Category: Publications

Published 1 August 2025 in Nature Communications (doi 10.1038/s41467-025-61932-6):

Cryo-EM structures of ρ1 GABA_A receptors with antagonist and agonist drugs

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

The family of ρ-type GABA_A receptors includes potential therapeutic targets in several neurological conditions, and features distinctive pharmacology compared to other subtypes. Here we report four cryo-EM structures with previously unresolved ligands, electrophysiology recordings, and molecular dynamics simulations to characterize binding and conformational impact of the drugs THIP (a non-opioid analgesic), CGP36742 (a phosphinic acid) and GABOB (an anticonvulsant) on a human ρ1 GABA_A receptor. A distinctive binding pose of THIP in ρ1 versus α4β3δ GABA_A receptors offers a rationale for its inverse effects on these subtypes. CGP36742 binding is similar to the canonical ρ-type inhibitor TPMPA, supporting a shared mechanism of action among phosphinic acids. Binding of GABOB is similar to GABA, but produces a mixture of partially-locked and desensitized states, likely underlying weaker agonist activity. Together, these results elucidate interactions of a ρ-type GABA_A receptor with therapeutic drugs, offering mechanistic insights and a basis for further pharmaceutical development.

°Co-corresponding authors

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Published 3 July 2025 in Nature Communications (doi 10.1038/s41467-025-61447-0):

A single main-chain hydrogen bond required to keep GABA_A receptors closed

Cecilia M Borghese, Jason D Galpin, Samuel Eriksson Lidbrink, Yuxuan Zhuang, Netrang G Desai, Rebecca J Howard, Erik Lindahl, Christopher A Ahern, Marcel P Goldschen-Ohm

GABA_A receptors (GABA_ARs) are the primary inhibitory neurotransmitter receptors throughout the central nervous system. Genetic mutations causing their dysfunction are related to a broad spectrum of human disorders such as epilepsy, neurodevelopment and intellectual disability, autism spectrum disorder, schizophrenia, and depression. GABA_ARs are also important drug targets for anxiolytics, anticonvulsants, antidepressants, and anesthetics. Despite significant progress in understanding their three-dimensional structure, a critical gap remains in determining the molecular basis for channel gating. We recently identified mutations in the M2-M3 linkers that suggest linker flexibility has asymmetric subunit-specific correlations with channel opening. Here we use non-canonical amino acids (ncAAs) to investigate the role of main-chain H-hydrogen bonds (H-bonds) that may stabilize the M2-M3 linkers. We show that a single main-chain H-bond within the β2 subunit M2-M3 linker inhibits pore opening and is required to keep the unliganded channel closed. Furthermore, breaking this H-bond accounts for approximately one third of the energy used to open the channel during activation by GABA. In contrast, the analogous H-bond in the α1 subunit has no effect on gating. Our molecular simulations support the idea that channel opening involves the state-dependent breakage/disruption of a specific main-chain H-bond within the β2 subunit M2-M3 linker.

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Published 27 June 2025 in PLoS Computational Biology (doi 10.1371/journal.pcbi.1013187):

Resolving the conformational ensemble of a membrane protein by integrating small-angle scattering with AlphaFold

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

The function of a protein is enabled by its conformational landscape. For non-rigid proteins, a complete characterization of this landscape requires understanding the protein’s structure in all functional states, the stability of these states under target conditions, and the transition pathways between them. Several strategies have recently been developed to drive the machine learning algorithm AlphaFold2 (AF) to sample multiple conformations, but it is more challenging to a priori predict what states are stabilized in particular conditions and how the transition occurs. Here, we combine AF sampling with small-angle scattering curves to obtain a weighted conformational ensemble of functional states under target environmental conditions. We apply this to the pentameric ion channel GLIC using small-angle neutron scattering (SANS) curves, and identify apparent closed and open states. By comparing experimental SANS data under resting and activating conditions, we can quantify the subpopulation of closed channels that open upon activation, matching both experiments and extensive simulation sampling using Markov state models. The predicted closed and open states closely resemble crystal structures determined under resting and activating conditions respectively, and project to predicted basins in free energy landscapes calculated from the Markov state models. Further, without using any structural information, the AF sampling also correctly captures intermediate conformations and projects onto the transition pathway resolved in the extensive sampling. This combination of machine learning algorithms and low-dimensional experimental data appears to provide an efficient way to predict not only stable conformations but also accurately sample the transition pathways several orders of magnitude faster than simulation-based sampling.

°Co-corresponding authors

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Version of Record 30 April 2025 in eLife (doi 10.7554/eLife.104237.3):

Engineering cardiolipin binding to an artificial membrane protein reveals determinants for lipid-mediated stabilization

Mia L Abramsson, Robin A Corey,° Jan L Skerle, Louise J Persson, Olivia Andén, Abraham O Oluwole, Rebecca J Howard, Erik Lindahl, Carol V Robinson, Kvido Strisovsky, Erik G Marklund, David Drew, Phillip J Stansfeld,° Michael Landreh°

Integral membrane proteins carry out essential functions in the cell, and their activities are often modulated by specific protein-lipid interactions in the membrane. Here, we elucidate the intricate role of cardiolipin (CDL), a regulatory lipid, as a stabilizer of membrane proteins and their complexes. Using the in silico-designed model protein TMHC4_R (ROCKET) as a scaffold, we employ a combination of molecular dynamics simulations and native mass spectrometry to explore the protein features that facilitate preferential lipid interactions and mediate stabilization. We find that the spatial arrangement of positively charged residues as well as local conformational flexibility are factors that distinguish stabilizing from non-stabilizing CDL interactions. However, we also find that even in this controlled, artificial system, a clear-cut distinction between binding and stabilization is difficult to attain, revealing that overlapping lipid contacts can partially compensate for the effects of binding site mutations. Extending our insights to naturally occurring proteins, we identify a stabilizing CDL site within the E. coli rhomboid intramembrane protease GlpG and uncover its regulatory influence on enzyme substrate preference. In this work, we establish a framework for engineering functional lipid interactions, paving the way for the design of proteins with membrane-specific properties or functions.

°Co-corresponding authors

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Published 11 April 2025 in Science Advances (doi 10.1126/sciadv.adr0797):

Discovering cryptic pocket opening and binding of a stimulant derivative in a vestibular site of the 5-HT_3A receptor

Nandan Haloi, Emelia Karlsson, Mark Delarue, Rebecca J Howard,° Erik Lindahl°

A diverse set of modulators, including stimulants and anesthetics, regulates ion channel function in our nervous system. However, structures of ligand-bound complexes can be difficult to capture by experimental methods, particularly when binding is dynamic. Here, we used computational methods and electrophysiology to identify a possible bound state of a modulatory stimulant derivative in a cryptic vestibular pocket of a mammalian serotonin-3 receptor. We first applied a molecular dynamics simulation–based goal-oriented adaptive sampling method to identify possible open-pocket conformations, followed by Boltzmann docking that combines traditional docking with Markov state modeling. 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.

°Co-corresponding authors

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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 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 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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