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- Bertaccini, E. J., et al.
(author)
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Assessment of homology templates and an anesthetic binding site within the ?-aminobutyric acid receptor
- 2013
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In: Anesthesiology. - 0003-3022 .- 1528-1175. ; 119:5, s. 1087-1095
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Journal article (peer-reviewed)abstract
- Background: Anesthetics mediate portions of their activity via modulation of the ?-aminobutyric acid receptor (GABAaR). Although its molecular structure remains unknown, significant progress has been made toward understanding its interactions with anesthetics via molecular modeling. Methods: The structure of the torpedo acetylcholine receptor (nAChR?), the structures of the ?4 and ?2 subunits of the human nAChR, the structures of the eukaryotic glutamate-gated chloride channel (GluCl), and the prokaryotic pH-sensing channels, from Gloeobacter violaceus and Erwinia chrysanthemi, were aligned with the SAlign and 3DMA algorithms. A multiple sequence alignment from these structures and those of the GABAaR was performed with ClustalW. The Modeler and Rosetta algorithms independently created three-dimensional constructs of the GABAaR from the GluCl template. The CDocker algorithm docked a congeneric series of propofol derivatives into the binding pocket and scored calculated binding affinities for correlation with known GABAaR potentiation EC50s. Results: Multiple structure alignments of templates revealed a clear consensus of residue locations relevant to anesthetic effects except for torpedo nAChR. Within the GABAaR models generated from GluCl, the residues notable for modulating anesthetic action within transmembrane segments 1, 2, and 3 converged on the intersubunit interface between ? and ? subunits. Docking scores of a propofol derivative series into this binding site showed strong linear correlation with GABAaR potentiation EC50. Conclusion: Consensus structural alignment based on homologous templates revealed an intersubunit anesthetic binding cavity within the transmembrane domain of the GABAaR, which showed a correlation of ligand docking scores with experimentally measured GABAaR potentiation.
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| 2. |
- Heusser, Stephanie A., et al.
(author)
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Functional characterization of neurotransmitter activation and modulation in a nematode model ligand-gated ion channel
- 2016
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In: Journal of Neurochemistry. - : Wiley. - 0022-3042 .- 1471-4159. ; 138:2, s. 243-253
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Journal article (peer-reviewed)abstract
- The superfamily of pentameric ligand-gated ion channels includes neurotransmitter receptors that mediate fast synaptic transmission in vertebrates, and are targets for drugs including alcohols, anesthetics, benzodiazepines, and anticonvulsants. However, the mechanisms of ion channel opening, gating, and modulation in these receptors leave many open questions, despite their pharmacological importance. Subtle conformational changes in both the extracellular and transmembrane domains are likely to influence channel opening, but have been difficult to characterize given the limited structural data available for human membrane proteins. Recent crystal structures of a modified Caenorhabditis elegans glutamate-gated chloride channel (GluCl) in multiple states offer an appealing model system for structure-function studies. However, the pharmacology of the crystallographic GluCl construct is not well established. To establish the functional relevance of this system, we used two-electrode voltage-clamp electrophysiology in Xenopus oocytes to characterize activation of crystallographic and native-like GluCl constructs by L-glutamate and ivermectin. We also tested modulation by ethanol and other anesthetic agents, and used site-directed mutagenesis to explore the role of a region of Loop F which was implicated in ligand gating by molecular dynamics simulations. Our findings indicate that the crystallographic construct functionally models concentration-dependent agonism and allosteric modulation of pharmacologically relevant receptors. Specific substitutions at residue Leu174 in loop F altered direct L-glutamate activation, consistent with computational evidence for this region's role in ligand binding. These insights demonstrate conservation of activation and modulation properties in this receptor family, and establish a framework for GluCl as a model system, including new possibilities for drug discovery. In this study, we elucidate the validity of a modified glutamate-gated chloride channel (GluCl(cryst)) as a structurally accessible model for GABA(A) receptors. In contrast to native-like controls, GluCl(cryst) exhibits classical activation by its neurotransmitter ligand L-glutamate. The modified channel is also sensitive to allosteric modulators associated with human GABA(A) receptors, and to site-directed mutations predicted to alter channel opening.
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| 5. |
- Nys, Mieke, et al.
(author)
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Allosteric binding site in a Cys-loop receptor ligand-binding domain unveiled in the crystal structure of ELIC in complex with chlorpromazine
- 2016
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In: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 113:43, s. E6696-E6703
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Journal article (peer-reviewed)abstract
- Pentameric ligand-gated ion channels or Cys-loop receptors are responsible for fast inhibitory or excitatory synaptic transmission. The antipsychotic compound chlorpromazine is a widely used tool to probe the ion channel pore of the nicotinic acetylcholine receptor, which is a prototypical Cys-loop receptor. In this study, we determine the molecular determinants of chlorpromazine binding in the Erwinia ligand-gated ion channel (ELIC). We report the X-ray crystal structures of ELIC in complex with chlorpromazine or its brominated derivative bromopromazine. Unexpectedly, we do not find a chlorpromazine molecule in the channel pore of ELIC, but behind the beta 8-beta 9 loop in the extracellular ligand-binding domain. The beta 8-beta 9 loop is localized downstream from the neurotransmitter binding site and plays an important role in coupling of ligand binding to channel opening. In combination with electrophysiological recordings from ELIC cysteine mutants and a thiol-reactive derivative of chlorpromazine, we demonstrate that chlorpromazine binding at the beta 8-beta 9 loop is responsible for receptor inhibition. We further use molecular-dynamics simulations to support the X-ray data and mutagenesis experiments. Together, these data unveil an allosteric binding site in the extracellular ligand-binding domain of ELIC. Our results extend on previous observations and further substantiate our understanding of a multisite model for allosteric modulation of Cys-loop receptors.
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| 6. |
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| 7. |
- Orellana, Laura, et al.
(author)
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Prediction and Validation of Protein Intermediate States from Structurally Rich Ensembles and Coarse-Grained Simulations
- 2016
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In: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 7
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Journal article (peer-reviewed)abstract
- Protein conformational changes are at the heart of cell functions, from signaling to ion transport. However, the transient nature of the intermediates along transition pathways hampers their experimental detection, making the underlying mechanisms elusive. Here, we retrieve dynamic information on the actual transition routes from Principal Component Analysis (PCA) of structurally-rich ensembles and, in combination with coarse-grained simulations, explore the conformational landscapes of five well-studied proteins. Modeling them as elastic networks in a hybrid Elastic-Network Brownian Dynamics simulation (eBDIMS), we generate trajectories connecting stable end-states that spontaneously sample the crystallographic motions, predicting the structures of known intermediates along thepaths. We also show that the explored non-linear routes can delimit the lowest energy passages between end-states sampled by atomistic molecular dynamics. The integrative methodology presented here provides a powerful framework to extract and expand dynamic pathway information from the Protein Data Bank, as well as to validate sampling methods in general.
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| 10. |
- Yoluk, Özge, 1986-
(author)
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Elucidating the Gating Mechanism of Cys-Loop Receptors
- 2016
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Doctoral thesis (other academic/artistic)abstract
- Cys-loop receptors are membrane proteins that are key players for the fast synaptic neurotransmission. Their ion transport initiates new nerve signals after activation by small agonist molecules, but this function is also highly sensitive to allosteric modulation by a number of compounds such as anesthetics, alcohol or anti-parasitic agents. For a long time, these modulators were believed to act primarily on the membrane, but the availability of high- resolution structures has made it possible to identify several binding sites in the transmembrane domains of the ion channels. It is known that ligand binding in the extracellular domain causes a conformational earthquake that interacts with the transmembrane domain, which leads to channel opening. The investigations carried out in this thesis aim at understanding the connection between ligand binding and channel opening.I present new models of the mammalian GABAA receptor based on the eukaryotic structure GluCl co-crystallized with an anti-parasitic agent, and show how these models can be used to study receptor-modulator interactions. I also show how removal of the bound modulator leads to gradual closing of the channel in molecular dynamics simulations. In contrast, simulations of the receptor with both the agonist and the modulator remain stable in an open-like conformation. This makes it possible to extract several key interactions, and I propose mechanisms for how the extracellular domain motion is initiated. The rapid increase in the number of cys-loop receptor structures the last few years has further made it possible to use principal component analysis (PCA) to create low-dimensional descriptions of the conformational landscape. By performing PCA on the crystal structure ensemble, I have been able to divide the structures into functional clusters and sample the transitions between them using various sampling methods.The studies presented in this thesis contribute to our understanding of the gating mechanism and the functional clustering of the cys-loop receptor structures, which both are important to design new allosteric modulator drugs that influence the channel function, in particular to treat neurological disorders.
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