Biology subjects

Lindahl, E.

Publications and source records attributed to Lindahl, E..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Divergent mechanisms of steroid inhibition in the human {rho}1 GABA(A) receptor

{rho}-type {gamma}-aminobutyric acid-A (GABAA) receptors are widely distributed in the retina and brain, and are potential drug targets for the treatment of visual, sleep and cognitive disorders. Endogenous neuroactive steroids including {beta}-estradiol and pregnenolone sulfate negatively modulate the function of {rho}1 GABAA receptors, but their inhibitory mechanisms are not clear. By combining four new cryo-EM structures with electrophysiology and molecular dynamics simulations, we characterize binding sites and negative modulation mechanisms of {beta}-estradiol and pregnenolone sulfate at the human {rho}1 GABAA receptor. {beta}-estradiol binds in a pocket at the interface between extracellular and transmembrane domains, apparently specific to the {rho} subfamily, and disturbs allosteric conformational transitions linking GABA binding to pore opening. In contrast, pregnenolone sulfate binds inside the pore to block ion permeation, with a preference for activated structures. These results illuminate contrasting mechanisms of {rho}1 inhibition by two different neuroactive steroids, with potential implications for subtype-specific gating and pharmacological design.

biophysics↗

bioRxiv2023

Symmetry-adapted Markov state models of closing, opening, and desensitizing in α7 nicotinic acetylcholine receptors

7 nicotinic acetylcholine receptors (nAChRs) are homopentameric ligand-gated ion channels gated by acetylcholine. These receptors play crucial roles in controlling electrical signaling within the nervous system by facilitating the passage of cations across the membrane. Recent studies have resolved and functionally annotated closed, open, and desensitized states of 7 nAChRs, providing insight into ion permeation and lipid modulation. However, the process by which 7 nAChRs transition between states remains unclear. To understand gating and lipid modulation, we generated two ensembles of molecular dynamics simulations of the apo form of 7 nAChRs, with or without cholesterol. Using symmetry-adapted Markov state modeling, we developed a five-state gating model. Free energies recapitulated functional behavior, with the closed state dominating in the absence of agonists. Notably, the transition rate from open to a non-conductive intermediate (flipped) state corresponded to experimentally measured open durations around 0.1 ms. The introduction of cholesterol relatively stabilized the desensitized state and reduced barriers between desensitized and open states. These results establish plausible asymmetric transition pathways between functionally important states, they define lipid modulation effects in the 7 nAChR conformational cycle, and provide an ensemble of structural models that could be utilized for guiding rational design strategies to develop lipidic pharmaceuticals targeting these receptors.

biophysics↗

bioRxiv2023

Constant-pH molecular dynamics simulations of progressive activation in a proton-gated ion channel

Although traditional molecular dynamics simulations successfully capture a variety of different molecular interactions, the protonation states of titratable residues are kept static. A recent constant-pH molecular dynamics implementation in the GROMACS package allows pH effects to be captured dynamically, and promises to provide both the accuracy and computational performance required for studying pH-mediated conformational dynamics in large, complex systems containing hundreds of titratable residues. Here, we demonstrate the applicability of this constant-pH implementation by simulating the proton-gated ion channel GLIC at resting and activating pH, starting from closed and open structures. Our simulations identify residues E26 and E35 as especially pH-sensitive and reveal state-dependent pKa shifts at multiple residues, as well as side chain and domain rearrangements in line with the early stages of gating. Our results are consistent with several previous experimental findings, demonstrating the applicability of constant-pH simulations to elucidate pH-mediated activation mechanisms in multidomain membrane proteins, likely extensible to other complex systems. Significance statementElectrostatic interactions play important roles in protein structure and function. Since changes in pH will (de)protonate residues and thereby modify such interactions, pH itself is a critical environmental parameter. However, protonation states of titratable residues are static during classical molecular dynamics simulations. Recently, a constant-pH algorithm was implemented in the GROMACS package, allowing pH effects to be captured dynamically. Here, we used this implementation to perform constant-pH simulations of the proton-gated ion channel GLIC, providing insight into its activation mechanism by revealing state-dependent shifts in protonation as well as pH-dependent side chain and domain-level rearrangements. The results show that constant-pH simulations are accurate, efficient, and capable of modeling dozens of titratable sites, with important implications for e.g. drug design.

biophysics↗

bioRxiv2023

Discovering cryptic pocket opening and ligandbinding in a vestibular site of the 5-HT3A receptor

Ligand-gated ion channels propagate electrochemical signals in the nervous system. A diverse set of modulators including stimulants, anesthetics, and lipids regulate their function; however, structures of ligand-bound complexes can be difficult to capture by experimental methods, particularly when binding is dynamic or transient. Here, we used computational methods and electrophysiology to identify a possible bound state of a modulatory stimulant derivative in a cryptic vestibular pocket, distinct from the orthosteric neurotransmitter binding site, of a mammalian serotonin-3A receptor. Starting from a closed-pocket experimental structure, we first applied a molecular dynamics simulations-based goal-oriented adaptive sampling method to identify possible open-pocket conformations. To find plausible ligand-binding poses, we performed Boltzmann docking, which combines traditional docking with Markov state modeling, of the newly sampled conformations. Clustering and analysis of stability and accessibility of docked poses supported a preferred binding site; we further validated this site by mutagenesis and electrophysiology, suggesting a mechanism of potentiation by stabilizing intersubunit contacts. Given the pharmaceutical relevance of serotonin-3 receptors in emesis, psychiatric and gastrointestinal diseases, characterizing relatively unexplored modulatory sites such as these could open valuable avenues to understanding conformational cycling and designing state-dependent drugs. Significance5-HT3A receptors receive the chemical signals of excitatory neurotransmission across the synapse in the central and peripheral nervous systems, and are involved in conditions including emesis, pain, psychiatric disorders, drug abuse, and irritable bowel syndrome. Given their pharmaceutical importance, there is great interest in understanding how and where ligands interact with these receptors. A pocket facing the extracellular vestibule of this membrane protein has been proposed as a modulatory site, but it remains largely uncharacterized in the context of structural modeling or pharmacologically relevant ligands. Here, we are able to identify and investigate binding of a stimulant derivative, 4-bromoamphetamine, in this site by using an integrative computational and experimental approach that is able to account for conformational flexibility.

biophysics↗

bioRxiv2023

Structure and dynamics of differential ligand binding in the human {rho}-type GABA(A) receptor

The neurotransmitter {gamma}-aminobutyric acid (GABA) drives critical inhibitory processes in and beyond the nervous system, partly via ionotropic type-A receptors (GABAARs). Pharmacological properties of {rho}-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 {rho}1 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 {rho}1 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 {rho}-specific pharmaceutical design, and to our biophysical understanding of ligand-gated ion channels.

biophysics↗

bioRxiv2023

MDverse: Shedding Light on the Dark Matter of Molecular Dynamics Simulations

The rise of open science and the absence of a global dedicated data repository for molecular dynamics (MD) simulations has led to the accumulation of MD [fi]les in generalist data repositories, constituting the dark matter of MD -- data that is technically accessible, but neither indexed, curated, or easily searchable. Leveraging an original search strategy, we found and indexed about 250,000 [fi]les and 2,000 datasets from Zenodo, Figshare and Open Science Framework. With a focus on [fi]les produced by the Gromacs MD software, we illustrate the potential offered by the mining of publicly available MD data. We identi[fi]ed systems with speci[fi]c molecular composition and were able to characterize essential parameters of MD simulation such as temperature and simulation length, and could identify model resolution, such as all-atom and coarse-grain. Based on this analysis, we inferred metadata to propose a search engine prototype to explore the MD data. To continue in this direction, we call on the community to pursue the effort of sharing MD data, and to report and standardize metadata to reuse this valuable matter.

bioinformatics↗

bioRxiv2023

Interactive computational and experimental approaches improve the sensitivity of periplasmic binding protein-based nicotine biosensors for measurements in biofluids

We developed fluorescent protein sensors for nicotine with improved sensitivity. For iNicSnFR12 at pH 7.4, the proportionality constant for {Delta}F/F0 vs [nicotine] ({delta}-slope, 2.7 M-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.

bioengineering↗

bioRxiv2023

Discovery of lipid binding sites in a ligand-gated ion channel by integrating simulations and cryo-EM

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.

biophysics↗

bioRxiv2022

Automated simulation-based membrane-protein refinement into cryo-EM data

I.The resolution revolution has increasingly enabled single-particle cryogenic electron microscopy (cryo-EM) reconstructions of previously inaccessible systems, including membrane proteins - a category that constitutes a disproportionate share of drug targets. We present a protocol for using density-guided molecular dynamics simulations to automatically refine atomistic models into membrane-protein cryo-EM maps. Using adaptive-force density-guided simulations as implemented in the GROMACS molecular dynamics package, we show how automated model refinement of a membrane protein is achieved without the need to manually tune the fitting force ad hoc. We also present selection criteria to choose the best fit model which balances stereochemistry and goodness-of-fit. The proposed protocol was used to refine models into a new cryo-EM density of the membrane protein maltoporin, either in a lipid bilayer or detergent micelle, and we found that results do not substantially differ from fitting in solution. Fitted structures satisfied classical model-quality metrics and improved the quality and the model-to-map correlation of the X-ray starting structure. Additionally, the density-guided fitting in combination with generalized orientation-dependent all-atom potential (GOAP) was used to correct the pixel-size estimation of the experimental cryo-EM density map. This work demonstrates the applicability of a straightforward automated approach to fitting membrane-protein cryo-EM densities. Such computational approaches promise to facilitate rapid refinement of proteins under different conditions or with various ligands present, including targets in the highly relevant superfamily of membrane proteins. II. STATEMENT OF SIGNIFICANCECryo-EM is an increasingly critical method of structure determination. As data collection and model generation become more efficient, iteratively fitting an experimental density can still require considerable time and expertise. Membrane proteins are particularly important targets in pharmacology and bioengineering, but can present distinctive challenges to data quality and modeling. Here, we tested a new tool to drive density fitting with molecular dynamics simulations, in context of a new structure of the membrane protein maltoporin. Fitting performed well in detergent, lipids, or solution, offering simpler options for fully automated simulation protocols. We were also able to apply fitting to adjust the microscopes pixel size. The approach described here should be applicable to rapid, accurate refinement of a variety of membrane-protein structures.

biophysics↗

bioRxiv2022

Gentle and fast all-atom model refinement to cryo-EM densities via Bayes' approach

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 Bayes approach by formulating the problem as identifying the structure most likely to have produced the observed density map. This allows us to derive a new type 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 Bayesian refinement potential outperforms alternatives, and the adaptive force scaling appears to also aid existing refinement potentials. The method is available as a component in the GROMACS molecular simulation toolkit.

biophysics↗

bioRxiv2022

Biophysical characterization of calcium-binding and modulatory-domain dynamics in a pentameric ligand-gated ion channel

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

biophysics↗

bioRxiv2022

Differential interactions of resting, activated, and desensitized states of the α7 nicotinic acetylcholine receptor with lipidic modulators

The 7 nicotinic acetylcholine receptor is a pentameric ligand-gated ion channel that modulates neuronal excitability, largely by allowing Ca2+ permeation. Agonist binding promotes transition from a resting state to an activated state, and then rapidly to a desensitized state. Recently, cryo-EM structures of the human 7 receptor in nanodiscs were reported in multiple conformations. These were selectively stabilized by inhibitory, activating, or potentiating compounds. However, the functional annotation of these structures, and their differential interactions with unresolved lipids and ligands, remain incomplete. Here, we characterized their ion permeation, membrane interactions, and ligand binding using computational electrophysiology, free-energy calculations, and coarse-grained molecular dynamics. In contrast to non-conductive structures in apparent resting and desensitized states, the structure determined in the presence of the potentiator PNU-120596 was consistent with an activated state permeable to Ca2+. Transition to this state was associated with compression and rearrangement of the membrane, particularly in the vicinity of the peripheral MX helix. An intersubunit transmembrane site was implicated in selective binding of either PNU-120596 in the activated state, or cholesterol in the desensitized state. This substantiates functional assignment of all three lipid-embedded 7-receptor structures with ion permeation simulations. It also proposes testable models of their state-dependent interactions with lipophilic ligands, including a mechanism for allosteric modulation at the transmembrane subunit interface.

biophysics↗

bioRxiv2022

Predictions of Skin Permeability Using Molecular Dynamics Simulation from Two-Dimensional Sampling of Spatial and Alchemical Perturbation Reaction Coordinates

A molecular level understanding of skin permeation may rationalize and streamline product development, and improve quality and control, of transdermal and topical drug delivery systems. It may also facilitate toxicity and safety assessment of cosmetics, skin care products. Here, we present new molecular dynamics simulation approaches that make it possible to efficiently sample the free energy and local diffusion coefficient across the skins barrier structure and predict skin permeability and the effects of chemical penetration enhancers. In particular, we introduce a new approach to use two-dimensional reaction coordinates with so-called Accelerated Weight Histograms, where we combine sampling along spatial coordinates with an alchemical perturbation virtual coordinate. We present predicted properties for twenty permeants, and demonstrate how our approach improves correlation with ex vivo/in vitro skin permeation data. For the compounds included in this study, the ob-tained [Formula] mean square difference was 0.8 cm2 h-2.

biophysics↗

bioRxiv2021

Biophysical characterization of the SARS-CoV-2 E protein

SARS-CoV-2 contains four structural proteins in its genome. These proteins aid in the assembly and budding of new virions at the ER-Golgi intermediate compartment (ERGIC). Current fundamental research efforts largely focus on one of these proteins - the spike (S) protein. Since successful antiviral therapies are likely to target multiple viral components, there is considerable interest in understanding the biophysical role of its other structural proteins, in particular structural membrane proteins. Here, we have focused our efforts on the characterization of the full-length envelope (E) protein from SARS-CoV-2, combining experimental and computational approaches. Recombinant expression of the full-length E protein from SARS-CoV-2 reveals that this membrane protein is capable of independent multimerization, possibly as a tetrameric or smaller species. Fluorescence microscopy shows that the protein localizes intracellularly, and coarse-grained MD simulations indicate it causes bending of the surrounding lipid bilayer, corroborating a potential role for the E protein in viral budding. Although we did not find robust electrophysiological evidence of ion-channel activity, cells transfected with the E protein exhibited reduced intracellular Ca2+, which may further promote viral replication. However, our atomistic MD simulations revealed that previous NMR structures are relatively unstable, and result in models incapable of ion conduction. Our study highlights the importance of using high-resolution structural data obtained from a full-length protein to gain detailed molecular insights, and eventually permitting virtual drug screening.

biophysics↗

bioRxiv2021

Probing solution structure of the pentameric ligand-gated ion channel GLIC by small-angle neutron scattering

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

biophysics↗

bioRxiv2021

A cationic lipid site at the outward transmembrane face of a pentameric ligand-gated ion channel

Pentameric ligand-gated ion channels (pLGICs) are crucial mediators of electrochemical signal transduction from bacteria to humans. Lipids play an important role in regulating pLGIC function, yet the structural basis for specific pLGIC-lipid interactions remains poorly understood. The bacterial channel ELIC recapitulates several properties of eukaryotic pLGICs, including activation by the neurotransmitter GABA and sensitivity to lipids, offering a simplified model system for structure-function studies. In this study, functional effects of non-canonical amino acid substitution of W206 at the top of the M1-helix, combined with detergent interactions observed in recent X-ray structures, are consistent with this region being the location of a lipid binding site on the outward face of the ELIC transmembrane domain. Coarse-grained and atomistic molecular dynamics simulations revealed preferential binding of lipids containing a positive charge, particularly involving interactions with residue W206 consistent with cation-{pi} binding. Polar contacts from the principal subunit, particularly M3 residue Q264, further supported lipid binding via headgroup ester linkages. Aromatic residues were identified at analogous sites in a handful of eukaryotic family members, including the human GABAA receptor subunit {varepsilon}, suggesting conservation of relevant interactions in other evolutionary branches. Further mutagenesis experiments indicated that mutations at this site in {varepsilon}-containing GABAA receptors can change the apparent affinity of the agonist response to GABA, consistent with a potential role of this site in channel gating. In conclusion, this work is a detailed case study in type-specific lipid interactions at an evolutionarily distinctive pLGIC site, with implications for lipid modulation and lipophilic drug design.

biophysics↗

bioRxiv2021

Markov state models of proton- and gate-dependent activation in a pentameric ligand-gated ion channel

Ligand-gated ion channels conduct currents in response to chemical stimuli, mediating electrochemical signaling in neurons and other excitable cells. For many channels the mechanistic 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 transitions. Consistent with new functional recordings reported here 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 in each condition, while higher-order clustering affirmed conformational trends in gating. Furthermore, our models uncovered state- and protonation-dependent symmetrization among subunits. This demonstrates the applicability of MSMs to map energetic and conformational transitions between ion-channel functional states, and how they correctly reproduce shifts upon activation or mutation, with implications for modeling neuronal function and developing state-selective drugs.

biophysics↗

bioRxiv2021

Dynamic closed states of a ligand-gated ion channel captured by cryo-EM and simulations

Ligand-gated ion channels are critical mediators of electrochemical signal transduction across evolution. Biophysical and pharmacological characterization of these receptor proteins relies on high-quality structures in multiple, subtly distinct functional states. However, structural data in this family remain limited, particularly for resting and intermediate states on the activation pathway. Here we report cryo-electron microscopy (cryo-EM) structures of the proton-activated Gloeobacter violaceus ligand-gated ion channel (GLIC) under three pH conditions. Decreased pH was associated with improved resolution and sidechain rearrangements at the subunit/domain interface, particularly involving functionally important residues in the {beta}1-{beta}2 and M2-M3 loops. Molecular dynamics simulations substantiated flexibility in the closed-channel extracellular domains relative to the transmembrane ones, and supported electrostatic remodeling around E35 and E243 in proton-induced gating. Exploration of secondary cryo-EM classes further indicated a low-pH population with an expanded pore. These results support a dissection of protonation and activation steps in pH-stimulated conformational cycling in GLIC, including interfacial rearrangements largely conserved in the pentameric channel family.

biochemistry↗