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Elucidating the Gating Mechanism of Cys-Loop Receptors
KTH, School of Engineering Sciences (SCI), Theoretical Physics, Theoretical Biological Physics.ORCID iD: 0000-0001-8354-0253
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2016. , 72 p.
Series
TRITA-FYS, ISSN 0280-316X ; 2016:26
Keyword [en]
ion channel, gating, simulation, molecular dynamics, receptor, cys-loop, modelling
National Category
Biophysics
Research subject
Theoretical Chemistry and Biology; Biological Physics
Identifiers
URN: urn:nbn:se:kth:diva-187230ISBN: 978-91-7729-009-4 (print)OAI: oai:DiVA.org:kth-187230DiVA: diva2:929445
Public defence
2016-06-13, sal F3, Lindstedtsvägen 26, Stockholm, 14:00 (English)
Opponent
Supervisors
Note

QC 20160518

Available from: 2016-05-18 Created: 2016-05-18 Last updated: 2016-05-20Bibliographically approved
List of papers
1. Assessment of homology templates and an anesthetic binding site within the ?-aminobutyric acid receptor
Open this publication in new window or tab >>Assessment of homology templates and an anesthetic binding site within the ?-aminobutyric acid receptor
2013 (English)In: Anesthesiology, ISSN 0003-3022, E-ISSN 1528-1175, Vol. 119, no 5, 1087-1095 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
4 aminobutyric acid receptor, chloride channel, glutamic acid, nicotinic receptor alpha4, nicotinic receptor beta2, propofol, algorithm, analgesic activity, article, binding affinity, consensus, drug binding site, drug effect, drug protein binding, Gloeobacter violaceus, human, molecular docking, molecular model, nonhuman, Pectobacterium chrysanthemi, pH, priority journal, prokaryote, protein structure, sequence alignment
National Category
Biophysics Anesthesiology and Intensive Care
Identifiers
urn:nbn:se:kth:diva-139979 (URN)10.1097/ALN.0b013e31829e47e3 (DOI)000329797900015 ()2-s2.0-84888298803 (Scopus ID)
Note

QC 20140121

Available from: 2014-01-21 Created: 2014-01-16 Last updated: 2017-12-06Bibliographically approved
2. Stabilization of the GluCl Ligand-Gated Ion Channel in the Presence and Absence of Ivermectin
Open this publication in new window or tab >>Stabilization of the GluCl Ligand-Gated Ion Channel in the Presence and Absence of Ivermectin
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2013 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 105, no 3, 640-647 p.Article in journal (Refereed) Published
Abstract [en]

Improving our understanding of the mechanisms and effects of anesthetics is a critically important part of neuroscience. The currently dominant theory is that anesthetics and similar molecules act by binding to Cys-loop receptors in the postsynaptic terminal of nerve cells and potentiate or inhibit their function. Although structures for some of the most important mammalian channels have still not been determined, a number of important results have been derived from work on homologous cationic channels in bacteria. However, partly due to the lack of a nervous system in bacteria, there are a number of questions about how these results relate to higher organisms. The recent determination of a structure of the eukaryotic chloride channel, GluCl, is an important step toward accurate modeling of mammalian channels, because it is more similar in function to human Cys-loop receptors such as GABA(A)R or GlyR. One potential issue with using GluCl to model other receptors is the presence of the large ligand ivermectin (IVM) positioned between all five subunits. Here, we have performed a series of microsecond molecular simulations to study how the dynamics and structure of GluCl change in the presence versus absence of IVM. When the ligand is removed, subunits move at least 2 angstrom closer to each other compared to simulations with IVM bound. In addition, the pore radius shrinks to 1.2 angstrom, all of which appears to support a model where IVM binding between subunits stabilizes an open state, and that the relaxed nonIVM conformations might be suitable for modeling other channels. Interestingly, the presence of IVM also has an effect on the structure of the important loop C located at the neurotransmitter-binding pocket, which might help shed light on its partial agonist behavior.

Keyword
Receptor Chloride Channel, X-Ray-Structure, Molecular-Dynamics, Glycine Receptor, Gaba(A) Receptor, Binding, Conformation, Simulations, Permeation, Activation
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-127748 (URN)10.1016/j.bpj.2013.06.037 (DOI)000323141100014 ()2-s2.0-84881394637 (Scopus ID)
Funder
EU, European Research Council, 209825Swedish Research Council, 2010-491 2010-5107Swedish Foundation for Strategic Research Swedish e‐Science Research CenterScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20140623

Available from: 2013-09-09 Created: 2013-09-05 Last updated: 2017-12-06Bibliographically approved
3. Conformational Gating Dynamics in the GluCl Anion-Selective Chloride Channel
Open this publication in new window or tab >>Conformational Gating Dynamics in the GluCl Anion-Selective Chloride Channel
2015 (English)In: ACS Chemical Neuroscience, ISSN 1948-7193, E-ISSN 1948-7193, Vol. 6, no 8, 1459-1467 p.Article in journal (Refereed) Published
Abstract [en]

Cys-loop receptors are central to propagation of signals in the nervous system. The gating of the membrane-spanning pore is triggered by structural rearrangements in the agonist-binding site, located some so A away from the pore. A sequential conformational change, propagating from the ligand-binding site to the pore, has been proposed to govern gating in all Cys-loop receptors. Here, we identify structural and dynamic components of the conformational gating in the eukaryotic glutamate-gated chloride channel (GluCl) by means of molecular dynamics (MD) simulations with and without the L-glutamate agonist bound. A significant increase in pore opening and accompanying hydration is observed in the presence of glutamate. Potential of mean force calculations reveal that the barrier for ion passage drops from 15 kcal/mol to 5-10 kcal/mol with the agonist bound. This appears to be explained by agonist binding that leads to significant changes in the intersubunit hydrogen-bonding pattern, which induce a slight tilt of the extracellular domain relative to the transmembrane domain in the simulations. This rearrangement is subtle, but correspond to the direction of the quaternary twist observed as a key difference between open and closed X-ray structures. While the full reversible gating is still a much slower process, the observed structural dynamics sheds new light on the early stages of how the agonist influences the extracellular domain, how the extracellular domain interacts with the transmembrane domain, and how changes in the transmembrane domain alter the free energy of ion passage.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2015
Keyword
Membrane protein, ligand-gated ion channel, cys-loop receptor, molecular dynamics simulations
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-173445 (URN)10.1021/acschemneuro.5b00111 (DOI)000359967300022 ()25992588 (PubMedID)2-s2.0-84939864190 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20171128

Available from: 2015-09-18 Created: 2015-09-11 Last updated: 2017-11-28Bibliographically approved
4. Functional Characterization of Neurotransmitter Activation and Modulation in a Nematode Model Ligand-gated Ion Channel
Open this publication in new window or tab >>Functional Characterization of Neurotransmitter Activation and Modulation in a Nematode Model Ligand-gated Ion Channel
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2016 (English)In: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 138, no 2, 243-253 p.Article in journal (Refereed) Published
Abstract [en]

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 modifiedCaenorhabditis 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.

Place, publisher, year, edition, pages
John Wiley & Sons, 2016
Keyword
anesthetic, cys-loop receptor, GluCl, Ion channel, pLGIC
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-187250 (URN)10.1111/jnc.13644 (DOI)000380263700004 ()27102368 (PubMedID)2-s2.0-84979036020 (Scopus ID)
External cooperation:
Funder
Swedish Research Council, 2013-5901Swedish e‐Science Research Center
Note

QC 20160518

Available from: 2016-05-18 Created: 2016-05-18 Last updated: 2017-11-30Bibliographically approved
5. Prediction and Validation of Protein Intermediate States from Structurally Rich Ensembles and Coarse-Grained Simulations
Open this publication in new window or tab >>Prediction and Validation of Protein Intermediate States from Structurally Rich Ensembles and Coarse-Grained Simulations
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

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. 

National Category
Structural Biology
Identifiers
urn:nbn:se:kth:diva-187252 (URN)10.1038/ncomms12575 (DOI)
Note

QC 20160518

Available from: 2016-05-18 Created: 2016-05-18 Last updated: 2016-11-30Bibliographically approved

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Output format
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