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Yvonnesdotter, L., Rovšnik, U., Blau, C., Lycksell, M., Howard, R. J. & Lindahl, E. (2023). Automated simulation-based membrane protein refinement into cryo-EM data. Biophysical Journal, 122(13), 2773-2781
Open this publication in new window or tab >>Automated simulation-based membrane protein refinement into cryo-EM data
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2023 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 122, no 13, p. 2773-2781Article in journal (Refereed) Published
Abstract [en]

The resolution revolution has increasingly enabled single-particle cryogenic electron microscopy (cryo-EM) reconstructions of previously inaccessible systems, including membrane proteins—a category that constitutes a disproportionate share of drug targets. We present a protocol for using density-guided molecular dynamics simulations to automatically refine atomistic models into membrane protein cryo-EM maps. Using adaptive force density-guided simulations as implemented in the GROMACS molecular dynamics package, we show how automated model refinement of a membrane protein is achieved without the need to manually tune the fitting force ad hoc. We also present selection criteria to choose the best-fit model that balances stereochemistry and goodness of fit. The proposed protocol was used to refine models into a new cryo-EM density of the membrane protein maltoporin, either in a lipid bilayer or detergent micelle, and we found that results do not substantially differ from fitting in solution. Fitted structures satisfied classical model-quality metrics and improved the quality and the model-to-map correlation of the x-ray starting structure. Additionally, the density-guided fitting in combination with generalized orientation-dependent all-atom potential was used to correct the pixel-size estimation of the experimental cryo-EM density map. This work demonstrates the applicability of a straightforward automated approach to fitting membrane protein cryo-EM densities. Such computational approaches promise to facilitate rapid refinement of proteins under different conditions or with various ligands present, including targets in the highly relevant superfamily of membrane proteins.

National Category
Biophysics
Identifiers
urn:nbn:se:su:diva-225410 (URN)10.1016/j.bpj.2023.05.033 (DOI)001122880200001 ()37277992 (PubMedID)2-s2.0-85162918774 (Scopus ID)
Available from: 2024-01-17 Created: 2024-01-17 Last updated: 2024-01-17Bibliographically approved
Bergh, C., Rovšnik, U., Howard, R. J. & Lindahl, E. (2023). Discovery of lipid binding sites in a ligand-gated ion channel by integrating simulations and cryo-EM. eLIFE, 12, Article ID RP86016.
Open this publication in new window or tab >>Discovery of lipid binding sites in a ligand-gated ion channel by integrating simulations and cryo-EM
2023 (English)In: eLIFE, E-ISSN 2050-084X, Vol. 12, article id RP86016Article in journal (Refereed) Published
Abstract [en]

Ligand-gated ion channels transduce electrochemical signals in neurons and other excitable cells. Aside from canonical ligands, phospholipids are thought to bind specifically to the transmembrane domain of several ion channels. However, structural details of such lipid contacts remain elusive, partly due to limited resolution of these regions in experimental structures. Here, we discovered multiple lipid interactions in the channel GLIC by integrating cryo-electron microscopy and large-scale molecular simulations. We identified 25 bound lipids in the GLIC closed state, a conformation where none, to our knowledge, were previously known. Three lipids were associated with each subunit in the inner leaflet, including a buried interaction disrupted in mutant simulations. In the outer leaflet, two intrasubunit sites were evident in both closed and open states, while a putative intersubunit site was preferred in open-state simulations. This work offers molecular details of GLIC-lipid contacts particularly in the ill-characterized closed state, testable hypotheses for state-dependent binding, and a multidisciplinary strategy for modeling protein-lipid interactions.

National Category
Biophysics Structural Biology
Identifiers
urn:nbn:se:su:diva-226504 (URN)10.7554/eLife.86016 (DOI)001154973600001 ()38289224 (PubMedID)2-s2.0-85184345538 (Scopus ID)
Available from: 2024-02-19 Created: 2024-02-19 Last updated: 2024-02-19Bibliographically approved
Blau, C., Yvonnesdotter, L. & Lindahl, E. (2023). Gentle and fast all-atom model refinement to cryo-EM densities via a maximum likelihood approach. PloS Computational Biology, 19(7), Article ID e1011255.
Open this publication in new window or tab >>Gentle and fast all-atom model refinement to cryo-EM densities via a maximum likelihood approach
2023 (English)In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 19, no 7, article id e1011255Article in journal (Refereed) Published
Abstract [en]

Better detectors and automated data collection have generated a flood of high-resolution cryo-EM maps, which in turn has renewed interest in improving methods for determining structure models corresponding to these maps. However, automatically fitting atoms to densities becomes difficult as their resolution increases and the refinement potential has a vast number of local minima. In practice, the problem becomes even more complex when one also wants to achieve a balance between a good fit of atom positions to the map, while also establishing good stereochemistry or allowing protein secondary structure to change during fitting. Here, we present a solution to this challenge using a maximum likelihood approach by formulating the problem as identifying the structure most likely to have produced the observed density map. This allows us to derive new types of smooth refinement potential-based on relative entropy-in combination with a novel adaptive force scaling algorithm to allow balancing of force-field and density-based potentials. In a low-noise scenario, as expected from modern cryo-EM data, the relative-entropy based refinement potential outperforms alternatives, and the adaptive force scaling appears to aid all existing refinement potentials. The method is available as a component in the GROMACS molecular simulation toolkit. Author summaryCryo-electron microscopy has gone through a revolution and now regularly produces data with 2 & ANGS; resolution. However, this data comes in the shape of density maps, and fitting atomic coordinates into these maps can be a labor-intensive and challenging problem. This is particularly valid when there are multiple conformations, flexible regions, or parts of the structure with lower resolution. In many cases it is also desirable to to understand how a molecule moves between such conformations. This can be addressed with molecular dynamics simulations using densities as target restraints, but the refinement potentials commonly used can distort protein structure or get stuck in local minima when the cryo-EM map has high resolution. This work derives new refinement potentials based on models of the cryo-EM scattering process that provide a gentle way to fit protein structures to densities in simulations, and we also suggest an automated heuristic way to balance the influence of the map and simulation force field.

National Category
Biophysics
Identifiers
urn:nbn:se:su:diva-220842 (URN)10.1371/journal.pcbi.1011255 (DOI)001041056800002 ()37523411 (PubMedID)2-s2.0-85168221746 (Scopus ID)
Available from: 2023-09-13 Created: 2023-09-13 Last updated: 2023-09-13Bibliographically approved
Wieczór, M., Genna, V., Aranda, J., Badia, R. M., Gelpí, J. L., Gapsys, V., . . . Orozco, M. (2023). Pre-exascale HPC approaches for molecular dynamics simulations. Covid-19 research: A use case. Wiley Interdisciplinary Reviews. Computational Molecular Science, 13(1), Article ID e1622.
Open this publication in new window or tab >>Pre-exascale HPC approaches for molecular dynamics simulations. Covid-19 research: A use case
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2023 (English)In: Wiley Interdisciplinary Reviews. Computational Molecular Science, ISSN 1759-0876, E-ISSN 1759-0884, Vol. 13, no 1, article id e1622Article in journal (Refereed) Published
Abstract [en]

Exascale computing has been a dream for ages and is close to becoming a reality that will impact how molecular simulations are being performed, as well as the quantity and quality of the information derived for them. We review how the biomolecular simulations field is anticipating these new architectures, making emphasis on recent work from groups in the BioExcel Center of Excellence for High Performance Computing. We exemplified the power of these simulation strategies with the work done by the HPC simulation community to fight Covid-19 pandemics. 

Keywords
BioExcel, COVID19, exascale, molecular dynamics, Computer programming, Expert systems, Monte Carlo methods, Statistical mechanics, Biomolecular Simulation, Centers of excellence, Exascale computing, Molecular simulations, Performance computing, Power, Simulation strategies
National Category
Computer and Information Sciences
Identifiers
urn:nbn:se:su:diva-206314 (URN)10.1002/wcms.1622 (DOI)000802572900001 ()2-s2.0-85130993494 (Scopus ID)
Available from: 2022-06-22 Created: 2022-06-22 Last updated: 2023-02-23Bibliographically approved
Bondarenko, V., Chen, Q., Singewald, K., Haloi, N., Tillman, T. S., Howard, R. J., . . . Tang, P. (2023). Structural Elucidation of Ivermectin Binding to α7nAChR and the Induced Channel Desensitization. ACS Chemical Neuroscience, 14(6), 1156-1165
Open this publication in new window or tab >>Structural Elucidation of Ivermectin Binding to α7nAChR and the Induced Channel Desensitization
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2023 (English)In: ACS Chemical Neuroscience, E-ISSN 1948-7193, Vol. 14, no 6, p. 1156-1165Article in journal (Refereed) Published
Abstract [en]

The α7 nicotinic acetylcholine receptor (α7nAChR) mediates signaling in the central nervous system and cholinergic anti-inflammatory pathways. Ivermectin is a positive allosteric modulator of a full-length α7nAChR and an agonist of the α7nAChR construct containing transmembrane (TMD) and intracellular (ICD) domains, but structural insights of the binding have not previously been determined. Here, combining nuclear magnetic resonance as a primary experimental tool with Rosetta comparative modeling and molecular dynamics simulations, we have revealed details of ivermectin binding to the α7nAChR TMD + ICD and corresponding structural changes in an ivermectin-induced desensitized state. Ivermectin binding was stabilized predominantly by hydrophobic interactions from interfacial residues between adjacent subunits near the extracellular end of the TMD, where the inter-subunit gap was substantially expanded in comparison to the apo structure. The ion-permeation pathway showed a profile distinctly different from the resting-state profile but similar to profiles of desensitized α7nAChR. The ICD also exhibited structural changes, including reorientation of the MX and h3 helices relative to the channel axis. The resulting structures of the α7nAChR TMD + ICD in complex with ivermectin provide opportunities for discovering new modulators of therapeutic potential and exploring the structural basis of cytoplasmic signaling under different α7nAChR functional states. 

Keywords
ivermectin binding, α7 nicotinic acetylcholine receptor, α7nAChR, channel desensitization, NMR, modeling
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-215815 (URN)10.1021/acschemneuro.2c00783 (DOI)000936888300001 ()36821490 (PubMedID)2-s2.0-85149024934 (Scopus ID)
Available from: 2023-03-29 Created: 2023-03-29 Last updated: 2023-08-28Bibliographically approved
Legesse, D. H., Fan, C., Teng, J., Zhuang, Y., Howard, R. J., Noviello, C. M., . . . Hibbs, R. E. (2023). Structural insights into opposing actions of neurosteroids on GABAA receptors. Nature Communications, 14(1)
Open this publication in new window or tab >>Structural insights into opposing actions of neurosteroids on GABAA receptors
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2023 (English)In: Nature Communications, E-ISSN 2041-1723, Vol. 14, no 1Article in journal (Refereed) Published
Abstract [en]

γ-Aminobutyric acid type A (GABAA) receptors mediate fast inhibitory signaling in the brain and are targets of numerous drugs and endogenous neurosteroids. A subset of neurosteroids are GABAA receptor positive allosteric modulators; one of these, allopregnanolone, is the only drug approved specifically for treating postpartum depression. There is a consensus emerging from structural, physiological and photolabeling studies as to where positive modulators bind, but how they potentiate GABA activation remains unclear. Other neurosteroids are negative modulators of GABAA receptors, but their binding sites remain debated. Here we present structures of a synaptic GABAA receptor bound to allopregnanolone and two inhibitory sulfated neurosteroids. Allopregnanolone binds at the receptor-bilayer interface, in the consensus potentiator site. In contrast, inhibitory neurosteroids bind in the pore. MD simulations and electrophysiology support a mechanism by which allopregnanolone potentiates channel activity and suggest the dominant mechanism for sulfated neurosteroid inhibition is through pore block.

Legesse et al. present structural studies of a human GABAA receptor in complex with positive and negative modulator neurosteroids, uncovering mechanisms of potentiation and inhibition.

National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-220877 (URN)10.1038/s41467-023-40800-1 (DOI)001053269200022 ()37607940 (PubMedID)2-s2.0-85168677110 (Scopus ID)
Available from: 2023-09-18 Created: 2023-09-18 Last updated: 2023-09-18Bibliographically approved
Cowgill, J., Fan, C., Haloi, N., Tobiasson, V., Zhuang, Y., Howard, R. J. & Lindahl, E. (2023). Structure and dynamics of differential ligand binding in the human ρ-type GABAA receptor. Neuron, 111(21), 3450-3464
Open this publication in new window or tab >>Structure and dynamics of differential ligand binding in the human ρ-type GABAA receptor
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2023 (English)In: Neuron, ISSN 0896-6273, E-ISSN 1097-4199, Vol. 111, no 21, p. 3450-3464Article in journal (Refereed) Published
Abstract [en]

The neurotransmitter γ-aminobutyric acid (GABA) drives critical inhibitory processes in and beyond the nervous system, partly via ionotropic type-A receptors (GABAARs). Pharmacological properties of ρ-type GABAARs are particularly distinctive, yet the structural basis for their specialization remains unclear. Here, we present cryo-EM structures of a lipid-embedded human ρ1 GABAAR, including a partial intracellular domain, under apo, inhibited, and desensitized conditions. An apparent resting state, determined first in the absence of modulators, was recapitulated with the specific inhibitor (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid and blocker picrotoxin and provided a rationale for bicuculline insensitivity. Comparative structures, mutant recordings, and molecular simulations with and without GABA further explained the sensitized but slower activation of ρ1 relative to canonical subtypes. Combining GABA with picrotoxin also captured an apparent uncoupled intermediate state. This work reveals structural mechanisms of gating and modulation with applications to ρ-specific pharmaceutical design and to our biophysical understanding of ligand-gated ion channels.

National Category
Biophysics Structural Biology
Identifiers
urn:nbn:se:su:diva-225080 (URN)10.1016/j.neuron.2023.08.006 (DOI)001109101000001 ()37659407 (PubMedID)2-s2.0-85175088643 (Scopus ID)
Available from: 2024-01-09 Created: 2024-01-09 Last updated: 2024-01-15Bibliographically approved
Wennberg, C., Lundborg, M., Lindahl, E. & Norlen, L. (2023). Understanding Drug Skin Permeation Enhancers Using Molecular Dynamics Simulations. Journal of Chemical Information and Modeling, 63(15), 4900-4911
Open this publication in new window or tab >>Understanding Drug Skin Permeation Enhancers Using Molecular Dynamics Simulations
2023 (English)In: Journal of Chemical Information and Modeling, ISSN 1549-9596, E-ISSN 1549-960X, Vol. 63, no 15, p. 4900-4911Article in journal (Refereed) Published
Abstract [en]

Our skin constitutes an effective permeability barrier that protects the body from exogenous substances but concomitantly severely limits the number of pharmaceutical drugs that can be delivered transdermally. In topical formulation design, chemical permeation enhancers (PEs) are used to increase drug skin permeability. In vitro skin permeability experiments can measure net effects of PEs on transdermal drug transport, but they cannot explain the molecular mechanisms of interactions between drugs, permeation enhancers, and skin structure, which limits the possibility to rationally design better new drug formulations. Here we investigate the effect of the PEs water, lauric acid, geraniol, stearic acid, thymol, ethanol, oleic acid, and eucalyptol on the transdermal transport of metronidazole, caffeine, and naproxen. We use atomistic molecular dynamics (MD) simulations in combination with developed molecular models to calculate the free energy difference between 11 PE-containing formulations and the skin’s barrier structure. We then utilize the results to calculate the final concentration of PEs in skin. We obtain an RMSE of 0.58 log units for calculated partition coefficients from water into the barrier structure. We then use the modified PE-containing barrier structure to calculate the PEs’ permeability enhancement ratios (ERs) on transdermal metronidazole, caffeine, and naproxen transport and compare with the results obtained from in vitro experiments. We show that MD simulations are able to reproduce rankings based on ERs. However, strict quantitative correlation with experimental data needs further refinement, which is complicated by significant deviations between different measurements. Finally, we propose a model for how to use calculations of the potential of mean force of drugs across the skin’s barrier structure in a topical formulation design.

National Category
Pharmaceutical Sciences Theoretical Chemistry
Identifiers
urn:nbn:se:su:diva-221279 (URN)10.1021/acs.jcim.3c00625 (DOI)001031268800001 ()37462219 (PubMedID)2-s2.0-85166537534 (Scopus ID)
Available from: 2023-09-26 Created: 2023-09-26 Last updated: 2023-09-26Bibliographically approved
Lycksell, M., Rovšnik, U., Hanke, A., Martel, A., Howard, R. J. & Lindahl, E. (2022). Biophysical characterization of calcium-binding and modulatory-domain dynamics in a pentameric ligand-gated ion channel. Proceedings of the National Academy of Sciences of the United States of America, 119(50), Article ID e2210669119.
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: 2023-12-07Bibliographically approved
Zhuang, Y., Noviello, C. M., Hibbs, R. E., Howard, R. J. & Lindahl, E. (2022). Differential interactions of resting, activated, and desensitized states of the α7 nicotinic acetylcholine receptor with lipidic modulators. Proceedings of the National Academy of Sciences of the United States of America, 119(43), Article ID e2208081119.
Open this publication in new window or tab >>Differential interactions of resting, activated, and desensitized states of the α7 nicotinic acetylcholine receptor with lipidic modulators
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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 43, article id e2208081119Article in journal (Refereed) Published
Abstract [en]

The α7 nicotinic acetylcholine receptor is a pentameric ligand-gated ion channel that modulates neuronal excitability, largely by allowing Ca2+ permeation. Agonist binding promotes transition from a resting state to an activated state, and then rapidly to a desensitized state. Recently, cryogenic electron microscopy (cryo-EM) structures of the human α7 receptor in nanodiscs were reported in multiple conformations. These were selectively stabilized by inhibitory, activating, or potentiating compounds. However, the functional annotation of these structures and their differential interactions with unresolved lipids and ligands remain incomplete. Here, we characterized their ion permeation, membrane interactions, and ligand binding using computational electrophysiology, free-energy calculations, and coarse-grained molecular dynamics. In contrast to nonconductive structures in apparent resting and desensitized states, the structure determined in the presence of the potentiator PNU-120596 was consistent with an activated state permeable to Ca2+. Transition to this state was associated with compression and rearrangement of the membrane, particularly in the vicinity of the peripheral MX helix. An intersubunit transmembrane site was implicated in selective binding of either PNU-120596 in the activated state or cholesterol in the desensitized state. This substantiates functional assignment of all three lipid-embedded α7-receptor structures with ion-permeation simulations. It also proposes testable models of their state-dependent interactions with lipophilic ligands, including a mechanism for allosteric modulation at the transmembrane subunit interface.

Place, publisher, year, edition, pages
National Academy of Sciences, 2022
Keywords
cholesterol, coarse-grained simulations, computational electrophysiology, ligand-gated ion channel, nicotinic acetylcholine receptor, 1-(5-chloro-2, 4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea, bungarotoxin receptor, carbanilamide derivative, isoxazole derivative, ligand, ligand gated ion channel, lipid, nicotinic receptor, allosterism, human, metabolism, Allosteric Regulation, alpha7 Nicotinic Acetylcholine Receptor, Humans, Isoxazoles, Ligand-Gated Ion Channels, Ligands, Lipids, Phenylurea Compounds, Receptors, Nicotinic
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-211900 (URN)10.1073/pnas.2208081119 (DOI)36251999 (PubMedID)2-s2.0-85140271549 (Scopus ID)
Available from: 2022-12-08 Created: 2022-12-08 Last updated: 2023-09-05Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-2734-2794

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