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Jalalypour, F., Howard, R. J. & Lindahl, E. (2024). Allosteric Cholesterol Site in Glycine Receptors Characterized through Molecular Simulations. Journal of Physical Chemistry B, 128(20), 4996-5007
Open this publication in new window or tab >>Allosteric Cholesterol Site in Glycine Receptors Characterized through Molecular Simulations
2024 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 128, no 20, p. 4996-5007Article in journal (Refereed) Published
Abstract [en]

Glycine receptors are pentameric ligand-gated ion channels that conduct chloride ions across postsynaptic membranes to facilitate fast inhibitory neurotransmission. In addition to gating by the glycine agonist, interactions with lipids and other compounds in the surrounding membrane environment modulate their function, but molecular details of these interactions remain unclear, in particular, for cholesterol. Here, we report coarse-grained simulations in a model neuronal membrane for three zebrafish glycine receptor structures representing apparent resting, open, and desensitized states. We then converted the systems to all-atom models to examine detailed lipid interactions. Cholesterol bound to the receptor at an outer-leaflet intersubunit site, with a preference for the open and desensitized versus resting states, indicating that it can bias receptor function. Finally, we used short atomistic simulations and iterative amino acid perturbations to identify residues that may mediate allosteric gating transitions. Frequent cholesterol contacts in atomistic simulations clustered with residues identified by perturbation analysis and overlapped with mutations influencing channel function and pathology. Cholesterol binding at this site was also observed in a recently reported pig heteromeric glycine receptor. These results indicate state-dependent lipid interactions relevant to allosteric transitions of glycine receptors, including specific amino acid contacts applicable to biophysical modeling and pharmaceutical design.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2024
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-347167 (URN)10.1021/acs.jpcb.4c01703 (DOI)001225969500001 ()38747451 (PubMedID)2-s2.0-85193511578 (Scopus ID)
Note

QC 20240603

Available from: 2024-06-03 Created: 2024-06-03 Last updated: 2024-06-03Bibliographically approved
Bergh, C., Rovsnik, U., Howard, R. J. & Lindahl, E. (2024). Discovery of lipid binding sites in a ligand-gated ion channel by integrating simulations and cryo-EM. eLife, 12, 2023-01
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
2024 (English)In: eLife, ISSN 2050-084X, Vol. 12, p. 2023-01Article in journal (Refereed) Published
Abstract [en]

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

Place, publisher, year, edition, pages
eLife Sciences Publications, Ltd, 2024
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-326989 (URN)10.7554/eLife.86016 (DOI)001154973600001 ()38289224 (PubMedID)2-s2.0-85184345538 (Scopus ID)
Note

QC 20230522

Available from: 2023-05-16 Created: 2023-05-23 Last updated: 2024-03-05Bibliographically approved
Haloi, N., Huang, S., Nichols, A. L., Fine, E. J., Friesenhahn, N. J., Marotta, C. B., . . . Lester, H. A. (2024). Interactive computational and experimental approaches improve the sensitivity of periplasmic binding protein-based nicotine biosensors for measurements in biofluids. Protein Engineering Design & Selection, 37, Article ID gzae003.
Open this publication in new window or tab >>Interactive computational and experimental approaches improve the sensitivity of periplasmic binding protein-based nicotine biosensors for measurements in biofluids
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2024 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 37, article id gzae003Article in journal (Refereed) Published
Abstract [en]

We developed fluorescent protein sensors for nicotine with improved sensitivity. For iNicSnFR12 at pH 7.4, the proportionality constant for ∆F/F0 vs [nicotine] (δ-slope, 2.7 μM−1) is 6.1-fold higher than the previously reported iNicSnFR3a. The activated state of iNicSnFR12 has a fluorescence quantum yield of at least 0.6. We measured similar dose-response relations for the nicotine-induced absorbance increase and fluorescence increase, suggesting that the absorbance increase leads to the fluorescence increase via the previously described nicotine-induced conformational change, the ‘candle snuffer’ mechanism. Molecular dynamics (MD) simulations identified a binding pose for nicotine, previously indeterminate from experimental data. MD simulations also showed that Helix 4 of the periplasmic binding protein (PBP) domain appears tilted in iNicSnFR12 relative to iNicSnFR3a, likely altering allosteric network(s) that link the ligand binding site to the fluorophore. In thermal melt experiments, nicotine stabilized the PBP of the tested iNicSnFR variants. iNicSnFR12 resolved nicotine in diluted mouse and human serum at 100 nM, the peak [nicotine] that occurs during smoking or vaping, and possibly at the decreasing levels during intervals between sessions. NicSnFR12 was also partially activated by unidentified endogenous ligand(s) in biofluids. Improved iNicSnFR12 variants could become the molecular sensors in continuous nicotine monitors for animal and human biofluids.

Place, publisher, year, edition, pages
Oxford University Press (OUP), 2024
Keywords
absorption, biosensor, computation, fluorescence, iNicSnFR
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-344189 (URN)10.1093/protein/gzae003 (DOI)001173155400001 ()38302088 (PubMedID)2-s2.0-85186123039 (Scopus ID)
Note

QC 20240318

Available from: 2024-03-06 Created: 2024-03-06 Last updated: 2024-03-18Bibliographically approved
Jalalypour, F., Howard, R. J. J. & Lindahl, E. (2023). An Allosteric Cholesterol Site in Glycine Receptors Defined Through Molecular Simulations. European Biophysics Journal, 52(SUPPL 1), S136-S136
Open this publication in new window or tab >>An Allosteric Cholesterol Site in Glycine Receptors Defined Through Molecular Simulations
2023 (English)In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 52, no SUPPL 1, p. S136-S136Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
SPRINGER, 2023
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-335964 (URN)001029235400439 ()
Note

QC 20230911

Available from: 2023-09-11 Created: 2023-09-11 Last updated: 2023-09-11Bibliographically approved
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.

Place, publisher, year, edition, pages
Elsevier BV, 2023
National Category
Biophysics Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-334870 (URN)10.1016/j.bpj.2023.05.033 (DOI)001122880200001 ()37277992 (PubMedID)2-s2.0-85162918774 (Scopus ID)
Note

QC 20231123

Available from: 2023-08-28 Created: 2023-08-28 Last updated: 2024-01-03Bibliographically approved
Wingbermühle, S. & Lindahl, E. (2023). Fully Automated Screening of Compound Libraries in Drug Discovery Using Docking and Molecular Dynamics. European Biophysics Journal, 52(SUPPL 1), S214-S214
Open this publication in new window or tab >>Fully Automated Screening of Compound Libraries in Drug Discovery Using Docking and Molecular Dynamics
2023 (English)In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 52, no SUPPL 1, p. S214-S214Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
SPRINGER, 2023
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-335874 (URN)001029235400747 ()
Note

QC 20230911

Available from: 2023-09-11 Created: 2023-09-11 Last updated: 2023-09-11Bibliographically approved
Wingbermühle, S. & Lindahl, E. (2023). Fully automated screening of compound libraries in drug discovery using docking and molecular dynamics. Biophysical Journal, 122(3S1)
Open this publication in new window or tab >>Fully automated screening of compound libraries in drug discovery using docking and molecular dynamics
2023 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 122, no 3S1Article in journal, Meeting abstract (Refereed) Published
Place, publisher, year, edition, pages
Elsevier BV, 2023
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-332150 (URN)10.1016/j.bpj.2022.11.1126 (DOI)000989629701014 ()
Note

QC 20230720

Available from: 2023-07-20 Created: 2023-07-20 Last updated: 2024-01-23Bibliographically 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), e1011255, 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, p. e1011255-, 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.

Place, publisher, year, edition, pages
Public Library of Science (PLoS), 2023
National Category
Bioinformatics (Computational Biology)
Identifiers
urn:nbn:se:kth:diva-334702 (URN)10.1371/journal.pcbi.1011255 (DOI)001041056800002 ()37523411 (PubMedID)
Note

QC 20230824

Available from: 2023-08-24 Created: 2023-08-24 Last updated: 2023-08-24Bibliographically approved
Shugaeva, T., Haloi, N., Howard, R. J. & Lindahl, E. (2023). Improving flexible protein fitting into cryo-EM maps using multiple conformers generated by AlphaFold 2. European Biophysics Journal, 52(SUPPL 1), S207-S207
Open this publication in new window or tab >>Improving flexible protein fitting into cryo-EM maps using multiple conformers generated by AlphaFold 2
2023 (English)In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 52, no SUPPL 1, p. S207-S207Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
SPRINGER, 2023
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-335958 (URN)001029235400721 ()
Note

QC 20230911

Available from: 2023-09-11 Created: 2023-09-11 Last updated: 2023-09-11Bibliographically approved
Bergh, C., Rovsnik, U., Howard, R. & Lindahl, E. (2023). Integrating simulations and cryo-EM reveal lipid binding sites in a ligand-gated ion channel. European Biophysics Journal, 52(SUPPL 1), S118-S118
Open this publication in new window or tab >>Integrating simulations and cryo-EM reveal lipid binding sites in a ligand-gated ion channel
2023 (English)In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 52, no SUPPL 1, p. S118-S118Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
SPRINGER, 2023
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-335945 (URN)001029235400370 ()
Note

QC 20230911

Available from: 2023-09-11 Created: 2023-09-11 Last updated: 2023-09-11Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-2734-2794

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