Competitive dewetting underlies site-specific binding of general anesthetics to GABA(A) receptors

AbstractGABA(A) receptors are pentameric ligand-gated ion channels playing a critical role in the modulation of neuronal excitability. These inhibitory receptors, gated by γ-aminobutyric acid (GABA), can be potentiated and even directly activated by intravenous and inhalational anesthetics. Intersubunit cavities in the transmembrane domain have been consistently identified as putative binding sites by numerous experiment and simulation results. Synaptic GABA(A) receptors are predominantly found in a 2α:2β:1γ stoichiometry, with four unique inter-subunit interfaces. Experimental and computational results have suggested a perplexing specificity, given that cavity-lining residues are highly conserved, and the functional effects of general anesthetics are only weakly sensitive to most mutations of cavity residues. Here we use Molecular Dynamics simulations and thermodynamically rigorous alchemical free energy perturbation (AFEP) techniques to calculate affinities of the intravenous anesthetic propofol and the inhaled anesthetic sevoflurane to all intersubunit sites in a heteromeric GABA(A) receptor. We find that the best predictor of general anesthetic affinity for the intersubunit cavity sites is water displacement: combinations of anesthetic and binding site that displace more water molecules have higher affinities than those that displace fewer. The amount of water displacement is, in turn, a function of size of the general anesthetic, successful competition of the general anesthetic with water for the few hydrogen bonding partners in the site, and inaccessibility of the site to lipid acyl chains. The latter explains the surprisingly low affinity of GAs for the γ − α intersubunit site, which is missing a bulky methionine residue at the cavity entrance and can be occupied by acyl chains in the unbound state. Simulations also identify sevoflurane binding sites in the β subunit centers and in the pore, but predict that these are lower affinity than the intersubunit sites.SignificanceAfter over a century of research, it is established that general anesthetics interact directly with hydrophobic cavities in proteins. We still do not know why not all small hydrophobic molecules can act as general anesthetics, or why not all hydrophobic cavities bind these molecules. General anesthetics can even select among homologous sites on one critical target, the GABA(A) heteropentamer, although the origins of selectivity are unknown. Here we used rigorous free energy calculations to find that binding affinity correlates with the number of released water molecules, which in turn depends upon the lipid content of the cavity without bound anesthetic. Results suggest a mechanism that reconciles lipid-centered and protein-centered theories, and which can directly inform design of new anesthetics.

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Identification of a putative binding site critical for general anesthetic activation of TRPA1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1618144114 ◽

2017 ◽

Vol 114 (14) ◽

pp. 3762-3767 ◽

Cited By ~ 25

Author(s):

Hoai T. Ton ◽

Thieu X. Phan ◽

Ara M. Abramyan ◽

Lei Shi ◽

Gerard P. Ahern

Keyword(s):

Ion Channels ◽

Transient Receptor Potential ◽

Transmembrane Domain ◽

Halogen Bond ◽

Critical Role ◽

Sensory Nerves ◽

Structural Basis ◽

General Anesthetics ◽

General Anesthetic ◽

Potential Binding

General anesthetics suppress CNS activity by modulating the function of membrane ion channels, in particular, by enhancing activity of GABAA receptors. In contrast, several volatile (isoflurane, desflurane) and i.v. (propofol) general anesthetics excite peripheral sensory nerves to cause pain and irritation upon administration. These noxious anesthetics activate transient receptor potential ankyrin repeat 1 (TRPA1), a major nociceptive ion channel, but the underlying mechanisms and site of action are unknown. Here we exploit the observation that pungent anesthetics activate mammalian but not Drosophila TRPA1. Analysis of chimeric Drosophila and mouse TRPA1 channels reveal a critical role for the fifth transmembrane domain (S5) in sensing anesthetics. Interestingly, we show that anesthetics share with the antagonist A-967079 a potential binding pocket lined by residues in the S5, S6, and the first pore helix; isoflurane competitively disrupts A-967079 antagonism, and introducing these mammalian TRPA1 residues into dTRPA1 recapitulates anesthetic agonism. Furthermore, molecular modeling predicts that isoflurane and propofol bind to this pocket by forming H-bond and halogen-bond interactions with Ser-876, Met-915, and Met-956. Mutagenizing Met-915 or Met-956 selectively abolishes activation by isoflurane and propofol without affecting actions of A-967079 or the agonist, menthol. Thus, our combined experimental and computational results reveal the potential binding mode of noxious general anesthetics at TRPA1. These data may provide a structural basis for designing drugs to counter the noxious and vasorelaxant properties of general anesthetics and may prove useful in understanding effects of anesthetics on related ion channels.

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Free Energy Calculations on the Two Drug Binding Sites in the M2 Proton Channel

Journal of the American Chemical Society ◽

10.1021/ja1114198 ◽

2011 ◽

Vol 133 (28) ◽

pp. 10817-10825 ◽

Cited By ~ 27

Author(s):

Ruo-Xu Gu ◽

Limin Angela Liu ◽

Dong-Qing Wei ◽

Jian-Guo Du ◽

Lei Liu ◽

...

Keyword(s):

Free Energy ◽

Binding Sites ◽

Free Energy Calculations ◽

Drug Binding ◽

Proton Channel ◽

Energy Calculations ◽

Drug Binding Sites ◽

M2 Proton Channel

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Enhancing Water Sampling of Buried Binding Sites Using Nonequilibrium Candidate Monte Carlo

10.26434/chemrxiv.12429464.v1 ◽

2020 ◽

Author(s):

Teresa Danielle Bergazin ◽

Ido Ben-Shalom ◽

Nathan M. Lim ◽

Samuel C. Gill ◽

Michael K. Gilson ◽

...

Keyword(s):

Molecular Dynamics ◽

Monte Carlo ◽

Binding Sites ◽

Water Exchange ◽

Free Energy Calculations ◽

Water Molecules ◽

Water Sampling ◽

Computer Aided Drug Design ◽

Practical Applications ◽

Computer Aided

<div>Water molecules can be found interacting with the surface and within cavities in proteins. However, water exchange between bulk and buried hydration sites can be slow compared to simulation timescales, thus leading to the inefficient sampling of the locations of water. This can pose problems for free energy calculations for computer-aided drug design. Here, we apply a hybrid method that combines nonequilibrium candidate Monte Carlo (NCMC) simulations and molecular dynamics (MD) to enhance sampling of water in specific areas of a system, such as the binding site of a protein. Our approach uses NCMC to gradually remove interactions between a selected water molecule and its environment, then translates the water to a new region, before turning the interactions back on. This approach of gradual removal of interactions, followed by a move and then reintroduction of interactions, allows the environment relax in response to the proposed water translation, improving acceptance of moves and thereby accelerating water exchange and sampling. We validate this approach on several test systems including the ligand-bound MUP-1 and HSP90 proteins with buried crystallographic waters removed. We show that our NCMC/MD method enhances water sampling relative to normal MD when applied to these systems. Thus, this approach provides a strategy to improve water sampling in molecular simulations which may be useful in practical applications in drug discovery and biomolecular design.</div>

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Intriguing Role of Water in Plant Hormone Perception

10.1101/2021.10.04.462894 ◽

2021 ◽

Author(s):

Chuankai Zhao ◽

Diego Eduardo Kleiman ◽

Diwakar Shukla

Keyword(s):

Free Energy ◽

Plant Growth ◽

Binding Site ◽

Plant Hormones ◽

Plant Hormone ◽

Water Molecules ◽

Water Displacement ◽

Dynamics Simulations ◽

Free Energy Of Binding

Plant hormones are small molecules that regulate plant growth, development, and responses to biotic and abiotic stresses. Plant hormones are specifically recognized by the binding site of their receptors. In this work, we investigated the role of water displacement and reorganization at the binding site of plant receptors on the binding of eight classes of phytohormones (auxin, jasmonate, gibberellin, strigolactone, brassinosteroid, cytokinin, salicylic acid, and abscisic acid) using extensive molecular dynamics simulations and inhomogeneous solvation theory. Our findings demonstrated that displacement of water molecules by phytohormones contributes to free energy of binding via entropy gain and is associated with free energy barriers. Also, our results have shown that displacement of unfavorable water molecules in the binding site can be exploited in rational agrochemical design. Overall, this study uncov- ers the role of water molecules in plant hormone perception, which creates new avenues for agrochemical design to target plant growth and development.

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Involvement of Tuberomamillary Histaminergic Neurons in Isoflurane Anesthesia

Anesthesiology ◽

10.1097/aln.0b013e3182207655 ◽

2011 ◽

Vol 115 (1) ◽

pp. 36-43 ◽

Cited By ~ 33

Author(s):

Tao Luo ◽

L. Stan Leung

Keyword(s):

Critical Role ◽

Cell Loss ◽

General Anesthetics ◽

General Anesthetic ◽

Isoflurane Anesthesia ◽

Histaminergic Neurons ◽

Loss Of Righting Reflex ◽

Tuberomamillary Nucleus ◽

Righting Reflex ◽

H1 Receptor Antagonist

Background The brain histaminergic system plays a critical role in maintenance of arousal. Previous studies suggest that histaminergic neurotransmission might be a potential mediator of general anesthetic actions. However, it is not clear whether histaminergic tuberomamillary nucleus (TMN) is necessarily involved in the sedative/hypnotic effects of general anesthetics. Methods Male Long Evans rats underwent either TMN orexin-saporin/sham lesion or implantation of intracerebroventricular cannula 2 weeks before the experiment. The behavioral endpoint of loss of righting reflex was used to assess the hypnotic property of isoflurane, propofol, pentobarbital, and ketamine in animals. Histaminergic cell loss was assessed by adenosine deaminase expression in the TMN using immunohistochemistry. Results Rats with bilateral TMN orexin-saporin lesion induced an average 72% loss of histaminergic cells compared with sham-lesion rats. TMN orexin-saporin lesion or intracerebroventricular administration of triprolidine (an H1 receptor antagonist) decreased the 50% effective concentration for loss of righting reflex value and prolonged emergence time to isoflurane anesthesia. However, TMN orexin-saporin lesion had no significant effect on the anesthetic sensitivity to propofol, pentobarbital, and ketamine. Conclusions These findings suggest a role of the TMN histaminergic neurons in modulating isoflurane anesthesia and that the neural circuits for isoflurane-induced hypnosis may differ from those of γ-aminobutyric acid-mediated anesthetics and ketamine.

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Molecular Basis of the Activation and Dissociation of Dimeric PYL2 Receptor in Abscisic Acid Signaling

10.1101/721761 ◽

2019 ◽

Author(s):

Chuankai Zhao ◽

Diwakar Shukla

Keyword(s):

Free Energy ◽

Abscisic Acid ◽

Critical Role ◽

Water Molecules ◽

Plant Responses ◽

Free Energy Barrier ◽

Conformational Plasticity ◽

Dynamics Simulations ◽

Key Aspects ◽

Aba Receptors

Phytohormone abscisic acid (ABA) is essential for plant responses to biotic and abiotic stresses. Dimeric receptors are a class of ABA receptors that are important for various ABA responses. While extensive experimental and computational studies have investigated these receptors, it remains not fully understood how ABA leads to their activation and dissociation for interaction with downstream phosphatase. It also remains unknown how networks of water molecules present in the binding site affect ABA perception despite its critical role in protein-ligand binding. Here, we study the activation and the homodimeric association processes of PYL2 receptor as well as its heterodimeric association with the phosphatase HAB1 using molecular dynamics simulations. Free energy landscapes from ~223 μs simulations show that dimerization substantially constrains PYL2 conformational plasticity and stabilizes inactive state, resulting in lower ABA affinity. Using hydration site analysis to characterize receptor solvation thermodynamics, we show that the displacement and reorganization of water molecules upon ABA binding contribute to binding affinity via gain of entropy and enthalpy, respectively. The penalty for expelling water molecules into the bulk causes the free energy barrier to binding (~4-5 kcal/mol). Finally, we establish the thermodynamic model for competitive binding between homodimeric PYL2 association and heterodimeric PYL2-HAB1 association in the absence and presence of ABA. Our results suggest that the binding of ABA destabilizes PYL2 complex and further stabilizes PYL2-HAB1 association, thereby promoting PYL2 dissociation. Overall, this study explains several key aspects on activation of dimeric ABA receptors, which provide new avenues for selective regulation of these receptors.

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Accounting for the Central Role of Interfacial Water in Protein-Ligand Binding Free Energy Calculations

10.26434/chemrxiv.12668816 ◽

2020 ◽

Author(s):

Ido Ben-Shalom ◽

Zhixiong Lin ◽

Brian Radak ◽

Charles Lin ◽

Woody Sherman ◽

...

Keyword(s):

Free Energy ◽

Binding Site ◽

Binding Free Energy ◽

Md Simulations ◽

Simulation Software ◽

Free Energy Calculations ◽

Computer Hardware ◽

Water Molecules ◽

Energy Calculations ◽

Binding Free Energy Calculations

Rigorous binding free energy methods in drug discovery are growing in popularity due to a combination of methodological advances, improvements in computer hardware, and workflow automation. These calculations typically use molecular dynamics (MD) to sample from the Boltzmann distribution of conformational states. However, when part or all the binding site is inaccessible to bulk solvent, the time needed for water molecules to equilibrate between bulk solvent and the binding site can be well beyond what is practical with standard MD. This sampling limitation is problematic in relative binding free energy calculations, which compute the reversible work of converting Ligand 1 to Ligand 2 within the binding site. Thus, if Ligand 1 is smaller and/or more polar than Ligand 2, the perturbation may allow additional water molecules to occupy a region of the binding site. However, this change in hydration may not be captured by standard MD simulations and may therefore lead to errors in the computed free energy. We recently developed a hybrid Monte Carlo/MD (MC/MD) method, which speeds the equilibration of water between bulk solvent and buried cavities, while sampling from the intended distribution of states. Here, we report on the use of this approach in the context of alchemical binding free energy calculations. We find that using MC/MD markedly improves the accuracy of the calculations and also reduces hysteresis between the forward and reverse perturbations, relative to matched calculations using only MD with or without the crystallographic water molecules. The present method is available for use in the AMBER simulation software.<br>

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Proton-coupled electron transfer and the role of water molecules in proton pumping by cytochrome c oxidase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1409543112 ◽

2015 ◽

Vol 112 (7) ◽

pp. 2040-2045 ◽

Cited By ~ 45

Author(s):

Vivek Sharma ◽

Giray Enkavi ◽

Ilpo Vattulainen ◽

Tomasz Róg ◽

Mårten Wikström

Keyword(s):

Electron Transfer ◽

Free Energy ◽

Proton Transfer ◽

Short Circuit ◽

Proton Pumping ◽

Free Energy Calculations ◽

Water Molecules ◽

Transfer Event ◽

Proton Coupled Electron Transfer ◽

Dynamics Simulations

Molecular oxygen acts as the terminal electron sink in the respiratory chains of aerobic organisms. Cytochrome c oxidase in the inner membrane of mitochondria and the plasma membrane of bacteria catalyzes the reduction of oxygen to water, and couples the free energy of the reaction to proton pumping across the membrane. The proton-pumping activity contributes to the proton electrochemical gradient, which drives the synthesis of ATP. Based on kinetic experiments on the O–O bond splitting transition of the catalytic cycle (A → PR), it has been proposed that the electron transfer to the binuclear iron–copper center of O2 reduction initiates the proton pump mechanism. This key electron transfer event is coupled to an internal proton transfer from a conserved glutamic acid to the proton-loading site of the pump. However, the proton may instead be transferred to the binuclear center to complete the oxygen reduction chemistry, which would constitute a short-circuit. Based on atomistic molecular dynamics simulations of cytochrome c oxidase in an explicit membrane–solvent environment, complemented by related free-energy calculations, we propose that this short-circuit is effectively prevented by a redox-state–dependent organization of water molecules within the protein structure that gates the proton transfer pathway.

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Redox-coupled quinone dynamics in the respiratory complex I

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1805468115 ◽

2018 ◽

Vol 115 (36) ◽

pp. E8413-E8420 ◽

Cited By ~ 32

Author(s):

Judith Warnau ◽

Vivek Sharma ◽

Ana P. Gamiz-Hernandez ◽

Andrea Di Luca ◽

Outi Haapanen ◽

...

Keyword(s):

Free Energy ◽

Binding Sites ◽

Complex I ◽

Redox State ◽

Effective Diffusion ◽

Proton Pumping ◽

Free Energy Calculations ◽

Dependent Manner ◽

Membrane Domain ◽

State Dependent

Complex I couples the free energy released from quinone (Q) reduction to pump protons across the biological membrane in the respiratory chains of mitochondria and many bacteria. The Q reduction site is separated by a large distance from the proton-pumping membrane domain. To address the molecular mechanism of this long-range proton-electron coupling, we perform here full atomistic molecular dynamics simulations, free energy calculations, and continuum electrostatics calculations on complex I from Thermus thermophilus. We show that the dynamics of Q is redox-state-dependent, and that quinol, QH2, moves out of its reduction site and into a site in the Q tunnel that is occupied by a Q analog in a crystal structure of Yarrowia lipolytica. We also identify a second Q-binding site near the opening of the Q tunnel in the membrane domain, where the Q headgroup forms strong interactions with a cluster of aromatic and charged residues, while the Q tail resides in the lipid membrane. We estimate the effective diffusion coefficient of Q in the tunnel, and in turn the characteristic time for Q to reach the active site and for QH2 to escape to the membrane. Our simulations show that Q moves along the Q tunnel in a redox-state-dependent manner, with distinct binding sites formed by conserved residue clusters. The motion of Q to these binding sites is proposed to be coupled to the proton-pumping machinery in complex I.

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