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  • 1. Abraham, Mark
    et al.
    Apostolov, Rossen
    Barnoud, Jonathan
    Bauer, Paul
    Blau, Christian
    Bonvin, Alexandre M. J. J.
    Chavent, Matthieu
    Chodera, John
    Condic-Jurkic, Karmen
    Delemotte, Lucie
    Grubmueller, Helmut
    Howard, Rebecca J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Jordan, E. Joseph
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Ollila, O. H. Samuli
    Selent, Jana
    Smith, Daniel G. A.
    Stansfeld, Phillip J.
    Tiemann, Johanna K. S.
    Trellet, Mikael
    Woods, Christopher
    Zhmurov, Artem
    Sharing Data from Molecular Simulations2019In: Journal of Chemical Information and Modeling, ISSN 1549-9596, E-ISSN 1549-960X, Vol. 59, no 10, p. 4093-4099Article in journal (Refereed)
    Abstract [en]

    Given the need for modern researchers to produce open, reproducible scientific output, the lack of standards and best practices for sharing data and workflows used to produce and analyze molecular dynamics (MD) simulations has become an important issue in the field. There are now multiple well-established packages to perform molecular dynamics simulations, often highly tuned for exploiting specific classes of hardware, each with strong communities surrounding them, but with very limited interoperability/transferability options. Thus, the choice of the software package often dictates the workflow for both simulation production and analysis. The level of detail in documenting the workflows and analysis code varies greatly in published work, hindering reproducibility of the reported results and the ability for other researchers to build on these studies. An increasing number of researchers are motivated to make their data available, but many challenges remain in order to effectively share and reuse simulation data. To discuss these and other issues related to best practices in the field in general, we organized a workshop in November 2018 (https://bioexcel.eu/events/workshop-on-sharing-data-from-molecular-simulations/). Here, we present a brief overview of this workshop and topics discussed. We hope this effort will spark further conversation in the MD community to pave the way toward more open, interoperable, and reproducible outputs coming from research studies using MD simulations.

  • 2. Azuara, Cyril
    et al.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Koehl, Patrice
    Orland, Henri
    Delarue, Marc
    PDB_Hydro: incorporating dipolar solvents with variable density in the Poisson-Boltzmann treatment of macromolecule electrostatics.2006In: Nucleic Acids Res, ISSN 1362-4962, Vol. 34, no Web Server issue, p. W38-42Article in journal (Refereed)
  • 3. Azuara, Cyril
    et al.
    Lindahl, Erik
    Stockholm University.
    Koehl, Patrice
    Orland, Henri
    Delarue, Marc
    PDB_Hydro: incorporating dipolar solvents with variable density in the Poisson-Boltzmann treatment of macromolecule electrostatics.2006In: Nucleic Acids Res, ISSN 1362-4962, Vol. 34, no Web Server issue, p. W38-42Article in journal (Refereed)
  • 4. Bergh, Cathrine
    et al.
    Heusser, Stephanie A.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Howard, Rebecca J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Markov state models of proton- and pore-dependent activation in a pentameric ligand-gated ion channel2021In: eLIFE, E-ISSN 2050-084X, Vol. 10, article id e68369Article in journal (Refereed)
    Abstract [en]

    Ligand-gated ion channels conduct currents in response to chemical stimuli, mediating electrochemical signaling in neurons and other excitable cells. For many channels, the details of gating remain unclear, partly due to limited structural data and simulation timescales. Here, we used enhanced sampling to simulate the pH-gated channel GLIC, and construct Markov state models (MSMs) of gating. Consistent with new functional recordings, we report in oocytes, our analysis revealed differential effects of protonation and mutation on free-energy wells. Clustering of closed- versus open-like states enabled estimation of open probabilities and transition rates, while higher-order clustering affirmed conformational trends in gating. Furthermore, our models uncovered state- and protonation-dependent symmetrization. This demonstrates the applicability of MSMs to map energetic and conformational transitions between ion-channel functional states, and how they reproduce shifts upon activation or mutation, with implications for modeling neuronal function and developing state-selective drugs.

  • 5.
    Bergh, Cathrine
    et al.
    KTH Royal Institute of Technology, Sweden.
    Rovsnik, Urska
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Howard, Rebecca J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. KTH Royal Institute of Technology, Sweden.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. KTH Royal Institute of Technology, Sweden.
    Discovery of lipid binding sites in a ligand-gated ion channel by integrating simulations and cryo-EMManuscript (preprint) (Other academic)
    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.

  • 6. Bergh, Cathrine
    et al.
    Rovšnik, Urška
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Howard, Rebecca J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Discovery of lipid binding sites in a ligand-gated ion channel by integrating simulations and cryo-EM2023In: eLIFE, E-ISSN 2050-084X, Vol. 12, article id RP86016Article in journal (Refereed)
    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.

  • 7.
    Bernsel, Andreas
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Viklund, Håkan
    Falk, Jenny
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Elofsson, Arne
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Prediction of membrane-protein topology from first principles2008In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 20, p. 7177-7181Article in journal (Refereed)
  • 8. Bertaccini, Edward J.
    et al.
    Trudell, James R.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Normal Mode Gating Motions of a Ligand-Gated Ion Channel Persist in a Fully Hydrated Lipid Bilayer Model2010In: ACS chemical neuroscience, ISSN 1948-7193, Vol. 1, no 8, p. 552-558Article in journal (Refereed)
    Abstract [en]

    We have previously used molecular modeling and normal-mode analyses combined with experimental data to visualize a plausible model of a transmembrane ligand-gated ion channel. We also postulated how the gating motion of the channel may be affected by the presence of various ligands, especially anesthetics. As is typical for normal-mode analyses, those studies were performed ut vacuo to reduce the computational complexity of the problem. While such calculations constitute an efficient way to model the large scale structural flexibility of transmembrane proteins, they can be criticized for neglecting the effects of an explicit phospholipid bilayer or hydrated environment. Here, we show the successful calculation of normal-mode motions for our model of a glycine alpha-1 receptor, now suspended in a fully hydrated lipid bilayer. Despite the almost uniform atomic density, the introduction of water and lipid does not grossly distort the overall gating motion. Normal-mode analysis revealed that even a fully immersed glycine alpha-1 receptor continues to demonstrate an iris-like channel gating motion as a low-frequency, high-amplitude natural harmonic vibration consistent with channel gating. Furthermore, the introduction of periodic boundary conditions allows the examination of simultaneous harmonic vibrations of lipid in synchrony with the protein gating motions that are compatible with reasonable lipid bilayer perturbations. While these perturbations tend to influence the overall protein motion, this work provides continued support for the iris-like motion model that characterizes gating within the family of ligand-gated ion channels.

  • 9. Bertaccini, Edward J
    et al.
    Trudell, James R
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Normal-mode analysis of the glycine alpha1 receptor by three separate methods.2007In: J Chem Inf Model, ISSN 1549-9596, Vol. 47, no 4, p. 1572-9Article in journal (Other academic)
  • 10. Bertaccini, Edward J
    et al.
    Trudell, James R
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sixma, Titia
    The Effects of Cobratoxin Ligand Binding on the Normal Mode Vibration of Acetycholine Binding Protein2007In: American Association University Anesthesiologists meeting, 2007Conference paper (Refereed)
  • 11. Bertaccini, Edward J
    et al.
    Wallner, Björn
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Trudell, James R
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Modeling Anesthetic Binding Sites within the Glycine Alpha One Receptor Based on Prokaryotic Ion Channel Templates: The Problem with TM42010In: Journal of chemical information and modeling, ISSN 1549-960X, Vol. 50, no 12, p. 2248-2255Article in journal (Refereed)
    Abstract [en]

    Ligand-gated ion channels (LGICs) significantly modulate anesthetic effects. Their exact molecular structure remains unknown. This has led to ambiguity regarding the proper amino acid alignment within their 3D structure and, in turn, the location of any anesthetic binding sites. Current controversies suggest that such a site could be located in either an intra- or intersubunit locale within the transmembrane domain of the protein. Here, we built a model of the glycine alpha one receptor (GlyRa1) based on the open-state structures of two new high-resolution ion channel templates from the prokaryote, Gloebacter violaceus (GLIC). Sequence scoring suggests reasonable homology between GlyRa1 and GLIC. Three of the residues notable for modulating anesthetic action are on transmembrane segments 1-3 (TM1-3): (ILE229, SER 267, and ALA 288). They line an intersubunit interface, in contrast to previous models. However, residues from the fourth transmembrane domain (TM4) that are known to modulate a variety of anesthetic effects are quite distant from this putative anesthetic binding site. While this model can account for a large proportion of the physicochemical data regarding such proteins, it cannot readily account for the alterations on anesthetic effects that are due to mutations within TM4.

  • 12.
    Bertaccini, Edward
    et al.
    Stanford University.
    Trudell, James
    Stanford University.
    Murail, Samuel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A Model of Putative Anesthetic Binding Sites within the Glycine Alpha One Receptor Based on Two New Prokaryotic Ion Channel Templates in the Open State2009Conference paper (Refereed)
    Abstract [en]

    INTRODUCTION:  Ligand-gated ion channels (LGICs) are thought to mediate a significant proportion of anesthetic effects.  We have used molecular modeling to build atomic level models of the glycine alpha one receptor (GlyRa1) to examine its interactions with anesthetics.  We have previously built models of a GlyRa1 based on a prokaryotic pentameric ion channel in the closed state from Erwinia Chrysanthemi (ELIC) and demonstrated a possible intersubunit binding site for anesthetics.(1-3)  Here, we further validate such a model in the similar construction of a GlyRa1 model based on the open state structures of two new ion channels from the prokaryote Gloebacter violaceus (GLIC).(4-5) These new open state templates have become extremely relevant since anesthetics are thought to bind to and stabilize the open state of the GlyRa1.  METHODS: The amino acid sequences and 3D coordinates of the torpedofish nicotinic acetylcholine receptor alpha 1 subunit (nAChRa1 from 2BG9.pdb) and two forms of GLIC (3EHZ.pdb and 3EAM.pdb) were obtained from the RCSB database.  The sequence of the human GlyRa1 was obtained from the NCBI database.  An initial BLAST sequence search was performed at the NCBI using the GLIC sequences.  Among the best scored homologous human sequences were those of the GlyRa1.  The three known structures underwent structural alignment for optimum coordinate overlap within Discovery Studio 2.0.1 (Accelrys, San Diego, CA).  The amino acid sequence of each structural subunit was then aligned with the sequence of the GlyRa1 using the Align123 algorithm (a derivative of ClustalW).  The Modeler module was used for assignment of coordinates for aligned amino acids, the construction of possible loops, and the initial refinement of amino acid sidechains using the averaged coordinates of 3EAM and 3EHZ.  Five GlyRa1 subunits were merged to form the final homomeric pentamer.

    RESULTS:  The BLAST derived scores suggest a close homology between the LGICs, GLIC and ELIC.  Subsequent CLUSTALW alignment of the GLIC and GlyRa1 sequences demonstrates reasonable sequence similarity. The model of the GlyRa1 is a homomer with pentameric symmetry about a central ion pore and shows significant transmembrane alpha helical and extracellular beta sheet content.  Unlike our previous model based on the ELIC template, the current model based on the GLIC templates shows a continuously open pore with a partial restriction within the transmembrane region.  Three of the residues notable for modulating anesthetic action are on transmembrane segments 1-3 (TM1-3) (ILE229, SER 267, ALA 288).  They now line the intersubunit interface, in contrast to our previous models.  However, residues from TM4 that are known to modulate a variety of anesthetic effects on this or homologous LGICs are present but could only indirectly influence an intersubunit anesthetic binding site.

    CONCLUSIONS:  A reasonable model of the GlyRa1 was constructed using homology modeling based on the GLIC templates.  This model posits an intersubunit site for anesthetic binding that may communicate with the intrasubunit region of each TMD.  However, distinct questions remain regarding an explanation for the effects of TM4 mutations on anesthetic modulation of these channels.

    1. Trudell JR, Bertaccini E: Comparative modeling of a GABAA alpha1 receptor using three crystal structures as templates. J Mol Graph Model 2004; 23: 39-49

    2. Bertaccini EJ, Shapiro J, Brutlag DL, Trudell JR: Homology modeling of a human glycine alpha 1 receptor reveals a plausible anesthetic binding site. J Chem Inf Model 2005; 45: 128-35

    3. Bertaccini E, Trudell JR: Putative Anesthetic Binding Sites within a GlyR alpha 1 Receptor Model Based on a Prokaryotic Template. Anesthesiology 2008: A644

    4. Bocquet N, Nury H, Baaden M, Le Poupon C, Changeux JP, Delarue M, Corringer PJ: X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation. Nature 2009; 457: 111-4

    5. Hilf RJ, Dutzler R: Structure of a potentially open state of a proton-activated pentameric ligand-gated ion channel. Nature 2009; 457: 115-8

  • 13.
    Bjelkmar, Pär
    et al.
    Theoretical and Computational Biophysics, Department of Theoretical Physics, Royal Institute of Technology.
    Larsson, Per
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cuendet, Michel
    Swiss Institute of Bioinformatics.
    Hess, Berk
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Implementation of the CHARMM Force Field in GROMACS: Analysis of Protein Stability Effects from Correction Maps, Virtual Interaction Sites, and Water Models2010In: Journal of Chemical Theory and Computation, ISSN 1549-9618, E-ISSN 1549-9626, Vol. 6, no 2, p. 459-466Article in journal (Refereed)
    Abstract [en]

    CHARMM27 is a widespread and popular force field for biomolecular simulation, and several recent algorithms such as implicit solvent models have been developed specifically for it. We have here implemented the CHARMM force field and all necessary extended functional forms in the GROMACS molecular simulation package, to make CHARMM-specific features available and to test them in combination with techniques for extended time steps, to make all major force fields available for comparison studies in GROMACS, and to test various solvent model optimizations, in particular the effect of Lennard-Jones interactions on hydrogens. The implementation has full support both for CHARMM-specific features such as multiple potentials over the same dihedral angle and the grid-based energy correction map on the , ψ protein backbone dihedrals, as well as all GROMACS features such as virtual hydrogen interaction sites that enable 5 fs time steps. The medium-to-long time effects of both the correction maps and virtual sites have been tested by performing a series of 100 ns simulations using different models for water representation, including comparisons between CHARMM and traditional TIP3P. Including the correction maps improves sampling of near native-state conformations in our systems, and to some extent it is even able to refine distorted protein conformations. Finally, we show that this accuracy is largely maintained with a new implicit solvent implementation that works with virtual interaction sites, which enables performance in excess of 250 ns/day for a 900-atom protein on a quad-core desktop computer.

  • 14.
    Bjelkmar, Pär
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Niemelä, Perttu S
    Vattulainen, Ilpo
    Tampere universitet.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Conformational changes and slow dynamics through microsecond polarized atomistic molecular simulation of an integral Kv1.2 ion channel.2009In: PLoS computational biology, ISSN 1553-7358, Vol. 5, no 2, p. e1000289-Article in journal (Refereed)
    Abstract [en]

    Structure and dynamics of voltage-gated ion channels, in particular the motion of the S4 helix, is a highly interesting and hotly debated topic in current membrane protein research. It has critical implications for insertion and stabilization of membrane proteins as well as for finding how transitions occur in membrane proteins-not to mention numerous applications in drug design. Here, we present a full 1 micros atomic-detail molecular dynamics simulation of an integral Kv1.2 ion channel, comprising 120,000 atoms. By applying 0.052 V/nm of hyperpolarization, we observe structural rearrangements, including up to 120 degrees rotation of the S4 segment, changes in hydrogen-bonding patterns, but only low amounts of translation. A smaller rotation ( approximately 35 degrees ) of the extracellular end of all S4 segments is present also in a reference 0.5 micros simulation without applied field, which indicates that the crystal structure might be slightly different from the natural state of the voltage sensor. The conformation change upon hyperpolarization is closely coupled to an increase in 3(10) helix contents in S4, starting from the intracellular side. This could support a model for transition from the crystal structure where the hyperpolarization destabilizes S4-lipid hydrogen bonds, which leads to the helix rotating to keep the arginine side chains away from the hydrophobic phase, and the driving force for final relaxation by downward translation is partly entropic, which would explain the slow process. The coordinates of the transmembrane part of the simulated channel actually stay closer to the recently determined higher-resolution Kv1.2 chimera channel than the starting structure for the entire second half of the simulation (0.5-1 micros). Together with lipids binding in matching positions and significant thinning of the membrane also observed in experiments, this provides additional support for the predictive power of microsecond-scale membrane protein simulations.

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  • 15. Blau, Christian
    et al.
    Yvonnesdotter, Linnea
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Gentle and fast all-atom model refinement to cryo-EM densities via a maximum likelihood approach2023In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 19, no 7, article id e1011255Article in journal (Refereed)
    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.

  • 16. Bondarenko, Vasyl
    et al.
    Chen, Qiang
    Singewald, Kevin
    Haloi, Nandan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Tillman, Tommy S.
    Howard, Rebecca J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Xu, Yan
    Tang, Pei
    Structural Elucidation of Ivermectin Binding to α7nAChR and the Induced Channel Desensitization2023In: ACS Chemical Neuroscience, E-ISSN 1948-7193, Vol. 14, no 6, p. 1156-1165Article in journal (Refereed)
    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. 

  • 17. Bondarenko, Vasyl
    et al.
    Wells, Marta M.
    Chen, Qiang
    Tillman, Tommy S.
    Singewald, Kevin
    Lawless, Matthew J.
    Caporoso, Joel
    Brandon, Nicole
    Coleman, Jonathan A.
    Saxena, Sunil
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Xu, Yan
    Tang, Pei
    Structures of highly flexible intracellular domain of human α7 nicotinic acetylcholine receptor2022In: Nature Communications, E-ISSN 2041-1723, Vol. 13, article id 793Article in journal (Refereed)
    Abstract [en]

    The intracellular domain (ICD) of Cys-loop receptors mediates diverse functions. To date, no structure of a full-length ICD is available due to challenges stemming from its dynamic nature. Here, combining nuclear magnetic resonance (NMR) and electron spin resonance experiments with Rosetta computations, we determine full-length ICD structures of the human α7 nicotinic acetylcholine receptor in a resting state. We show that ~57% of the ICD residues are in highly flexible regions, primarily in a large loop (loop L) with the most mobile segment spanning ~50 Å from the central channel axis. Loop L is anchored onto the MA helix and virtually forms two smaller loops, thereby increasing its stability. Previously known motifs for cytoplasmic binding, regulation, and signaling are found in both the helices and disordered flexible regions, supporting the essential role of the ICD conformational plasticity in orchestrating a broad range of biological processes. 

  • 18.
    Bromstrup, Torben
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Howard, Rebecca J.
    Trudell, James R.
    Harris, R. Adron
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Inhibition versus Potentiation of Ligand-Gated Ion Channels Can Be Altered by a Single Mutation that Moves Ligands between Intra- and Intersubunit Sites2013In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 21, no 8, p. 1307-1316Article in journal (Refereed)
    Abstract [en]

    Pentameric ligand-gated ion channels (pLGICs) are similar in structure but either inhibited or potentiated by alcohols and anesthetics. This dual modulation has previously not been understood, but the determination of X-ray structures of prokaryotic GLIC provides an ideal model system. Here, we show that a single-site mutation at the F14' site in the GLIC transmembrane domain turns desflurane and chloroform from inhibitors to potentiators, and that this is explained by competing allosteric sites. The F14'A mutation opens an intersubunit site lined by N239 (15'), 1240 (16'), and Y263. Free energy calculations confirm this site is the preferred binding location for desflurane and chloroform in GLIC F14'A. In contrast, both anesthetics prefer an intrasubunit site in wild-type GLIC. Modulation is therefore the net effect of competitive binding between the intersubunit potentiating site and an intrasubunit inhibitory site. This provides direct evidence for a dual-site model of allosteric regulation of pLGICs.

  • 19. Conti, Luca
    et al.
    Renhorn, Jakob
    Gabrielsson, Anders
    Turesson, Fredrik
    Liin, Sara I.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Elinder, Fredrik
    Reciprocal voltage sensor-to-pore coupling leads to potassium channel C-type inactivation2016In: Scientific Reports, E-ISSN 2045-2322, Vol. 6, article id 27562Article in journal (Refereed)
    Abstract [en]

    Voltage-gated potassium channels open at depolarized membrane voltages. A prolonged depolarization causes a rearrangement of the selectivity filter which terminates the conduction of ions - a process called slow or C-type inactivation. How structural rearrangements in the voltage-sensor domain (VSD) cause alteration in the selectivity filter, and vice versa, are not fully understood. We show that pulling the pore domain of the Shaker potassium channel towards the VSD by a Cd2+ bridge accelerates C-type inactivation. Molecular dynamics simulations show that such pulling widens the selectivity filter and disrupts the K+ coordination, a hallmark for C-type inactivation. An engineered Cd2+ bridge within the VSD also affect C-type inactivation. Conversely, a pore domain mutation affects VSD gating-charge movement. Finally, C-type inactivation is caused by the concerted action of distant amino acid residues in the pore domain. All together, these data suggest a reciprocal communication between the pore domain and the VSD in the extracellular portion of the channel.

  • 20.
    Cowgill, John
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Fan, Chen
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Haloi, Nandan
    Tobiasson, Victor
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Zhuang, Yuxuan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Howard, Rebecca J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Structure and dynamics of differential ligand binding in the human ρ-type GABAA receptor2023In: Neuron, ISSN 0896-6273, E-ISSN 1097-4199, Vol. 111, no 21, p. 3450-3464Article in journal (Refereed)
    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.

  • 21.
    de la Rosa-Trevín, José Miguel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Viga, Pedro Alberto Hernández
    Otón, Joaquín
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Development of basic building blocks for cryo-EM: the emcore and emvis software libraries2020In: Acta Crystallographica Section D: Structural Biology , E-ISSN 2059-7983, Vol. 76, p. 350-356Article in journal (Refereed)
    Abstract [en]

    Image-processing software has always been an integral part of structure determination by cryogenic electron microscopy (cryo-EM). Recent advances in hardware and software are recognized as one of the key factors in the so-called cryo-EM resolution revolution. Increasing computational power has opened many possibilities to consider more demanding algorithms, which in turn allow more complex biological problems to be tackled. Moreover, data processing has become more accessible to many experimental groups, with computations that used to last for many days at supercomputing facilities now being performed in hours on personal workstations. All of these advances, together with the rapid expansion of the community, continue to pose challenges and new demands on the software-development side. In this article, the development of emcore and emvis, two basic software libraries for image manipulation and data visualization in cryo-EM, is presented. The main goal is to provide basic functionality organized in modular components that other developers can reuse to implement new algorithms or build graphical applications. An additional aim is to showcase the importance of following established practices in software engineering, with the hope that this could be a first step towards a more standardized way of developing and distributing software in the field.

  • 22.
    Forsberg, Björn
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Aibara, Shintaro
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Howard, Rebecca J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Mortezaei, Narges
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Assembly and symmetry of the fungal E3BP-containing core of the Pyruvate Dehydrogenase ComplexManuscript (preprint) (Other academic)
    Abstract [en]

    The pyruvate dehydrogenase complex (PDC) is a central component of all aerobic respiration, connecting glycolysis to mitochondrial oxidation of pyruvate. Despite its central metabolic role, its precise composition and means of regulation remain unknown. To explain the variation in stoichiometry reported for the E3-recruiting protein X (PX) in the fungal PDC, we established cryo-EM reconstructions of the native and recombinant PDC from the filamentous fungus and model organism Neurospora crassa. We find that the PX C-terminal domain localizes interior to the E2 core. Critically, we show that two distinct arrangements of a trimeric oligomer exists, which both result in strict tetrahedral symmetry of the PDC core interior. Both oligomerization and volume occlusion of the PDC interior by PX appears to limit its binding stoichiometry, which explains the variety of stoichiometries found previously for S. cerevisiae. This also suggests that the PX oligomer stability and size are potential mechanisms to dynamically adjust PDC compostion in response to external cues. Moreover, we find that the site where PX binds is conserved within fungi but not mammals, suggesting that it could be therapeutically targeted. To this end, we also show that a PX knockout results in loss of activity through dysfunctional E3 recruitment, leading to severely impaired N. crassa growth on sucrose. The fungal PDC is thus shown to be fundamentally similar to the mammalian PDC in function but subject to other conditions of possible regulation, conditioned by a steric restrictions imposed by the symmetry of the PDC and its components.

  • 23.
    Forsberg, Björn O.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Aibara, Shintaro
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Max Planck Institute for Biophysical Chemistry, Germany.
    Howard, Rebecca J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Mortezaei, Narges
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Vironova AB, Sweden.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Arrangement and symmetry of the fungal E3BP-containing core of the pyruvate dehydrogenase complex2020In: Nature Communications, E-ISSN 2041-1723, Vol. 11, no 1, article id 4667Article in journal (Refereed)
    Abstract [en]

    The pyruvate dehydrogenase complex (PDC) is a multienzyme complex central to aerobic respiration, connecting glycolysis to mitochondrial oxidation of pyruvate. Similar to the E3-binding protein (E3BP) of mammalian PDC, PX selectively recruits E3 to the fungal PDC, but its divergent sequence suggests a distinct structural mechanism. Here, we report reconstructions of PDC from the filamentous fungus Neurospora crassa by cryo-electron microscopy, where we find protein X (PX) interior to the PDC core as opposed to substituting E2 core subunits as in mammals. Steric occlusion limits PX binding, resulting in predominantly tetrahedral symmetry, explaining previous observations in Saccharomyces cerevisiae. The PX-binding site is conserved in (and specific to) fungi, and complements possible C-terminal binding motifs in PX that are absent in mammalian E3BP. Consideration of multiple symmetries thus reveals a differential structural basis for E3BP-like function in fungal PDC.

  • 24.
    Forsberg, Björn O.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Aibara, Shintaro
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Kimanius, Dari
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Paul, Bijoya
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Amunts, Alexey
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Cryo-EM reconstruction of the chlororibosome to 3.2 angstrom resolution within 24 h2017In: IUCrJ, E-ISSN 2052-2525, Vol. 4, p. 723-727Article in journal (Refereed)
    Abstract [en]

    The introduction of direct detectors and the automation of data collection in cryo-EM have led to a surge in data, creating new opportunities for advancing computational processing. In particular, on-the-fly workflows that connect data collection with three-dimensional reconstruction would be valuable for more efficient use of cryo-EM and its application as a sample-screening tool. Here, accelerated on-the-fly analysis is reported with optimized organization of the data-processing tools, image acquisition and particle alignment that make it possible to reconstruct the three-dimensional density of the 70S chlororibosome to 3.2 angstrom resolution within 24 h of tissue harvesting. It is also shown that it is possible to achieve even faster processing at comparable quality by imposing some limits to data use, as illustrated by a 3.7 angstrom resolution map that was obtained in only 80 min on a desktop computer. These on-the-fly methods can be employed as an assessment of data quality from small samples and extended to high-throughput approaches.

  • 25. Fourati, Zaineb
    et al.
    Howard, Rebecca J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Heusser, Stephanie A.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Hu, Haidai
    Ruza, Reinis R.
    Sauguet, Ludovic
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Delarue, Marc
    Structural Basis for a Bimodal Allosteric Mechanism of General Anesthetic Modulation in Pentameric Ligand-Gated Ion Channels2018In: Cell Reports, E-ISSN 2211-1247, Vol. 23, no 4, p. 993-1004Article in journal (Refereed)
    Abstract [en]

    Ion channel modulation by general anesthetics is a vital pharmacological process with implications for receptor biophysics and drug development. Functional studies have implicated conserved sites of both potentiation and inhibition in pentameric ligand-gated ion channels, but a detailed structural mechanism for these bimodal effects is lacking[1] . The prokaryotic model protein GLIC recapitulates anesthetic modulation of human ion channels, and is accessible to structure determination in both apparent open and closed states. Here, we report ten X-ray structures and electrophysiological characterization of GLIC variants in the presence and absence of general anesthetics, including the surgical agent propofol. We show that general anesthetics can allosterically favor closed channels by binding in the pore, or favor open channels via various subsites in the transmembrane domain. Our results support an integrated, multi-site mechanism for allosteric modulation, and provide atomic details of both potentiation and inhibition by one of the most common general anesthetics.

  • 26. Gharpure, Anant
    et al.
    Teng, Jinfeng
    Zhuang, Yuxuan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Noviello, Colleen M.
    Walsh, Richard M.
    Cabuco, Rico
    Howard, Rebecca J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Zaveri, Nurulain T.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Hibbs, Ryan E.
    Agonist Selectivity and Ion Permeation in the alpha 3 beta 4 Ganglionic Nicotinic Receptor2019In: Neuron, ISSN 0896-6273, E-ISSN 1097-4199, Vol. 104, no 3, p. 501-511Article in journal (Refereed)
    Abstract [en]

    Nicotinic acetylcholine receptors are pentameric ion channels that mediate fast chemical neurotransmission. The alpha 3 beta 4 nicotinic receptor subtype forms the principal relay between the central and peripheral nervous systems in the autonomic ganglia. This receptor is also expressed focally in brain areas that affect reward circuits and addiction. Here, we present structures of the alpha 3 beta 4 nicotinic receptor in lipidic and detergent environments, using functional reconstitution to define lipids appropriate for structural analysis. The structures of the receptor in complex with nicotine, as well as the alpha 3 beta 4-selective ligand AT-1001, complemented by molecular dynamics, suggest principles of agonist selectivity. The structures further reveal much of the architecture of the intracellular domain, where mutagenesis experiments and simulations define residues governing ion conductance.

  • 27. Gossen, Jonas
    et al.
    Albani, Simone
    Hanke, Anton
    Joseph, Benjamin P.
    Bergh, Cathrine
    Kuzikov, Maria
    Costanzi, Elisa
    Manelfi, Candida
    Storici, Paola
    Gribbon, Philip
    Beccari, Andrea R.
    Talarico, Carmine
    Spyrakis, Francesca
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Zaliani, Andrea
    Carloni, Paolo
    Wade, Rebecca C.
    Musiani, Francesco
    Kokh, Daria B.
    Rossetti, Giulia
    A Blueprint for High Affinity SARS-CoV-2 Mpro Inhibitors from Activity-Based Compound Library Screening Guided by Analysis of Protein Dynamics2021In: ACS Pharmacology & Translational Science, E-ISSN 2575-9108, Vol. 4, no 3, p. 1079-1095Article in journal (Refereed)
    Abstract [en]

    The SARS-CoV-2 coronavirus outbreak continues to spread at a rapid rate worldwide. The main protease (Mpro) is an attractive target for anti-COVID-19 agents. Unexpected difficulties have been encountered in the design of specific inhibitors. Here, by analyzing an ensemble of similar to 30 000 SARS-CoV-2 Mpro conformations from crystallographic studies and molecular simulations, we show that small structural variations in the binding site dramatically impact ligand binding properties. Hence, traditional druggability indices fail to adequately discriminate between highly and poorly druggable conformations of the binding site. By performing similar to 200 virtual screenings of compound libraries on selected protein structures, we redefine the protein's druggability as the consensus chemical space arising from the multiple conformations of the binding site formed upon ligand binding. This procedure revealed a unique SARS-CoV-2 Mpro blueprint that led to a definition of a specific structure-based pharmacophore. The latter explains the poor transferability of potent SARS-CoV Mpro inhibitors to SARS-CoV-2 Mpro, despite the identical sequences of the active sites. Importantly, application of the pharmacophore predicted novel high affinity inhibitors of SARS-CoV-2 Mpro, that were validated by in vitro assays performed here and by a newly solved X-ray crystal structure. These results provide a strong basis for effective rational drug design campaigns against SARS-CoV-2 Mpro and a new computational approach to screen protein targets with malleable binding sites.

  • 28. Grottesi, Alessandro
    et al.
    Bešker, Neva
    Emerson, Andrew
    Manelfi, Candida
    Beccari, Andrea R.
    Frigerio, Francesco
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Cerchia, Carmen
    Talarico, Carmine
    Computational Studies of SARS-CoV-2 3CLpro: Insights from MD Simulations2020In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 21, no 15, article id 5346Article in journal (Refereed)
    Abstract [en]

    Given the enormous social and health impact of the pandemic triggered by severe acute respiratory syndrome 2 (SARS-CoV-2), the scientific community made a huge effort to provide an immediate response to the challenges posed by Coronavirus disease 2019 (COVID-19). One of the most important proteins of the virus is an enzyme, called 3CLpro or main protease, already identified as an important pharmacological target also in SARS and Middle East respiratory syndrome virus (MERS) viruses. This protein triggers the production of a whole series of enzymes necessary for the virus to carry out its replicating and infectious activities. Therefore, it is crucial to gain a deeper understanding of 3CLpro structure and function in order to effectively target this enzyme. All-atoms molecular dynamics (MD) simulations were performed to examine the different conformational behaviors of the monomeric and dimeric form of SARS-CoV-2 3CLpro apo structure, as revealed by microsecond time scale MD simulations. Our results also shed light on the conformational dynamics of the loop regions at the entry of the catalytic site. Studying, at atomic level, the characteristics of the active site and obtaining information on how the protein can interact with its substrates will allow the design of molecules able to block the enzymatic function crucial for the virus.

  • 29. Grønberg, Christina
    et al.
    Sitsel, Oleg
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Gourdon, Pontus
    Andersson, Magnus
    Membrane Anchoring and Ion-Entry Dynamics in P-type ATPase Copper Transport2016In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 111, no 11, p. 2417-2429Article in journal (Refereed)
    Abstract [en]

    Cu+-specific P-type ATPase membrane protein transporters regulate cellular copper levels. The lack of crystal structures in Cu+-binding states has limited our understanding of how ion entry and binding are achieved. Here, we characterize the molecular basis of Cu+ entry using molecular-dynamics simulations, structural modeling, and in vitro and in vivo functional assays. Protein structural rearrangements resulting in the exposure of positive charges to bulk solvent rather than to lipid phosphates indicate a direct molecular role of the putative docking platform in Cu+ delivery. Mutational analyses and simulations in the presence and absence of Cu+ predict that the ion-entry path involves two ion-binding sites: one transient Met148-Cys382 site and one intramembranous site formed by trigonal coordination to Cys384, Asn689, and Met717. The results reconcile earlier biochemical and x-ray absorption data and provide a molecular understanding of ion entry in Cu+-transporting P-type ATPases.

  • 30. Gómez-Blanco, J.
    et al.
    de la Rosa-Trevín, José Miguel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Marabini, R.
    del Cano, L.
    Jimenez, A.
    Martinez, M.
    Melero, R.
    Majtner, T.
    Maluenda, D.
    Mota, J.
    Rancel, Y.
    Ramirez-Aportela, E.
    Vilas, J. L.
    Carroni, Marta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Fleischmann, Stefan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Ashton, A. W.
    Basham, M.
    Clare, D. K.
    Savage, K.
    Siebert, C. A.
    Sharov, G. G.
    Sorzano, C. O. S.
    Conesa, P.
    Carazo, J. M.
    Using Scipion for stream image processing at Cryo-EM facilities2018In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 204, no 3, p. 457-463Article in journal (Refereed)
    Abstract [en]

    Three dimensional electron microscopy is becoming a very data-intensive field in which vast amounts of experimental images are acquired at high speed. To manage such large-scale projects, we had previously developed a modular workflow system called Scipion (de la Rosa-Trevfn et al., 2016). We present here a major extension of Scipion that allows processing of EM images while the data is being acquired. This approach helps to detect problems at early stages, saves computing time and provides users with a detailed evaluation of the data quality before the acquisition is finished. At present, Scipion has been deployed and is in production mode in seven Cryo-EM facilities throughout the world.

  • 31. Haloi, Nandan
    et al.
    Huang, Shan
    Nichols, Aaron L.
    Fine, Eve J.
    Friesenhahn, Nicholas J.
    Marotta, Christopher B.
    Dougherty, Dennis A.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Howard, Rebecca J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Mayo, Stephen L.
    Lester, Henry A.
    Interactive computational and experimental approaches improve the sensitivity of periplasmic binding protein-based nicotine biosensors for measurements in biofluids2024In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 37, article id gzae003Article in journal (Refereed)
    Abstract [en]

    We developed fluorescent protein sensors for nicotine with improved sensitivity. For iNicSnFR12 at pH 7.4, the proportionality constant for ∆F/F0vs [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.

  • 32. Haug, Ferdinand M.
    et al.
    Pumroy, Ruth A.
    Sridhar, Akshay
    Pantke, Sebastian
    Dimek, Florian
    Fricke, Tabea C.
    Hage, Axel
    Herzog, Christine
    Echtermeyer, Frank G.
    de la Roche, Jeanne
    Koh, Adrian
    Kotecha, Abhay
    Howard, Rebecca J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Moiseenkova-Bell, Vera
    Leffler, Andreas
    Functional and structural insights into activation of TRPV2 by weak acids2024In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 43, no 11, p. 2264-2290Article in journal (Refereed)
    Abstract [en]

    Transient receptor potential (TRP) ion channels are involved in the surveillance or regulation of the acid-base balance. Here, we demonstrate that weak carbonic acids, including acetic acid, lactic acid, and CO2 activate and sensitize TRPV2 through a mechanism requiring permeation through the cell membrane. TRPV2 channels in cell-free inside-out patches maintain weak acid-sensitivity, but protons applied on either side of the membrane do not induce channel activation or sensitization. The involvement of proton modulation sites for weak acid-sensitivity was supported by the identification of titratable extracellular (Glu495, Glu561) and intracellular (His521) residues on a cryo-EM structure of rat TRPV2 (rTRPV2) treated with acetic acid. Molecular dynamics simulations as well as patch clamp experiments on mutant rTRPV2 constructs confirmed that these residues are critical for weak acid-sensitivity. We also demonstrate that the pore residue Glu609 dictates an inhibition of weak acid-induced currents by extracellular calcium. Finally, TRPV2-expression in HEK293 cells is associated with an increased weak acid-induced cytotoxicity. Together, our data provide new insights into weak acids as endogenous modulators of TRPV2.

  • 33.
    Hennerdal, Aron
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Falk, Jenny
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Elofsson, Arne
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Internal duplications in alpha-helical membrane protein topologies are common but the nonduplicated forms are rare2010In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 19, no 12, p. 2305-2318Article in journal (Refereed)
    Abstract [en]

    Many alpha-helical membrane proteins contain internal symmetries, indicating that they might have evolved through a gene duplication and fusion event Here, we have characterized internal duplications among membrane proteins of known structure and in three complete genomes We found that the majority of large transmembrane (TM) proteins contain an internal duplication The duplications found showed a large variability both in the number of TM-segments included and in their orientation Surprisingly, an approximately equal number of antiparallel duplications and parallel duplications were found However, of all 11 superfamilies with an internal duplication, only for one, the AcrB Multidrug Efflux Pump, the duplicated unit could be found in its nonduplicated form An evolutionary analysis of the AcrB homologs indicates that several independent fusions have occurred, including the fusion of the SecD and SecF proteins into the 12-TM-protein SecDF in Brucella and Staphylococcus aureus In one additional case, the Vitamin B-12 transporter-like ABC transporters, the protein had undergone an additional fusion to form protein with 20 TM-helices in several bacterial genomes Finally, homologs to all human membrane proteins were used to detect the presence of duplicated and nonduplicated proteins This confirmed that only in rare cases can homologs with different duplication status be found, although internal symmetry is frequent among these proteins One possible explanation is that it is frequent that duplication and fusion events happen simultaneously and that there is almost always a strong selective advantage for the fused form

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  • 34. Henrion, Ulrike
    et al.
    Renhorn, Jakob
    Börjesson, Sara I.
    Nelson, Erin M.
    Schwaiger, Christine S.
    Bjelkmar, Pär
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wallner, Björn
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Elinder, Fredrik
    Tracking a complete voltage-sensor cycle with metal-ion bridges2012In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 22, p. 8552-8557Article in journal (Refereed)
    Abstract [en]

    Voltage-gated ion channels open and close in response to changes in membrane potential, thereby enabling electrical signaling in excitable cells. The voltage sensitivity is conferred through four voltage-sensor domains (VSDs) where positively charged residues in the fourth transmembrane segment (S4) sense the potential. While an open state is known from the Kv1.2/2.1 X-ray structure, the conformational changes underlying voltage sensing have not been resolved. We present 20 additional interactions in one open and four different closed conformations based on metal-ion bridges between all four segments of the VSD in the voltage-gated Shaker K channel. A subset of the experimental constraints was used to generate Rosetta models of the conformations that were subjected to molecular simulation and tested against the remaining constraints. This achieves a detailed model of intermediate conformations during VSD gating. The results provide molecular insight into the transition, suggesting that S4 slides at least 12 angstrom along its axis to open the channel with a 3(10) helix region present that moves in sequence in S4 in order to occupy the same position in space opposite F290 from open through the three first closed states.

  • 35.
    Heusser, Stephanie A.
    et al.
    Swiss Federal Institute of Technology, Switzerland.
    Howard, Rebecca J.
    Borghese, Cecilia M.
    Cullins, Madeline A.
    Broemstrup, Torben
    Lee, Ui S.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Carlsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Harris, R. Adron
    Functional Validation of Virtual Screening for Novel Agents with General Anesthetic Action at Ligand-Gated Ion Channelss2013In: Molecular Pharmacology, ISSN 0026-895X, E-ISSN 1521-0111, Vol. 84, no 5, p. 670-678Article in journal (Refereed)
    Abstract [en]

    GABA(A) receptors play a crucial role in the actions of general anesthetics. The recently published crystal structure of the general anesthetic propofol bound to Gloeobacter violaceus ligand-gated ion channel (GLIC), a bacterial homolog of GABA(A) receptors, provided an opportunity to explore structure-based ligand discovery for pentameric ligand-gated ion channels (pLGICs). We used molecular docking of 153,000 commercially available compounds to identify molecules that interact with the propofol binding site in GLIC. In total, 29 compounds were selected for functional testing on recombinant GLIC, and 16 of these compounds modulated GLIC function. Active compounds were also tested on recombinant GABA(A) receptors, and point mutations around the presumed binding pocket were introduced into GLIC and GABA(A) receptors to test for binding specificity. The potency of active compounds was only weakly correlated with properties such as lipophilicity or molecular weight. One compound was found to mimic the actions of propofol on GLIC and GABA(A), and to be sensitive to mutations that reduce the action of propofol in both receptors. Mutant receptors also provided insight about the position of the binding sites and the relevance of the receptor's conformation for anesthetic actions. Overall, the findings support the feasibility of the use of virtual screening to discover allosteric modulators of pLGICs, and suggest that GLIC is a valid model system to identify novel GABA(A) receptor ligands.

  • 36.
    Heusser, Stephanie A
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Lycksell, Marie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wang, Xueqing
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Howard, Rebecca J
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Propofol potentiation in the pentameric ion channel GLIC is mediated by a deep membrane-facing cavityManuscript (preprint) (Other academic)
  • 37.
    Heusser, Stephanie A.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Lycksell, Marie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Wang, Xueqing
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Mc Comas, Sarah E.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Howard, Rebecca J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Allosteric potentiation of a ligand-gated ion channel is mediated by access to a deep membrane-facing cavity2018In: 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)
    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.

  • 38.
    Heusser, Stephanie A.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Yoluk, Özge
    Klement, Göran
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Riederer, Erika A.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Howard, Rebecca J.
    Functional characterization of neurotransmitter activation and modulation in a nematode model ligand-gated ion channel2016In: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 138, no 2, p. 243-253Article in journal (Refereed)
    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 modified Caenorhabditis 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. In this study, we elucidate the validity of a modified glutamate-gated chloride channel (GluCl(cryst)) as a structurally accessible model for GABA(A) receptors. In contrast to native-like controls, GluCl(cryst) exhibits classical activation by its neurotransmitter ligand L-glutamate. The modified channel is also sensitive to allosteric modulators associated with human GABA(A) receptors, and to site-directed mutations predicted to alter channel opening.

  • 39. Hu, Haidai
    et al.
    Howard, Rebecca J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Bastolla, Ugo
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Delarue, Marc
    Structural basis for allosteric transitions of a multidomain pentameric ligand-gated ion channel2020In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 117, no 24, p. 13437-13446Article in journal (Refereed)
    Abstract [en]

    Pentameric ligand-gated ion channels (pLGICs) are allosteric receptors that mediate rapid electrochemical signal transduction in the animal nervous system through the opening of an ion pore upon binding of neurotransmitters. Orthologs have been found and characterized in prokaryotes and they display highly similar structure-function relationships to eukaryotic pLGICs; however, they often encode greater architectural diversity involving additional amino-terminal domains (NTDs). Here we report structural, functional, and normal-mode analysis of two conformational states of a multidomain pLGIC, called DeCLIC, from a Desulfofustis deltaproteobacterium, including a periplasmic NTD fused to the conventional ligand-binding domain (LBD). X-ray structure determination revealed an NTD consisting of two jelly-roll domains interacting across each subunit interface. Binding of Ca2+ at the LBD subunit interface was associated with a closed transmembrane pore, with resolved monovalent cations intracellular to the hydrophobic gate. Accordingly, DeCLIC-injected oocytes conducted currents only upon depletion of extracellular Ca2+; these were insensitive to quaternary ammonium block. Furthermore, DeCLIC crystallized in the absence of Ca2+ with a wide-open pore and remodeled periplasmic domains, including increased contacts between the NTD and classic LBD agonist-binding sites. Functional, structural, and dynamical properties of DeCLIC paralleled those of sTeLIC, a pLGIC from another symbiotic prokaryote. Based on these DeCLIC structures, we would reclassify the previous structure of bacterial ELIC (the first high-resolution structure of a pLGIC) as a "locally closed" conformation. Taken together, structures of DeCLIC in multiple conformations illustrate dramatic conformational state transitions and diverse regulatory mechanisms available to ion channels in pLGICs, particularly involving Ca2+ modulation and periplasmic NTDs.

  • 40. Jalalypour, Farzaneh
    et al.
    Howard, Rebecca J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Allosteric Cholesterol Site in Glycine Receptors Characterized through Molecular Simulations2024In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 128, no 20, p. 4996-5007Article in journal (Refereed)
    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.

  • 41.
    Johansson, Anna C V
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The role of lipid composition for insertion and stabilization of amino acids in membranes2009In: Journal of Chemical Physics, ISSN 0021-9606, E-ISSN 1089-7690, Vol. 130, no 18, p. 185101-Article in journal (Refereed)
    Abstract [en]

    While most membrane protein helices are clearly hydrophobic, recent experiments have indicated that it is possible to insert marginally hydrophobic helices into bilayers and have suggested apparent in vivo free energies of insertion for charged residues that are low, e.g., a few kcals for arginine. In contrast, a number of biophysical simulation studies have predicted that the bilayer interior is close to a pure hydrophobic environment with large penalties for hydrophilic amino acids--and yet the experimental scales do significantly better at predicting actual membrane proteins from sequence. Here, we have systematically studied the dependence of the free energy profiles on lipid properties, including tail length, saturation, headgroup hydrogen bond strength, and charge, both to see to whether the in vivo insertion can be explained in whole or part from lipid composition of the endoplasmic reticulum (ER) membranes, and if the solvation properties can help interpret how protein function depends on the lipids. We find that lipid charge is important to stabilize charged amino acids inside the bilayer (with implications, e.g., for ion channels), that thicker bilayers have higher solvation costs for hydrophilic side chains, and that headgroup hydrogen bond strength determines how adaptive the lipids are as a hydrophobic/hydrophilic solvent. None of the different free energy profiles are even close to the low apparent in vivo insertion cost, which suggests that regardless of the specific ER membrane composition the current experimental results cannot be explained by normal lipid-type variation.

  • 42.
    Johansson, Anna CV
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Amino-acid solvation structure in transmembrane helices from molecular dynamics simulations2006In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 91, no 12, p. 4450-4463Article in journal (Refereed)
  • 43.
    Johansson, Anna CV
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Position-resolved free energy of solvation for amino acids in lipid membranes from molecular dynamics simulations2008In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 70, no 4, p. 1332-1344Article in journal (Refereed)
  • 44.
    Johansson, Anna CV
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Protein contents in biological membranes explain abnormal solvation of charged and polar residues2009In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 37, p. 15684-15689Article in journal (Refereed)
    Abstract [en]

    Transmembrane helices are generally believed to insert into membranes based on their hydrophobicity. Nevertheless, there are important exceptions where polar or titratable residues have great functional importance, for instance the S4 helix of voltage-gated ion channels. It has been shown experimentally that insertion can be accomplished by hydrophobic counterbalance, which enabled biological hydrophobicity scales that predict an arginine insertion cost of only 2.5 kcal/mol, compared to 14.9 kcal/mol in cyklohexane. Previous simulations of pure bilayers have produced values close to the pure hydrocarbon, which has lead to vivid discussion about the experimental conditions.  Here, we have performed computer simulations of models better mimicking biological membranes by explicitly including protein helices at mass fractions from 15% to 55%. This has a striking effect on the solvation free energy of arginine, which drops more than a factor of two even for purely hydrophobic extra helices. With some polar residues present, the solvation cost comes close to experimental observation around 30% mass fraction, and negligible at 40%. The effect is mainly due to the extra helices making it easier for arginine to retain hydration water, with increasing amounts at higher protein mass fraction. These results offer a possible explanation to the previous discrepancy between the in vivo hydrophobicity scale and computer simulations, and highlight the importance of the relatively high protein contents in biological membranes. While many membrane proteins are stable in pure bilayers, the simplified models might not be sufficiently accurate descriptions of insertion for polar or charged residues in biological membranes.

  • 45.
    Johansson, Anna CV
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Titratable amino acid solvation in lipid membranes as a function of protonation state2009In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 113, no 1, p. 245-253Article in journal (Refereed)
    Abstract [en]

    Knowledge about the insertion and stabilization of membrane proteins is a key step toward understanding their function and enabling membrane protein design. Transmembrane helices are normally quite hydrophobic so as to efficiently insert into membranes, but there are many exceptions with polar or titratable residues. An obvious example is the S4 helices of voltage-gated ion channels with up to 4 arginines, leading to vivid discussion about whether such helices can insert spontaneously, and if so, what their conformation, protonation state, and cost of insertion really are. To address this question, we have determined geometric and energetic solvation properties for different protonation states of the titrateable amino acids, including hydration, side chain orientation, free energy profiles, and effects on the membrane thickness. As expected, charged states are significantly more expensive to insert (8-16 kcal/mol) than neutral variants (1-3 kcal/mol). Although both sets of values exhibit quite high relative correlation with experimental in vivo hydrophobicity scales, the magnitudes of the in vivo hydrophobicity scales are much lower and strikingly appears as a compressed version of the calculated values. This agrees well with computational studies on longer lipids but results in an obvious paradox: the differences between in vivo insertion and simulations cannot be explained by methodological differences in force fields, possible limited hydrophobic thickness of the endoplasmic reticulum (ER) membrane, or parameters; even anionic lipid head groups (PG) only have limited effect on charged side chains, and virtually none for hydrophobic ones. This leads us to propose a model for in vivo insertion that could reconcile these differences and explain the correlation: if there are considerable hydrophobic barriers inside the translocon, the experimental reference state for the solvation free energy when comparing insertion/translocation in vivo would be quite close to the bilayer environment rather than water.

  • 46. Kasimova, Marina A.
    et al.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Delemotte, Lucie
    Determining the molecular basis of voltage sensitivity in membrane proteins2018In: The Journal of General Physiology, ISSN 0022-1295, E-ISSN 1540-7748, Vol. 150, no 10, p. 1444-1458Article in journal (Refereed)
    Abstract [en]

    Voltage-sensitive membrane proteins are united by their ability to transform changes in membrane potential into mechanical work. They are responsible for a spectrum of physiological processes in living organisms, including electrical signaling and cell-cycle progression. Although the mechanism of voltage-sensing has been well characterized for some membrane proteins, including voltage-gated ion channels, even the location of the voltage-sensing elements remains unknown for others. Moreover, the detection of these elements by using experimental techniques is challenging because of the diversity of membrane proteins. Here, we provide a computational approach to predict voltage-sensing elements in any membrane protein, independent of its structure or function. It relies on an estimation of the propensity of a protein to respond to changes in membrane potential. We first show that this property correlates well with voltage sensitivity by applying our approach to a set of voltage-sensitive and voltage-insensitive membrane proteins. We further show that it correctly identifies authentic voltage-sensitive residues in the voltage-sensor domain of voltage-gated ion channels. Finally, we investigate six membrane proteins for which the voltage-sensing elements have not yet been characterized and identify residues and ions that might be involved in the response to voltage. The suggested approach is fast and simple and enables a characterization of voltage sensitivity that goes beyond mere identification of charges. We anticipate that its application before mutagenesis experiments will significantly reduce the number of potential voltage-sensitive elements to be tested.

  • 47.
    Kasson, Peter M.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. University of Virginia. USA.
    Hess, Berk
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Probing microscopic material properties inside simulated membranes through spatially resolved three-dimensional local pressure fields and surface tensions2013In: Chemistry and Physics of Lipids, ISSN 0009-3084, E-ISSN 1873-2941, Vol. 169, p. 106-112Article in journal (Refereed)
    Abstract [en]

    Cellular lipid membranes are spatially inhomogeneous soft materials. Materials properties such as pressure and surface tension thus show important microscopic-scale variation that is critical to many biological functions. We present a means to calculate pressure and surface tension in a 3D-resolved manner within molecular-dynamics simulations and show how such measurements can yield important insight. We also present the first corrections to local virial and pressure fields to account for the constraints typically used in lipid simulations that otherwise cause problems in highly oriented systems such as bilayers. Based on simulations of an asymmetric bacterial ion channel in a POPC bilayer, we demonstrate how 3D-resolved pressure can probe for both short-range and long-range effects from the protein on the membrane environment. We also show how surface tension is a sensitive metric for inter-leaflet equilibrium and can be used to detect even subtle imbalances between bilayer leaflets in a membrane-protein simulation. Since surface tension is known to modulate the function of many proteins, this effect is an important consideration for predictions of ion channel function. We outline a strategy by which our local pressure measurements, which we make available within a version of the GROMACS simulation package, may be used to design optimally equilibrated membrane-protein simulations.

  • 48.
    Kasson, Peter M.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pande, Vijay S.
    Atomic-Resolution Simulations Predict a Transition State for Vesicle Fusion Defined by Contact of a Few Lipid Tails2010In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 6, no 6, p. e1000829-Article in journal (Refereed)
    Abstract [en]

    Membrane fusion is essential to both cellular vesicle trafficking and infection by enveloped viruses. While the fusion protein assemblies that catalyze fusion are readily identifiable, the specific activities of the proteins involved and nature of the membrane changes they induce remain unknown. Here, we use many atomic-resolution simulations of vesicle fusion to examine the molecular mechanisms for fusion in detail. We employ committor analysis for these million-atom vesicle fusion simulations to identify a transition state for fusion stalk formation. In our simulations, this transition state occurs when the bulk properties of each lipid bilayer remain in a lamellar state but a few hydrophobic tails bulge into the hydrophilic interface layer and make contact to nucleate a stalk. Additional simulations of influenza fusion peptides in lipid bilayers show that the peptides promote similar local protrusion of lipid tails. Comparing these two sets of simulations, we obtain a common set of structural changes between the transition state for stalk formation and the local environment of peptides known to catalyze fusion. Our results thus suggest that the specific molecular properties of individual lipids are highly important to vesicle fusion and yield an explicit structural model that could help explain the mechanism of catalysis by fusion proteins.

  • 49. Kasson, Peter M.
    et al.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pande, Vijay S.
    Water Ordering at Membrane Interfaces Controls Fusion Dynamics2011In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 133, no 11, p. 3812-3815Article in journal (Refereed)
    Abstract [en]

    Membrane interfaces are critical to many cellular functions, yet the vast array of molecular components involved make the fundamental physics of interaction difficult to define. Water has been shown to play an important role in the dynamics of small biological systems, for example when trapped in hydrophobic regions, but the molecular details of water have generally been thought dispensable when considering large membrane interfaces. Nevertheless, spectroscopic data indicate that water has distinct, ordered behavior near membrane surfaces. While coarse-grained simulations have achieved success recently in aiding understanding the dynamics of membrane assemblies, it is natural to ask, does the missing chemical nature of water play an important role? We have therefore performed atomic-resolution simulations of vesicle fusion to understand the role of chemical detail, particularly the molecular structure of water, in membrane fusion and at membrane interfaces more generally. These membrane interfaces present a form of hydrophilic confinement, yielding surprising, non-bulk-like water behavior.

  • 50. Kim, Jeong Joo
    et al.
    Gharpure, Anant
    Teng, Jinfeng
    Zhuang, Yuxuan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Howard, Rebecca J.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Zhu, Shaotong
    Noviello, Colleen M.
    Walsh, Richard M.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Hibbs, Ryan E.
    Shared structural mechanisms of general anaesthetics and benzodiazepines2020In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 585, p. 303-308Article in journal (Refereed)
    Abstract [en]

    Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of gamma-aminobutyric acid type A (GABA(A)) receptors to dampen neuronal activity in the brain(1-5). However, direct structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABA(A)receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABA(A)receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABA(A)receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain. Cryo-electron microscopy structures of GABA(A)receptors bound to intravenous anaesthetics and benzodiazepines reveal both common and distinct transmembrane binding sites, and show that the mechanisms of action of anaesthetics partially overlap with those of benzodiazepines.

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