Publications

GPCRs on Acid

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

Read the full publication here.

Publications

Computational Opening

Published 10 January 2025 in Science Advances (doi 10.1038/s41467-024-51904-7):

Adaptive sampling-based structural prediction reveals opening of a GABAA 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.

Read the full publication here.

News

Grattis Dr Chen

Members of Molecular Biophysics Stockholm joined family and friends in celebrating Yue Chen‘s successful spikning and defense of her PhD in Applied PhysicsDeciphering Activation Mechanisms of β2 Adrenergic and Dopamine D2 Receptors Using Enhanced Sampling Simulations. Yue nailed her thesis at SciLifeLab on 3 December, and defended it at the Karolinska Institute on 18 December, with Professor Phil Biggin (University of Oxford) as opponent. Professor Lucie Delemotte led a toast to her advisee of four years as she prepares to begin a postdoctoral fellowship abroad.

Publications

MSMs for alpha-7s

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.

Read the full publication here.

Publications

Steroids Here and There

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

Divergent mechanisms of steroid inhibition in the human ρ1 GABAA receptor

Chen Fan,* John Cowgill,* Rebecca J HowardErik Lindahl°

ρ-type γ-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 β-estradiol and pregnenolone sulfate negatively modulate the function of ρ1 GABAA 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 GABAA 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

Read the full publication here.

Publications

Across the MDverse

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

Read the full publication here.

Publications

Deactivation From Within

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.

Read the full publication here.

Publications

Charging Through Skin

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

Read the full publication here.

News

Grattis Dr Pellegrino

Members of Molecular Biophysics Stockholm joined family and friends in celebrating Michele Pellegrino‘s successful spikning and defense of his PhD in Applied PhysicsBridging the Molecular and the Continuous Pictures of Wetting Dynamics on Hydrophilic Surfaces. Michele nailed his thesis at SciLifeLab on 3 June, and defended it at KTH Royal Institute of Technology on 13 June, with Professor James E Sprittles (University of Warwick) as opponent. Professor Berk Hess led a toast to his advisee of over four years as he prepares to begin a postdoctoral fellowship abroad.

News

Grattis Dr Jansen

Members of Molecular Biophysics Stockholm joined family and friends in celebrating Anton Jansen‘s successful spikning and defense of his PhD in Applied PhysicsConstant-pH Molecular Dynamics and Applications. Anton nailed his thesis at SciLifeLab on 24 May, and defended it at KTH Royal Institute of Technology on 31 May, with Professor Ilpo Vattulainen (University of Helsinki, Finland) as opponent. Professor Berk Hess led a toast to his advisee of nearly four years.

Publications

Transporter Physics Meets Genomics

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.

Read the full publication here.

Publications

Allosteric Contacts of Cholesterol

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.

Read the full publication here.

News

Grattis Dr Mitrovic

Members of Molecular Biophysics Stockholm joined family and friends in celebrating Darko Mitrovic‘s successful spikning and defense of his PhD in Applied PhysicsCombining Evolution and Physics through Machine Learning to Decipher Molecular Mechanisms. Darko nailed his thesis at SciLifeLab on 2 May, and defended it at KTH Royal Institute of Technology on 15 May, with Professor Gerhard Hummer (Max Planck Institute, Frankfurt, Germany) as opponent. Associate Professor Lucie Delemotte led a toast to her advisee of nearly four years, as he transitions to a postdoctoral fellowship.

Publications

TRP on Acid

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

Read the full publication here.

Publications

A Correction to Droplet Friction

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.

Read the full publication here.

Publications

Simulating Biosensors

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.

Read the full publication here.

*These authors contributed equally.

News

Grattis Dr Rovšnik

Members of Molecular Biophysics Stockholm joined family and friends in celebrating Urška Rovšnik‘s successful spikning and defense of her PhD in Biochemistry, Structural transitions of proton-gated ion channels: Involving pH sensing, heterogeneity and lipid interactions. Urška nailed her thesis at SciLifeLab on 15 January, and defended it there on 31 January, with Dr Hugues Nury (Institut de Biologie Structurale, Grenoble, France) as opponent. Professor Erik Lindahl and Ligand-Gated Ion Channels team-lead Reba Howard led a toast to their advisee of over five years, as she prepares for a postdoctoral position at Gothenburg University.

Publications

Multidisciplinary Lipid Discovery

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.

Read the full publication here.

Publications

Automating CpHMD

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.

Read the full publication here.

*Co-corresponding authors

News

Grattis Dr McComas

Members of Molecular Biophysics Stockholm joined family and friends in celebrating Sarah McComas‘ successful spikning and defense of her PhD in Biochemistry, The molecular basis for substrate recognition and gating in sugar transporters. Sarah nailed her thesis at SciLifeLab on 27 November, and defended it 8 December at Stockholm University, with Professor Philip Biggin (University of Oxford) as opponent. Along with her supervisor Professor David Drew, the Membrane Protein Modeling team led a toast to their colleague of over five years.

Publications

Adrenergic AI

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.

Read the full publication here.

News

Grattis Dr Zhuang

Members and friends of Molecular Biophysics Stockholm celebrated Yuxuan Zhuang‘s successful spikning and defense of his PhD from Stockholm University in Biophysics, Simulating Functional Cycles and Drug Modulation in Ligand-Gated Ion Channels. Yuxuan nailed his thesis on 6 October, and defended it 19 October at SciLifeLab, with Professor Grace Brannigan (Rutgers University – Camden, NJ) as opponent. Professor Erik Lindahl and Integrative Structural Biology team-lead Reba Howard led a toast to their advisee of over five years, as he heads to a Wallenberg Foundation Postdoctoral Scholarship at Stanford University.

Publications

Resolving Rho1

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 GABAA 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

Read the full publication here.

Publications

Alchemical Atlas

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.

Read the full publication here.

Publications

New Sites for Steroids

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

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

Read the full publication here.