| 1. |
- Carnevale, Vincenzo, et al.
(författare)
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Molecular Dynamics Simulations of Ion Channels
- 2021
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Ingår i: TIBS -Trends in Biochemical Sciences. Regular ed.. - : Elsevier BV. - 0968-0004 .- 1362-4326. ; 46:7, s. 621-622
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 2. |
- Chen, Yue, et al.
(författare)
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Allosteric Effect of Nanobody Binding on Ligand-Specific Active States of the beta 2 Adrenergic Receptor
- 2021
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Ingår i: Journal of Chemical Information and Modeling. - : American Chemical Society (ACS). - 1549-9596 .- 1549-960X. ; 61:12, s. 6024-6037
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Tidskriftsartikel (refereegranskat)abstract
- Nanobody binding stabilizes G-protein-coupled receptors (GPCR) in a fully active state and modulates their affinity for bound ligands. However, the atomic-level basis for this allosteric regulation remains elusive. Here, we investigate the conformational changes induced by the binding of a nanobody (Nb80) on the active-like beta 2 adrenergic receptor (beta 2AR) via enhanced sampling molecular dynamics simulations. Dimensionality reduction analysis shows that Nb80 stabilizes structural features of the beta 2AR with an similar to 14 angstrom outward movement of transmembrane helix 6 and a close proximity of transmembrane (TM) helices 5 and 7, and favors the fully active-like conformation of the receptor, independent of ligand binding, in contrast to the conditions under which no intracellular binding partner is bound, in which case the receptor is only stabilized in an intermediateactive state. This activation is supported by the residues located at hotspots located on TMs 5, 6, and 7, as shown by supervised machine learning methods. Besides, ligand-specific subtle differences in the conformations assumed by intracellular loop 2 and extracellular loop 2 are captured from the trajectories of various ligand-bound receptors in the presence of Nb80. Dynamic network analysis further reveals that Nb80 binding triggers tighter and stronger local communication networks between the Nb80 and the ligand-binding sites, primarily involving residues around ICL2 and the intracellular end of TM3, TM5, TM6, as well as ECL2, ECL3, and the extracellular ends of TM6 and TM7. In particular, we identify unique allosteric signal transmission mechanisms between the Nb80-binding site and the extracellular domains in conformations modulated by a full agonist, BI167107, and a G-protein-biased partial agonist, salmeterol, involving mainly TM1 and TM2, and TM5, respectively. Altogether, our results provide insights into the effect of intracellular binding partners on the GPCR activation mechanism, which should be taken into account in structure-based drug discovery.
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| 3. |
- Delemotte, Lucie
(författare)
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Chapter 7 : Bridging the Gap between Atomistic Molecular Dynamics Simulations and Wet-lab Experimental Techniques: Applications to Membrane Proteins
- 2021
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Ingår i: RSC Theoretical and Computational Chemistry Series. - : Royal Society of Chemistry. ; , s. 247-286
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Bokkapitel (refereegranskat)abstract
- Molecular dynamics (MD) simulations provide atomistic insights into not only the structure, but also the dynamics and ensemble properties of (bio-)molecular systems, hence providing a direct link to functional characterization using wet-lab experiments. The models, algorithms and hardware needed to conduct MD simulations have matured, meaning that reliable estimates of ensemble properties can now be obtained. However, the choice of model and protocol is non-trivial and cannot be fully automated yet, therefore an understanding of the models, the algorithms and the insights that can be obtained, and of how they can be combined with the output of other techniques, is necessary. This chapter provides a description of the MD algorithm, including extensions of the methodology to generate conformational ensembles representing functional states. The insights that MD simulations can provide into membrane protein functions are then illustrated using case studies. They are classified according to whether they provide testable hypotheses, provide molecular-level interpretation of experimental observables, or they exploit experimental data to drive the sampling of simulations towards biological timescales.
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| 4. |
- Elbahnsi, Ahmad, et al.
(författare)
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Structure and Sequence-based Computational Approaches to Allosteric Signal Transduction : Application to Electromechanical Coupling in Voltage-gated Ion Channels
- 2021
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 433:17
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Forskningsöversikt (refereegranskat)abstract
- Allosteric signaling underlies the function of many biomolecules, including membrane proteins such as ion channels. Experimental methods have enabled specific quantitative insights into the coupling between the voltage sensing domain (VSD) and the pore gate of voltage-gated ion channels, located tens of Angstrom apart from one another, as well as pinpointed specific residues and domains that participate in electromechanical signal transmission. Nevertheless, an overall atomic-level resolution picture is difficult to obtain from these methods alone. Today, thanks to the cryo-EM resolution revolution, we have access to high resolution structures of many different voltage-gated ion channels in various conformational states, putting a quantitative description of the processes at the basis of these changes within our close reach. Here, we review computational methods that build on structures to detect and characterize allosteric signaling and pathways. We then examine what has been learned so far about electromechanical coupling between VSD and pore using such methods. While no general theory of electromechanical coupling in voltage-gated ion channels integrating results from all these methods is available yet, we outline the types of insights that could be achieved in the near future using the methods that have not yet been put to use in this field of application.
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| 5. |
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| 6. |
- Fleetwood, Oliver, 1990-, et al.
(författare)
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Identification of ligand-specific G protein-coupled receptor states and prediction of downstream efficacy via data-driven modeling
- 2021
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Ingår i: eLIFE. - : eLife Sciences Publications, Ltd. - 2050-084X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Ligand binding stabilizes different G protein-coupled receptor states via a complex allosteric process that is not completely understood. Here, we have derived free energy landscapes describing activation of the beta(2) adrenergic receptor bound to ligands with different efficacy profiles using enhanced sampling molecular dynamics simulations. These reveal shifts toward active-like states at the Gprotein-binding site for receptors bound to partial and full agonists, and that the ligands modulate the conformational ensemble of the receptor by tuning protein microswitches. We indeed find an excellent correlation between the conformation of the microswitches close to the ligand binding site and in the transmembrane region and experimentally reported cyclic adenosine monophosphate signaling responses. Dimensionality reduction further reveals the similarity between the unique conformational states induced by different ligands, and examining the output of classifiers highlights two distant hotspots governing agonism on transmembrane helices 5 and 7.
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| 7. |
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| 8. |
- Fleetwood, Oliver, 1990-
(författare)
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New approaches to data-driven analysis and enhanced sampling simulations of G protein-coupled receptors
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are large biomolecules that carry out specific functions within living organisms. Understanding how proteins function is a massive scientific challenge with a wide area of applications. In particular, by controlling protein function we may develop therapies for many diseases. To understand a protein’s function, we need to consider its full conformational ensemble, and not only a single snapshot of a structure. Allosteric signaling is a factor often driving protein conformation change, where the binding of a molecule to one site triggers a response in another part of the protein. G protein-coupled receptors (GPCRs) are transmembrane proteins that bind molecules outside the membrane, which enables coupling to a G protein in their intracellular domain. Understanding the complex allosteric process governing this mechanism could have a significant impact on the development of novel drugs.Molecular dynamics (MD) is a computational method that can capture protein conformational change at an atomistic level. However, MD is a computationally expensive approach to simulating proteins, and is thus infeasible for many applications. Enhanced sampling techniques have emerged to reduce the computational cost of standard MD. Another challenge with MD is to extract useful information and distinguish signal from noise in an MD trajectory. Data-driven methods can streamline analysis of protein simulations and improve our understanding of biomolecular systems.Paper 1 and 2 contain methodological developments to analyze the results of MD in a data-driven manner. We provide methods that create interpretable maps of important molecular features from protein simulations (Paper 1) and identify allosteric communication pathways in biological systems (Paper 2). As a result, more insights can be extracted from MD trajectories. Our approach is generalizable and can become useful to analyze complex simulations of various biomolecular systems. In Paper 3 and 4, we combine the aforementioned methodological advancements with enhanced sampling techniques to study a prototypical GPCR, the β2 adrenergic receptor. First, we make improvements to the string method with swarms of trajectories and derive the conformational change and free energy along the receptor’s activation pathway. Next, we identify key molecular microswitches directly or allosterically controlled by orthosteric ligands and show how these couple to a shift in probability of the receptor’s active state. In Paper 4, we also find that ligands induce ligand-specific states, and the molecular basis governing these states. These new approaches generate insights compatible with previous simulation and experimental studies at a relatively low computational cost. Our work also provides new insights into the molecular basis of allosteric communication in membrane proteins, and might become a useful tool in the design of novel GPCR drugs.
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| 9. |
- Galleano, Iacopo, et al.
(författare)
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Functional cross-talk between phosphorylation and disease-causing mutations in the cardiac sodium channel Na(v)1.5
- 2021
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 118:33
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Tidskriftsartikel (refereegranskat)abstract
- The voltage-gated sodium channel Nav1.5 initiates the cardiac action potential. Alterations of its activation and inactivation properties due to mutations can cause severe, life-threatening arrhythmias. Yet despite intensive research efforts, many functional aspects of this cardiac channel remain poorly understood. For instance, Nav1.5 undergoes extensive posttranslational modification in vivo, but the functional significance of these modifications is largely unexplored, especially under pathological conditions. This is because most conventional approaches are unable to insert metabolically stable posttranslational modification mimics, thus preventing a precise elucidation of the contribution by these modifications to channel function. Here, we overcome this limitation by using protein semisynthesis of Nav1.5 in live cells and carry out complementary molecular dynamics simulations. We introduce metabolically stable phosphorylation mimics on both wild-type (WT) and two pathogenic long-QT mutant channel backgrounds and decipher functional and pharmacological effects with unique precision. We elucidate the mechanism by which phosphorylation of Y1495 impairs steady-state inactivation in WT Nav1.5. Surprisingly, we find that while the Q1476R patient mutation does not affect inactivation on its own, it enhances the impairment of steady-state inactivation caused by phosphorylation of Y1495 through enhanced unbinding of the inactivation particle. We also show that both phosphorylation and patient mutations can impact Nav1.5 sensitivity toward the clinically used antiarrhythmic drugs quinidine and ranolazine, but not flecainide. The data highlight that functional effects of Nav1.5 phosphorylation can be dramatically amplified by patient mutations. Our work is thus likely to have implications for the interpretation of mutational phenotypes and the design of future drug regimens.
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| 10. |
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| 11. |
- Ghovanloo, Mohammad-Reza, et al.
(författare)
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Cannabidiol inhibits the skeletal muscle Nav1.4 by blocking its pore and by altering membrane elasticity
- 2021
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Ingår i: The Journal of General Physiology. - : Rockefeller University Press. - 0022-1295 .- 1540-7748. ; 153:5
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Tidskriftsartikel (refereegranskat)abstract
- Cannabidiol (CBD) is the primary nonpsychotropic phytocannabinoid found in Cannabis sativa, which has been proposed to be therapeutic against many conditions, including muscle spasms. Among its putative targets are voltage-gated sodium channels (Navs), which have been implicated in many conditions. We investigated the effects of CBD on Nav1.4, the skeletal muscle Nav subtype. We explored direct effects, involving physical block of the Nav pore, as well as indirect effects, involving modulation of membrane elasticity that contributes to Nav inhibition. MD simulations revealed CBD's localization inside the membrane and effects on bilayer properties. Nuclear magnetic resonance (NMR) confirmed these results, showing CBD localizing below membrane headgroups. To determine the functional implications of these findings, we used a gramicidinbased fluorescence assay to show that CBD alters membrane elasticity or thickness, which could alter Nav function through bilayer-mediated regulation. Site-directed mutagenesis in the vicinity of the Nav1.4 pore revealed that removing the local anesthetic binding site with F1586A reduces the block of INa by CBD. Altering the fenestrations in the bilayer-spanning domain with Nav1.4-WWWW blocked CBD access from the membrane into the Nav1.4 pore (as judged by MD). The stabilization of inactivation, however, persisted in WWWW, which we ascribe to CBD-induced changes in membrane elasticity. To investigate the potential therapeutic value of CBD against Nav1.4 channelopathies, we used a pathogenic Nav1.4 variant, P1158S, which causes myotonia and periodic paralysis. CBD reduces excitability in both wild-type and the P1158S variant. Our in vitro and in silico results suggest that CBD may have therapeutic value against Nav1.4 hyperexcitability.
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| 12. |
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| 13. |
- Perez-Conesa, Sergio, et al.
(författare)
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Informing NMR experiments with molecular dynamics simulations to characterize the dominant activated state of the KcsA ion channel
- 2021
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Ingår i: Journal of Chemical Physics. - : AIP Publishing. - 0021-9606 .- 1089-7690. ; 154:16
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Tidskriftsartikel (refereegranskat)abstract
- As the first potassium channel with an x-ray structure determined, and given its homology to eukaryotic channels, the pH-gated prokaryotic channel KcsA has been extensively studied. Nevertheless, questions related, in particular, to the allosteric coupling between its gates remain open. The many currently available x-ray crystallography structures appear to correspond to various stages of activation and inactivation, offering insights into the molecular basis of these mechanisms. Since these studies have required mutations, complexation with antibodies, and substitution of detergents in place of lipids, examining the channel under more native conditions is desirable. Solid-state nuclear magnetic resonance (SSNMR) can be used to study the wild-type protein under activating conditions (low pH), at room temperature, and in bacteriomimetic liposomes. In this work, we sought to structurally assign the activated state present in SSNMR experiments. We used a combination of molecular dynamics (MD) simulations, chemical shift prediction algorithms, and Bayesian inference techniques to determine which of the most plausible x-ray structures resolved to date best represents the activated state captured in SSNMR. We first identified specific nuclei with simulated NMR chemical shifts that differed significantly when comparing partially open vs fully open ensembles from MD simulations. The simulated NMR chemical shifts for those specific nuclei were then compared to experimental ones, revealing that the simulation of the partially open state was in good agreement with the SSNMR data. Nuclei that discriminate effectively between partially and fully open states belong to residues spread over the sequence and provide a molecular level description of the conformational change.
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| 14. |
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| 15. |
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| 16. |
- Silverå Ejneby, Malin, et al.
(författare)
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Resin-acid derivatives bind to multiple sites on the voltage-sensor domain of the Shaker potassium channel
- 2021
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Ingår i: The Journal of General Physiology. - : Rockefeller University Press. - 0022-1295 .- 1540-7748. ; 153:4
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Tidskriftsartikel (refereegranskat)abstract
- Voltage-gated potassium (K-V) channels can be opened by negatively charged resin acids and their derivatives. These resin acids have been proposed to attract the positively charged voltage-sensor helix (S4) toward the extracellular side of the membrane by binding to a pocket located between the lipid-facing extracellular ends of the transmembrane segments S3 and S4. By contrast to this proposed mechanism, neutralization of the top gating charge of the Shaker KV channel increased resin-acid-induced opening, suggesting other mechanisms and sites of action. Here, we explore the binding of two resin-acid derivatives, Wu50 and Wu161, to the activated/open state of the Shaker KV channel by a combination of in silico docking, molecular dynamics simulations, and electrophysiology of mutated channels. We identified three potential resin-acid-binding sites around S4: (1) the S3/S4 site previously suggested, in which positively charged residues introduced at the top of S4 are critical to keep the compound bound, (2) a site in the cleft between S4 and the pore domain (S4/pore site), in which a tryptophan at the top of S6 and the top gating charge of S4 keeps the compound bound, and (3) a site located on the extracellular side of the voltage-sensor domain, in a cleft formed by S1-S4 (the top-VSD site). The multiple binding sites around S4 and the anticipated helical-screw motion of the helix during activation make the effect of resin-acid derivatives on channel function intricate. The propensity of a specific resin acid to activate and open a voltage-gated channel likely depends on its exact binding dynamics and the types of interactions it can form with the protein in a state-specific manner.
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