supports that heterotropic CYP3A4 interactions are also relevant in vivo.(8) Cooperativity in CYP3A4 catalysis has been explained by mechanisms invoking simultaneous occupation of the active site by multiple ligands or binding to nearby allosteric sites. (9)(10)(11)(12)(13)(14)(15) The membrane environment can have a profound effec...

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Free energy landscape of G-protein coupled receptors, explored by accelerated molecular dynamics

Abstract: G-protein coupled receptors (GPCRs) mediate cellular responses to various hormones and neurotransmitters and are important targets for treating a wide spectrum of diseases.

Cited by 78 publications

References 66 publications

Accelerated structure-based design of chemically diverse allosteric modulators of a muscarinic G protein-coupled receptor

Accelerated structure-based design of chemically diverse allosteric modulators of a muscarinic G protein-coupled receptor

Unidirectional allostery in the regulatory subunit RIα facilitates efficient deactivation of protein kinase A

Membrane-embedded substrate recognition by cytochrome P450 3A4

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