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Combining low resolution, high resolution, functional, and simulation techniques: In the study of pentameric ligand-gated ion channels
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0003-3271-7973
2022 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Combining methods yielding different information is a powerful approach for understanding structural biology in general, and in particular systems such as pentameric ligand-gated ion channels (pLGICs). Pentameric ligand-gated ion channels are membrane proteins that sense chemical signals, which they convert to changes in membrane potential. 

pLGICs constitute important drug targets in humans, for example as sites of action for general anaesthetics. There are bacterial homologs, of which some - like the Gloeobacter violaceus ligand-gated channel GLIC - are suitable model systems, while others - like DeCLIC from a Desulfofustis deltaproteobacterium - demonstrate the structural range of this protein family.

This thesis presents how low resolution, high resolution, functional, and simulation techniques have been combined in the study of GLIC and DeCLIC. The methods covered are small-angle neutron scattering (low resolution structural method), cryogenic electron microscopy (high resolution structural method), electrophysiology (functional method), and molecular dynamics simulations (simulation method), with a particular focus on the scattering and simulation experiments. 

In the presented work, simulations and functional experiments are combined to elucidate modulation of GLIC by general anaesthetics, the average solution structure of both GLIC and DeCLIC is described from small-angle scattering utilizing conformational sampling from simulations, and new conformations of DeCLIC are found through cryogenic electron microscopy - including an open conformation consistent with scattering under corresponding solution conditions.

This work has contributed to both the understanding of complex allosteric modulation, and of the conformational range available to pentameric ligand-gated ion channels. On the methodological side, I have furthered approaches for integrating small-angle neutron scattering and molecular dynamics simulations to describe membrane protein structure in solution - which stands to increase the information gained from scattering experiments, and to promote the use of scattering as a complementary technique in structural studies.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2022. , p. 37
Keywords [en]
Neutrons, Small-angle scattering, Cryo-EM, Simulations, Ion channels
National Category
Biophysics
Research subject
Biophysics
Identifiers
URN: urn:nbn:se:su:diva-210421ISBN: 978-91-8014-060-7 (print)ISBN: 978-91-8014-061-4 (electronic)OAI: oai:DiVA.org:su-210421DiVA, id: diva2:1703958
Public defence
2022-12-01, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16B and online via Zoom, public link is available at the department website, Stockholm, 09:00 (English)
Opponent
Supervisors
Available from: 2022-11-08 Created: 2022-10-16 Last updated: 2022-10-28Bibliographically approved
List of papers
1. Allosteric potentiation of a ligand-gated ion channel is mediated by access to a deep membrane-facing cavity
Open this publication in new window or tab >>Allosteric potentiation of a ligand-gated ion channel is mediated by access to a deep membrane-facing cavity
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2018 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 42, p. 10672-10677Article in journal (Refereed) Published
Abstract [en]

Theories of general anesthesia have shifted in focus from bulk lipid effects to specific interactions with membrane proteins. Target receptors include several subtypes of pentameric ligand-gated ion channels; however, structures of physiologically relevant proteins in this family have yet to define anesthetic binding at high resolution. Recent cocrystal structures of the bacterial protein GLIC provide snapshots of state-dependent binding sites for the common surgical agent propofol (PFL), offering a detailed model system for anesthetic modulation. Here, we combine molecular dynamics and oocyte electrophysiology to reveal differential motion and modulation upon modification of a transmembrane binding site within each GLIC subunit. WT channels exhibited net inhibition by PFL, and a contraction of the cavity away from the pore-lining M2 helix in the absence of drug. Conversely, in GLIC variants exhibiting net PFL potentiation, the cavity was persistently expanded and proximal to M2. Mutations designed to favor this deepened site enabled sensitivity even to subclinical concentrations of PFL, and a uniquely prolonged mode of potentiation evident up to similar to 30 min after washout. Dependence of these prolonged effects on exposure time implicated the membrane as a reservoir for a lipid-accessible binding site. However, at the highest measured concentrations, potentiation appeared to be masked by an acute inhibitory effect, consistent with the presence of a discrete, water-accessible site of inhibition. These results support a multisite model of transmembrane allosteric modulation, including a possible link between lipid- and receptor-based theories that could inform the development of new anesthetics.

Keywords
ion channels, molecular dynamics, oocyte, general anesthetic, allostery
National Category
Biochemistry and Molecular Biology Biophysics
Identifiers
urn:nbn:se:su:diva-161934 (URN)10.1073/pnas.1809650115 (DOI)000447491300054 ()30275330 (PubMedID)
Available from: 2018-11-12 Created: 2018-11-12 Last updated: 2022-10-16Bibliographically approved
2. Probing solution structure of the pentameric ligand-gated ion channel GLIC by small-angle neutron scattering
Open this publication in new window or tab >>Probing solution structure of the pentameric ligand-gated ion channel GLIC by small-angle neutron scattering
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2021 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 118, no 37, article id e2108006118Article in journal (Refereed) Published
Abstract [en]

Pentameric ligand-gated ion channels undergo subtle conformational cycling to control electrochemical signal transduction in many kingdoms of life. Several crystal structures have now been reported in this family, but the functional relevance of such models remains unclear. Here, we used small-angle neutron scattering (SANS) to probe ambient solution-phase properties of the pH-gated bacterial ion channel GLIC under resting and activating conditions. Data collection was optimized by inline paused-flow size-exclusion chromatography, and exchanging into deuterated detergent to hide the micelle contribution. Resting-state GLIC was the best-fit crystal structure to SANS curves, with no evidence for divergent mechanisms. Moreover, enhanced-sampling molecular-dynamics simulations enabled differential modeling in resting versus activating conditions, with the latter corresponding to an intermediate ensemble of both the extracellular and transmembrane domains. This work demonstrates state-dependent changes in a pentameric ion channel by SANS, an increasingly accessible method for macromolecular characterization with the coming generation of neutron sources.

Keywords
Cys-loop receptors, gating, small-angle neutron scattering, molecular dynamics, deuterated detergent
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-198864 (URN)10.1073/pnas.2108006118 (DOI)000705153400018 ()34504004 (PubMedID)2-s2.0-85114750418 (Scopus ID)
Available from: 2021-11-17 Created: 2021-11-17 Last updated: 2023-12-07Bibliographically approved
3. Biophysical characterization of calcium-binding and modulatory-domain dynamics in a pentameric ligand-gated ion channel
Open this publication in new window or tab >>Biophysical characterization of calcium-binding and modulatory-domain dynamics in a pentameric ligand-gated ion channel
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2022 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 119, no 50, article id e2210669119Article in journal (Refereed) Published
Abstract [en]

Pentameric ligand-gated ion channels (pLGICs) perform electrochemical signal transduction in organisms ranging from bacteria to humans. Among the prokaryotic pLGICs, there is architectural diversity involving N-terminal domains (NTDs) not found in eukaryotic relatives, exemplified by the calcium-sensitive channel (DeCLIC) from a Desulfofustis deltaproteobacterium, which has an NTD in addition to the canonical pLGIC structure. Here, we have characterized the structure and dynamics of DeCLIC through cryoelectron microscopy (cryo-EM), small-angle neutron scattering (SANS), and molecular dynamics (MD) simulations. In the presence and absence of calcium, cryo-EM yielded structures with alternative conformations of the calcium-binding site. SANS profiles further revealed conformational diversity at room temperature beyond that observed in static structures, shown through MD to be largely attributable to rigid-body motions of the NTD relative to the protein core, with expanded and asymmetric conformations improving the fit of the SANS data. This work reveals the range of motion available to the DeCLIC NTD and calcium-binding site, expanding the conformational landscape of the pLGIC family. Further, these findings demonstrate the power of combining low-resolution scattering, high-resolution structural, and MD simulation data to elucidate interfacial interactions that are highly conserved in the pLGIC family. 

Keywords
ligand-gated ion channel, Cys-loop receptors, small-angle neutron scattering, calcium
National Category
Biophysics
Research subject
Biophysics; Structural Biology
Identifiers
urn:nbn:se:su:diva-210411 (URN)10.1073/pnas.2210669119 (DOI)000964667700001 ()36480474 (PubMedID)2-s2.0-85143563351 (Scopus ID)
Available from: 2022-10-13 Created: 2022-10-13 Last updated: 2024-06-10Bibliographically approved
4. Structural characterization of pH-modulated closed and open states in a pentameric ligand-gated ion channel
Open this publication in new window or tab >>Structural characterization of pH-modulated closed and open states in a pentameric ligand-gated ion channel
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(English)Manuscript (preprint) (Other academic)
Keywords
Cys-loop receptors, Cryo-EM, Small-angle neutron scattering, Molecular dynamics
National Category
Biophysics
Research subject
Biophysics; Structural Biology
Identifiers
urn:nbn:se:su:diva-210413 (URN)
Available from: 2022-10-13 Created: 2022-10-13 Last updated: 2023-12-07

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