Category: Publications

Published 19 March 2025 in Structure (doi 10.1016/j.str.2025.02.012):

Cryo-EM structure of the human monocarboxylate transporter 10

Viktoria Bågenholm, Karl Patric Nordlin, Andrea Pasquadibisceglie, Andrey Belinskiy, Caroline Marcher Holm, Hajira Ahmed Hotiana, Kamil Gotfryd, Lucie Delemotte, Hussam Hassan Nour-Eldin, Per Amstrup Pedersen, Pontus Gourdon

The monocarboxylate transporter (MCT) membrane protein family has 14 human members that perform key cellular functions, such as regulating metabolism. MCT8 and MCT10 have unique cargo specificity, transporting thyroid hormone and, in the case of MCT10, aromatic amino acids. Dysfunctional MCT8 causes the severe Allan-Herndon-Dudley syndrome, yet the (patho)physiology and function of MCT8 and MCT10 are not clearly understood, especially at a structural level. We present the cryoelectron microscopy (cryo-EM) structure of MCT10, displaying the classical major facilitator superfamily fold, caught in an inward-open configuration. Together with cargo docking models, the outward-open MCT10 AlphaFold model and validating functional analysis, cargo specificity and transport principles are proposed. These findings significantly enhance our understanding of the structure and function of MCTs, information that also may be valuable for the development of novel treatments against MCT-related disorders to address global challenges such as diabetes, obesity, and cancer.

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Published 11 March 2025 in British Journal of Pharmacology (doi 10.1111/bph.70011):

Vestibular modulation by stimulant derivatives in a pentameric ligand-gated ion channel

Emelia Karlsson, Olivia Andén, Chen Fan, Zaineb Fourati, Ahmed Haouz, Yuxuan Zhuang, Rebecca J Howard, Marc Delarue, Erik Lindahl

Background and Purpose

Allosteric modulation of pentameric ligand-gated ion channels (pLGICs) are critical for the action of neurotransmitters and many psychoactive drugs. However, details of their modulatory mechanisms remain unclear, especially beyond the orthosteric neurotransmitter-binding sites. The recently reported prokaryotic symbiont of Tevnia jerichonana ligand-gated ion channel (sTeLIC), a pH-gated homologue of eukaryotic receptors in the pLGIC family, is thought to be modulated by aromatic compounds via a relatively uncharacterised modulatory site in the extracellular vestibule.

Experimental Approach

We have characterised the effects of psychostimulant derivatives on sTeLIC using two-electrode voltage-clamp electrophysiology in the presence and absence of engineered mutations, and determined X-ray and cryo-EM structures of the channel in both closed and open states.

Key Results

We have shown that sTeLIC is sensitive to potentiation by several amphiphilic compounds, which preferentially bind to a vestibular pocket in the contracted open-state extracellular domain.

Conclusions and Implications

This work provides a detailed structure–function mechanism for allosteric potentiation via a noncanonical ligand site, with potential conservation of the eukaryotic pentameric ligand-gated ion channels.

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Published 6 February 2025 in Cell (doi 10.1016/j.cell.2024.11.036):

Molecular basis of proton sensing by G protein-coupled receptors

Matthew K Howard,* Nicholas Hoppe,* Xi-Ping Huang,° Darko Mitrovic,° Christian B Billesbølle, Christian B Macdonald, Eshan Mehrotra, Patrick Rockefeller Grimes, Donovan D Trinidad, Lucie Delemotte, Justin G English, Willow Coyote-Maestas,’ Aashish Manglik’

Three proton-sensing G protein-coupled receptors (GPCRs)-GPR4, GPR65, and GPR68-respond to extracellular pH to regulate diverse physiology. How protons activate these receptors is poorly understood. We determined cryogenic-electron microscopy (cryo-EM) structures of each receptor to understand the spatial arrangement of proton-sensing residues. Using deep mutational scanning (DMS), we determined the functional importance of every residue in GPR68 activation by generating ∼9,500 mutants and measuring their effects on signaling and surface expression. Constant-pH molecular dynamics simulations provided insights into the conformational landscape and protonation patterns of key residues. This unbiased approach revealed that, unlike other proton-sensitive channels and receptors, no single site is critical for proton recognition. Instead, a network of titratable residues extends from the extracellular surface to the transmembrane region, converging on canonical motifs to activate proton-sensing GPCRs. Our approach integrating structure, simulations, and unbiased functional interrogation provides a framework for understanding GPCR signaling complexity.

*Equal contributions; °equal contributions; ‘co-corresponding authors

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Published 10 January 2025 in Science Advances (doi 10.1126/sciadv.adq3788):

Adaptive sampling-based structural prediction reveals opening of a GABA_A receptor through the αβ interface

Nandan Haloi, Samuel Eriksson Lidbrink, Rebecca J Howard, Erik Lindahl

γ-Aminobutyric acid type A (GABAA) receptors are ligand-gated ion channels in the central nervous system with largely inhibitory function. Despite being a target for drugs including general anesthetics and benzodiazepines, experimental structures have yet to capture an open state of classical synaptic α1β2γ2 GABAA receptors. Here, we use a goal-oriented adaptive sampling strategy in molecular dynamics simulations followed by Markov state modeling to capture an energetically stable putative open state of the receptor. The model conducts chloride ions with comparable conductance as in electrophysiology measurements. Relative to experimental structures, our open model is relatively expanded at both the cytoplasmic (-2′) and central (9′) gates, coordinated with distinctive rearrangements at the transmembrane αβ subunit interface. Consistent with previous experiments, targeted substitutions disrupting interactions at this interface slowed the open-to-desensitized transition rate. This work demonstrates the capacity of advanced simulation techniques to investigate a computationally and experimentally plausible functionally critical of a complex membrane protein yet to be resolved by experimental methods.

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Published 3 January 2025 in Science Advances (doi 10.1126/sciadv.adr7427):

A propofol binding site in the voltage sensor domain mediates inhibition of HCN1 channel activity

Verena Burtscher, Lei Wang, John Cowgill, Zi-Wei Chen, Christopher Edge, Edward Smith, Yongchang Chang, Lucie Delemotte, Alex S. Evers, Baron Chanda

Hyperpolarization-activated and cyclic nucleotide–gated (HCN) ion channels are members of the cyclic nucleotide–binding family and are crucial for regulating cellular automaticity in many excitable cells. HCN channel activation contributes to pain perception, and propofol, a widely used anesthetic, acts as an analgesic by inhibiting the voltage-dependent activity of HCN channels. However, the molecular determinants of propofol action on HCN channels remain unknown. Here, we use a propofol-analog photoaffinity labeling reagent to identify propofol binding sites in the human HCN1 isoform. Mass spectrometry analyses combined with molecular dynamics simulations show that a binding pocket is formed by extracellularly facing residues in the S3 and S4 transmembrane segments in the resting voltage-sensor conformation. Mutations of residues within the putative binding pocket mitigate or eliminate voltage-dependent modulation of HCN1 currents by propofol. Together, these findings reveal a conformation-specific propofol binding site that underlies voltage-dependent inhibition of HCN currents and provides a framework for identifying highly specific modulators of HCN channel gating.

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Published 18 October 2024 in Nature Communications (doi 10.1038/s41467-024-53170-z):

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

Yuxuan Zhuang, Rebecca J Howard, Erik Lindahl

α7 nicotinic acetylcholine receptors (nAChRs) are homopentameric ligand-gated ion channels with critical roles in the nervous system. Recent studies have resolved and functionally annotated closed, open, and desensitized states of these receptors, providing insight into ion permeation and lipid binding. However, the process by which α7 nAChRs transition between states remains unclear. To understand gating and lipid modulation, we generated two ensembles of molecular dynamics simulations of apo α7 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 absence of agonist. Open-to-nonconducting transition rates corresponded to experimental open durations. Cholesterol relatively stabilized the desensitized state, and reduced open-desensitized barriers. These results establish plausible asymmetric transition pathways between states, define lipid modulation effects on the α7 nAChR conformational cycle, and provide an ensemble of structural models applicable to rational design of lipidic pharmaceuticals.

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Published 6 September 2024 in Nature Communications (doi 10.1038/s41467-024-51904-7):

Divergent mechanisms of steroid inhibition in the human ρ1 GABA_A receptor

Chen Fan,* John Cowgill,* Rebecca J Howard,° Erik Lindahl°

ρ-type γ-aminobutyric acid-A (GABA_A) 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 β-estradiol and pregnenolone sulfate negatively modulate the function of ρ1 GABA_A receptors, but their inhibitory mechanisms are not clear. By combining five cryo-EM structures with electrophysiology and molecular dynamics simulations, we characterize binding sites and negative modulation mechanisms of β-estradiol and pregnenolone sulfate at the human ρ1 GABA_A receptor. β-estradiol binds in a pocket at the interface between extracellular and transmembrane domains, apparently specific to the ρ 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 ρ1 inhibition by two different neuroactive steroids, with potential implications for subtype-specific gating and pharmacological design.

*Equal contributions; °co-corresponding authors

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Published 30 August 2024 in eLife (doi 10.7554/elife.90061):

MDverse, shedding light on the dark matter of molecular dynamics simulations

Johanna K S Tiemann,* Magdalena Szczuka, Lisa Bouarroudj, Mohamed Oussaren, Steven Garcia, Rebecca J Howard, Lucie Delemotte, Erik Lindahl, Marc Baaden, Kresten Lindorff-Larsen, Matthieu Chavent,* Pierre Poulain*

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 files 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 files and 2000 datasets from Zenodo, Figshare and Open Science Framework. With a focus on files produced by the Gromacs MD software, we illustrate the potential offered by the mining of publicly available MD data. We identified systems with specific 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.

*Co-corresponding authors

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Published 16 July 2024 in Biophysical Journal (doi 10.1016/j.bpj.2024.01.010):

An intracellular hydrophobic nexus critical for hERG1 channel slow deactivation

Whitney A Stevens-Sostre, Lisandra Flores-Aldama, Daniel Bustos, Jin Li, João H Morais-Cabral, Lucie Delemotte, Gail A Robertson

Slow deactivation is a critical property of voltage-gated K⁺ channels encoded by the human Ether-à-go-go-Related Gene 1 (hERG). hERG1 channel deactivation is modulated by interactions between intracellular N-terminal Per-Arnt-Sim (PAS) and C-terminal cyclic nucleotide-binding homology (CNBh) domains. The PAS domain is multipartite, comprising a globular domain (gPAS; residues 26-135) and an N-terminal PAS-cap that is further subdivided into an initial unstructured “tip” (residues 1-12) and an amphipathic α-helical region (residues 13-25). Although the PAS-cap tip has long been considered the effector of slow deactivation, how its position near the gating machinery is controlled has not been elucidated. Here, we show that a triad of hydrophobic interactions among the gPAS, PAS-cap α helix, and the CNBh domains is required to support slow deactivation in hERG1. The primary sequence of this “hydrophobic nexus” is highly conserved among mammalian ERG channels but shows key differences to fast-deactivating Ether-à-go-go 1 (EAG1) channels. Combining sequence analysis, structure-directed mutagenesis, electrophysiology, and molecular dynamics simulations, we demonstrate that polar serine substitutions uncover an intermediate deactivation mode that is also mimicked by deletion of the PAS-cap α helix. Molecular dynamics simulation analyses of the serine-substituted channels show an increase in distance among the residues of the hydrophobic nexus, a rotation of the intracellular gating ring, and a retraction of the PAS-cap tip from its receptor site near the voltage sensor domain and channel gate. These findings provide compelling evidence that the hydrophobic nexus coordinates the respective components of the intracellular gating ring and positions the PAS-cap tip to control hERG1 deactivation gating.

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Published 8 July 2024 in Journal of Chemical Information and Modeling (doi 10.1021/acs.jcim.4c00722):

Simulating the skin permeation process of ionizable molecules

Magnus Lundborg,* Christian Wennberg, Erik Lindahl, Lars Norlén*

It is commonly assumed that ionizable molecules, such as drugs, permeate through the skin barrier in their neutral form. By using molecular dynamics simulations of the charged and neutral states separately, we can study the dynamic protonation behavior during the permeation process. We have studied three weak acids and three weak bases and conclude that the acids are ionized to a larger extent than the bases, when passing through the headgroup region of the lipid barrier structure, at pH values close to their pKa. It can also be observed that even if these dynamic protonation simulations are informative, in the cases studied herein they are not necessary for the calculation of permeability coefficients. It is sufficient to base the calculations only on the neutral form, as is commonly done.

*Co-corresponding authors

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Published 16 May 2024 in Molecular Cell (doi 10.1016/j.molcel.2024.04.008):

The full spectrum of SLC22 OCT1 mutations illuminates the bridge between drug transporter biophysics and pharmacogenomics

Sook Wah Yee, Christian B Macdonald, Darko Mitrovic, Xujia Zhou, Megan L Koleske, Jia Yang, Dina Buitrago Silva, Patrick Rockefeller Grimes, Donovan D Trinidad, Swati S More, Linda Kachuri, John S Witte, Lucie Delemotte, Kathleen M Giacomini, Willow Coyote-Maestas

Mutations in transporters can impact an individual’s response to drugs and cause many diseases. Few variants in transporters have been evaluated for their functional impact. Here, we combine saturation mutagenesis and multi-phenotypic screening to dissect the impact of 11,213 missense single-amino-acid deletions, and synonymous variants across the 554 residues of OCT1, a key liver xenobiotic transporter. By quantifying in parallel expression and substrate uptake, we find that most variants exert their primary effect on protein abundance, a phenotype not commonly measured alongside function. Using our mutagenesis results combined with structure prediction and molecular dynamic simulations, we develop accurate structure-function models of the entire transport cycle, providing biophysical characterization of all known and possible human OCT1 polymorphisms. This work provides a complete functional map of OCT1 variants along with a framework for integrating functional genomics, biophysical modeling, and human genetics to predict variant effects on disease and drug efficacy.

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Published 15 May 2024 in the Journal of Physical Chemistry B (doi 10.1021/acs.jpcb.4c01703):

Allosteric cholesterol site in glycine receptors characterized through molecular simulations

Farzaneh Jalalypour, Rebecca J Howard, Erik Lindahl

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.

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Published 26 April 2024 in EMBO Journal (doi 10.1038/s44318-024-00106-4):

Functional and structural insights into activation of TRPV2 by weak acids

Ferdinand M Haug, Ruth A Pumroy, Akshay Sridhar, Sebastian Pantke, Florian Dimek, Tabea C Fricke, Axel Hage, Christine Herzog, Frank G Echtermeyer, Jeanne de la Roche, Adrian Koh, Abhay Kotecha, Rebecca J Howard, Erik Lindahl, Vera Moiseenkova-Bell°, Andreas Leffler°

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.

°Corresponding authors

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Published 19 March 2024 in Physical Review Fluids (doi 10.1103/PhysRevFluids.9.034002):

Near-wall depletion and layering affect contact line friction of multicomponent liquids

Michele Pellegrino, Berk Hess

The main causes of energy dissipation in micro- and nanoscale wetting are viscosity and liquid-solid friction localized in the three-phase contact line region. Theoretical models predict the contact line friction coefficient to correlate with the shear viscosity of the wetting fluid. Experiments conducted to investigate such correlation have not singled out a unique scaling law between the two coefficients. We perform molecular dynamics simulations of liquid water-glycerol droplets wetting silicalike surfaces, aimed to demystify the effect of viscosity on contact line friction. The viscosity of the fluid is tuned by changing the relative mass fraction of glycerol in the mixture and it is estimated both via equilibrium and nonequilibrium molecular dynamics simulations. Contact line friction is measured directly by inspecting the velocity of the moving contact line and the microscopic contact angle. It is found that the scaling between contact line friction and viscosity is sublinear, contrary to the prediction of molecular kinetic theory. The disagreement is explained by accounting for the depletion of glycerol in the near-wall region. A correction is proposed, based on multicomponent molecular kinetic theory and the definition of a rescaled interfacial friction coefficient.

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Published 1 February 2024 in Protein Engineering, Design and Selection (doi 10.1093/protein/gzae003):

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

Nandan Haloi*, Shan Huang*, Aaron L Nichols, Eve J Fine, Nicholas J Friesenhahn, Christopher B Marotta, Dennis A Dougherty, Erik Lindahl, Rebecca J Howard, Stephen L Mayo, Henry A Lester

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.

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*These authors contributed equally.

Published 30 January 2024 in eLife (doi 10.7554/eLife.86016.3):

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

Cathrine Bergh, Urška Rovšnik, Rebecca J Howard, Erik Lindahl

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.

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Published 12 January 2024 in The Journal of Chemical Information and Modeling (doi 10.1021/acs.jcim.3c01313):

phbuilder: A tool for efficiently setting up constant pH molecular dynamics simulations in GROMACS

Anton Jansen, Noora Aho, Gerrit Groenhof, Pavel Buslaev*, Berk Hess*

Constant pH molecular dynamics (MD) is a powerful technique that allows the protonation state of residues to change dynamically, thereby enabling the study of pH dependence in a manner that has not been possible before. Recently, a constant pH implementation was incorporated into the GROMACS MD package. Although this implementation provides good accuracy and performance, manual modification and the preparation of simulation input files are required, which can be complicated, tedious, and prone to errors. To simplify and automate the setup process, we present phbuilder, a tool that automatically prepares constant pH MD simulations for GROMACS by modifying the input structure and topology as well as generating the necessary parameter files. phbuilder can prepare constant pH simulations from both initial structures and existing simulation systems, and it also provides functionality for performing titrations and single-site parametrizations of new titratable group types. The tool is freely available at www.gitlab.com/gromacs-constantph. We anticipate that phbuilder will make constant pH simulations easier to set up, thereby making them more accessible to the GROMACS user community.

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*Co-corresponding authors

Published 10 November 2023 in The Journal of Physical Chemistry B (doi 10.1021/acs.jpcb.3c04897):

Coevolution-driven method for efficiently simulating conformational changes in proteins reveals molecular details of ligand effects in the β2AR receptor

Darko Mitrovic, Yue Chen, Antoni Marciniak, Lucie Delemotte

With the advent of AI-powered structure prediction, the scientific community is inching closer to solving protein folding. An unresolved enigma, however, is to accurately, reliably, and deterministically predict alternative conformational states that are crucial for the function of, e.g., transporters, receptors, or ion channels where conformational cycling is innately coupled to protein function. Accurately discovering and exploring all conformational states of membrane proteins has been challenging due to the need to retain atomistic detail while enhancing the sampling along interesting degrees of freedom. The challenges include but are not limited to finding which degrees of freedom are relevant, how to accelerate the sampling along them, and then quantifying the populations of each micro- and macrostate. In this work, we present a methodology that finds relevant degrees of freedom by combining evolution and physics through machine learning and apply it to the conformational sampling of the β2 adrenergic receptor. In addition to predicting new conformations that are beyond the training set, we have computed free energy surfaces associated with the protein’s conformational landscape. We then show that the methodology is able to quantitatively predict the effect of an array of ligands on the β2 adrenergic receptor activation through the discovery of new metastable states not present in the training set. Lastly, we also stake out the structural determinants of activation and inactivation pathway signaling through different ligands and compare them to functional experiments to validate our methodology and potentially gain further insights into the activation mechanism of the β2 adrenergic receptor.

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Published 1 September 2023 in Neuron (doi 10.1016/j.neuron.2023.08.006):

Structure and dynamics of differential ligand binding in the human ρ-type GABA_A receptor

John Cowgill*, Chen Fan*, Nandan Haloi, Yuxuan Zhuang, Rebecca J Howard°, Erik Lindahl°

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.

*Contributed equally
°Corresponding authors

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Published 31 August 2023 in The Protein Journal (doi 10.1007/s10930-023-10137-1):

On the path to optimal alchemistry

Magnus Lundborg, Jack Lidmar, Berk Hess

Alchemical free energy calculations have become a standard and widely used tool, in particular for calculating and comparing binding affinities of drugs. Although methods to compute such free energies have improved significantly over the last decades, the choice of path between the end states of interest is usually still the same as two decades ago. We will show that there is a fundamentally arbitrary, implicit choice of parametrization of this path. To address this, the notion of the length of a path or a metric is required. A metric recently introduced in the context of the accelerated weight histogram method also proves to be very useful here. We demonstrate that this metric can not only improve the efficiency of sampling along a given path, but that it can also be used to improve the actual choice of path. For a set of relevant use cases, the combination of these improvements can increase the efficiency of alchemical free energy calculations by up to a factor 16.

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Published 22 August 2023 in Nature Communications (doi 10.1038/s41467-023-40800-1):

Structural insights into opposing actions of neurosteroids on GABA_A receptors

Dagimhiwat H Legesse, Chen Fan, Jinfeng Teng, Yuxuan Zhuang, Rebecca J Howard, Colleen M Noviello, Erik Lindahl, Ryan E Hibbs

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

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Published online 31 July 2023 in PLOS Computational Biology (doi 10.1371/journal.pcbi.1011255):

Gentle and fast all-atom model refinement to cryo-EM densities via a maximum likelihood approach

Christian Blau, Linnea Yvonnesdotter, Erik Lindahl

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.

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Published online 18 July 2023 in Journal of Chemical Information and Modeling (doi 10.1021/acs.jcim.3c00625):

Understanding drug skin permeation enhancers using molecular dynamics simulations

Christian Wennberg, Magnus Lundborg, Erik Lindahl, Lars Norlén

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

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Published 11 July 2023 in Biophysical Journal (doi 10.1016/j.bpj.2023.05.033):

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

Linnea Yvonnesdotter, Urška Rovšnik, Christian Blau, Marie Lycksell, Rebecca J Howard, Erik Lindahl

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

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Published 5 July 2023 in eLife (doi 10.7554/eLife.84808):

Determinants of sugar-induced influx in the mammalian fructose transporter GLUT5

Sarah E McComas, Tom Reichenbach, Darko Mitrovic, Claudia Alleva, Marta Bonaccorsi, Lucie Delemotte, David Drew

In mammals, glucose transporters (GLUT) control organism-wide blood-glucose homeostasis. In human, this is accomplished by 14 different GLUT isoforms, that transport glucose and other monosaccharides with varying substrate preferences and kinetics. Nevertheless, there is little difference between the sugar-coordinating residues in the GLUT proteins and even the malarial Plasmodium falciparum transporter PfHT1, which is uniquely able to transport a wide range of different sugars. PfHT1 was captured in an intermediate ‘occluded’ state, revealing how the extracellular gating helix TM7b has moved to break and occlude the sugar-binding site. Sequence difference and kinetics indicated that the TM7b gating helix dynamics and interactions likely evolved to enable substrate promiscuity in PfHT1, rather than the sugar-binding site itself. It was unclear, however, if the TM7b structural transitions observed in PfHT1 would be similar in the other GLUT proteins. Here, using enhanced sampling molecular dynamics simulations, we show that the fructose transporter GLUT5 spontaneously transitions through an occluded state that closely resembles PfHT1. The coordination of D-fructose lowers the energetic barriers between the outward- and inward-facing states, and the observed binding mode for D-fructose is consistent with biochemical analysis. Rather than a substrate-binding site that achieves strict specificity by having a high affinity for the substrate, we conclude GLUT proteins have allosterically coupled sugar binding with an extracellular gate that forms the high-affinity transition-state instead. This substrate-coupling pathway presumably enables the catalysis of fast sugar flux at physiological relevant blood-glucose concentrations.

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