Category: Publications

Published 21 June 2023 in eLife (doi 10.7554/eLife.80303):

Interplay between VSD, pore, and membrane lipids in electromechanical coupling in HCN channels

Ahmad Elbahnsi, John Cowgill, Verena Burtscher, Linda Wedemann, Luise Zeckey, Baron Chanda, Lucie Delemotte

Hyperpolarized-activated and cyclic nucleotide-gated (HCN) channels are the only members of the voltage-gated ion channel superfamily in mammals that open upon hyperpolarization, conferring them pacemaker properties that are instrumental for rhythmic firing of cardiac and neuronal cells. Activation of their voltage-sensor domains (VSD) upon hyperpolarization occurs through a downward movement of the S4 helix bearing the gating charges, which triggers a break in the alpha-helical hydrogen bonding pattern at the level of a conserved Serine residue. Previous structural and molecular simulation studies had however failed to capture pore opening that should be triggered by VSD activation, presumably because of a low VSD/pore electromechanical coupling efficiency and the limited timescales accessible to such techniques. Here, we have used advanced modeling strategies, including enhanced sampling molecular dynamics simulations exploiting comparisons between non-domain swapped voltage-gated ion channel structures trapped in closed and open states to trigger pore gating and characterize electromechanical coupling in HCN1. We propose that the coupling mechanism involves the reorganization of the interfaces between the VSD helices, in particular S4, and the pore-forming helices S5 and S6, subtly shifting the balance between hydrophobic and hydrophilic interactions in a ‘domino effect’ during activation and gating in this region. Remarkably, our simulations reveal state-dependent occupancy of lipid molecules at this emergent coupling interface, suggesting a key role of lipids in hyperpolarization-dependent gating. Our model provides a rationale for previous observations and a possible mechanism for regulation of HCN channels by the lipidic components of the membrane.

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Published 23 February 2023 in ACS Chemical Neuroscience (doi 10.1021/acschemneuro.2c00783):

Structural elucidation of ivermectin binding to α7nAChR and the induced channel desensitization

Vasyl Bondarenko, Qiang Chen, Kevin Singewald, Nandan Haloi, Tommy S. Tillman, Rebecca J Howard, Erik Lindahl, Yan Xu, Pei Tang

The α7 nicotinic acetylcholine receptor (α7nAChR) mediates signaling in the central nervous system and cholinergic anti-inflammatory pathways. Ivermectin is a positive allosteric modulator of a full-length α7nAChR and an agonist of the α7nAChR construct containing transmembrane (TMD) and intracellular (ICD) domains, but structural insights of the binding have not previously been determined. Here, combining nuclear magnetic resonance as a primary experimental tool with Rosetta comparative modeling and molecular dynamics simulations, we have revealed details of ivermectin binding to the α7nAChR TMD + ICD and corresponding structural changes in an ivermectin-induced desensitized state. Ivermectin binding was stabilized predominantly by hydrophobic interactions from interfacial residues between adjacent subunits near the extracellular end of the TMD, where the inter-subunit gap was substantially expanded in comparison to the apo structure. The ion-permeation pathway showed a profile distinctly different from the resting-state profile but similar to profiles of desensitized α7nAChR. The ICD also exhibited structural changes, including reorientation of the MX and h3 helices relative to the channel axis. The resulting structures of the α7nAChR TMD + ICD in complex with ivermectin provide opportunities for discovering new modulators of therapeutic potential and exploring the structural basis of cytoplasmic signaling under different α7nAChR functional states.

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Published 14 December 2022 in Journal of General Physiology (doi 10.1085/jgp.202213214):

An α–π transition in S6 shapes the conformational cycle of the bacterial sodium channel NavAb

Koushik Choudhury, Rebecca J Howard, Lucie Delemotte

Voltage-gated sodium channels play an important role in electrical signaling in excitable cells. In response to changes in membrane potential, they cycle between nonconducting and conducting conformations. With recent advances in structural biology, structures of sodium channels have been captured in several distinct conformations, which are thought to represent different functional states. However, it has been difficult to capture the intrinsically transient open state. We recently showed that a proposed open state of the bacterial sodium channel NavMs was not conductive and that a conformational change involving a transition to a π-helix in the pore-lining S6 helix converted this structure into a conducting state. However, the relevance of this structural feature in other sodium channels, and its implications for the broader gating cycle, remained unclear. Here, we propose a comparable open state of another class of bacterial channel from Aliarcobacter butzleri (NavAb) with characteristic pore hydration, ion permeation, and drug binding properties. Furthermore, we show that a π-helix transition can lead to pore opening and that such a conformational change blocks fenestrations in the inner helix bundle. We also discover that a region in the C-terminal domain can undergo a disordering transition proposed to be important for pore opening. These results support a role for a π-helix transition in the opening of NavAb, enabling new proposals for the structural annotation and drug modulation mechanisms in this important sodium channel model.

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Published 8 December 2022 in Proceedings of the National Academy of Sciences of the USA (doi 10.1073/pnas.2210669119):

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

Marie Lycksell, Urška Rovšnik, Anton Hanke, Anne Martel, Rebecca J Howard, Erik Lindahl

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 cryoelectron 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.

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Published 18 October 2022 in Biophysical Journal (doi 10.1016/j.bpj.2022.09.009):

Skin permeability prediction with MD simulation sampling spatial and alchemical reaction coordinates

Magnus Lundborg, Christian Wennberg, Jack Lidmar, Berk Hess, Erik Lindahl, Lars Norlén

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 and 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 skin’s barrier structure to predict skin permeability and the effects of chemical penetration enhancers. In particular, we introduce a new approach to use two-dimensional reaction coordinates in the accelerated weight histogram method, where we combine sampling along spatial coordinates with an alchemical perturbation virtual coordinate. We present predicted properties for 20 permeants, and demonstrate how our approach improves correlation with ex vivo/in vitro skin permeation data. For the compounds included in this study, the obtained log K_Pexp-calc mean square difference was 0.9 cm² h⁻².

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Published 17 October 2022 in Proceedings of the National Academy of Sciences of the USA (doi 10.1073/pnas.2208081119):

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

Yuxuan Zhuang, Colleen M Noviello, Ryan E Hibbs, Rebecca J Howard, Erik Lindahl

The α7 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, cryogenic electron microscopy (cryo-EM) structures of the human α7 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 nonconductive 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 α7-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.

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Published 7 October 2022 in PLoS Computational Biology (doi 10.1371/journal.pcbi.1010583):

Markov state modelling reveals heterogeneous drug-inhibition mechanism of Calmodulin

Annie M Westerlund, Akshay Sridhar, Leo Dahl, Alma Andersson, Anna-Yaroslava Bodnar, Lucie Delemotte

Calmodulin (CaM) is a calcium sensor which binds and regulates a wide range of target-proteins. This implicitly enables the concentration of calcium to influence many downstream physiological responses, including muscle contraction, learning and depression. The antipsychotic drug trifluoperazine (TFP) is a known CaM inhibitor. By binding to various sites, TFP prevents CaM from associating to target-proteins. However, the molecular and state-dependent mechanisms behind CaM inhibition by drugs such as TFP are largely unknown. Here, we build a Markov state model (MSM) from adaptively sampled molecular dynamics simulations and reveal the structural and dynamical features behind the inhibitory mechanism of TFP-binding to the C-terminal domain of CaM. We specifically identify three major TFP binding-modes from the MSM macrostates, and distinguish their effect on CaM conformation by using a systematic analysis protocol based on biophysical descriptors and tools from machine learning. The results show that depending on the binding orientation, TFP effectively stabilizes features of the calcium-unbound CaM, either affecting the CaM hydrophobic binding pocket, the calcium binding sites or the secondary structure content in the bound domain. The conclusions drawn from this work may in the future serve to formulate a complete model of pharmacological modulation of CaM, which furthers our understanding of how these drugs affect signaling pathways as well as associated diseases.

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Available online 27 September 2022 in Physics of Fluids (doi 10.1063/5.0121144):

Asymmetry of wetting and de-wetting on high-friction surfaces originates from the same molecular physics

Michele Pellegrino, Berk Hess

The motion of three-phase contact lines is one of the most relevant research topics of micro- and nano-fluidics. According to many hydrodynamic and molecular models, the dynamics of contact lines is assumed overdamped and dominated by localised liquid-solid friction, entailing the existence of a mobility relation between contact line speed and microscopic contact angle. We present and discuss a set of non-equilibrium atomistic Molecular Dynamics simulations of water nanodroplets spreading on or confined between silica-like walls, showing the existence of the aforementioned relation and its invariance under wetting modes (‘spontaneous’ or ‘forced’). Upon changing the wettability of the walls, it has been noticed that more hydrophilic substrates are easier to wet rather than de-wet; we show how this asymmetry can be automatically captured by a contact line friction model that accounts for the molecular transport between liquid layers. A simple examination of the order and orientation of near-contact-line water molecules corroborates the physical foundation of the model. Lastly, we propose an approach to discriminate between contact line friction models which overcomes the limitations of experimental resolution. This work constitutes a stepping stone towards demystifying wetting dynamics on high-friction hydrophilic substrates and underlines the relevance of contact line friction in modelling the motion of three-phase contact lines.

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Published 21 September 2022 in Journal of Chemical Theory and Computation (doi 10.1021/acs.jctc.2c00516):

Scalable Constant pH Molecular Dynamics in GROMACS

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

Molecular dynamics (MD) computer simulations are used routinely to compute atomistic trajectories of complex systems. Systems are simulated in various ensembles, depending on the experimental conditions one aims to mimic. While constant energy, temperature, volume, and pressure are rather straightforward to model, pH, which is an equally important parameter in experiments, is more difficult to account for in simulations. Although a constant pH algorithm based on the λ-dynamics approach by Brooks and co-workers was implemented in a fork of the GROMACS molecular dynamics program, uptake has been rather limited, presumably due to the poor scaling of that code with respect to the number of titratable sites. To overcome this limitation, we implemented an alternative scheme for interpolating the Hamiltonians of the protonation states that makes the constant pH molecular dynamics simulations almost as fast as a normal MD simulation with GROMACS. In addition, we implemented a simpler scheme, called multisite representation, for modeling side chains with multiple titratable sites, such as imidazole rings. This scheme, which is based on constraining the sum of the λ-coordinates, not only reduces the complexity associated with parametrizing the intramolecular interactions between the sites but also is easily extendable to other molecules with multiple titratable sites. With the combination of a more efficient interpolation scheme and multisite representation of titratable groups, we anticipate a rapid uptake of constant pH molecular dynamics simulations within the GROMACS user community.

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*corresponding authors

Published 6 August 2022 in Nature Communications (doi 10.1038/s41467-022-32212-4):

Structural and dynamic mechanisms of GABA_A receptor modulators with opposing activities

Shaotong Zhu, Akshay Sridhar, Jinfeng Teng, Rebecca J Howard, Erik Lindahl & Ryan E Hibbs

γ-Aminobutyric acid type A (GABA_A) receptors are pentameric ligand-gated ion channels abundant in the central nervous system and are prolific drug targets for treating anxiety, sleep disorders and epilepsy. Diverse small molecules exert a spectrum of effects on γ-aminobutyric acid type A (GABA_A) receptors by acting at the classical benzodiazepine site. They can potentiate the response to GABA, attenuate channel activity, or counteract modulation by other ligands. Structural mechanisms underlying the actions of these drugs are not fully understood. Here we present two high-resolution structures of GABA_A receptors in complex with zolpidem, a positive allosteric modulator and heavily prescribed hypnotic, and DMCM, a negative allosteric modulator with convulsant and anxiogenic properties. These two drugs share the extracellular benzodiazepine site at the α/γ subunit interface and two transmembrane sites at β/α interfaces. Structural analyses reveal a basis for the subtype selectivity of zolpidem that underlies its clinical success. Molecular dynamics simulations provide insight into how DMCM switches from a negative to a positive modulator as a function of binding site occupancy. Together, these findings expand our understanding of how GABA_A receptor allosteric modulators acting through a common site can have diverging activities.

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Published 1 October 2022 in Biochimica et Biophysica Acta – Biomembranes (doi 10.1016/j.bbamem.2022.183994):

Probing effects of the SARS-CoV-2 E protein on membrane curvature and intracellular calcium

Aujan Mehregan, Sergio Pérez-Conesa, Yuxuan Zhuang, Ahmad Elbahnsi, Diletta Pasini, Erik Lindahl, Rebecca J Howard, Chris Ulens, Lucie Delemotte

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.

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Published 15 July 2022 in Nature Communications (doi 10.1038/s41467-022-29594-w):

Cryo-EM structure of the human Kv3.1 channel reveals gating control by the cytoplasmic T1 domain

Gamma Chi, Qiansheng Liang, Akshay Sridhar, John B Cowgill, Kasim Sader, Mazdak Radjainia, Pu Qian, Pablo Castro-Hartmann, Shayla Venkaya, Nanki Kaur Singh, Gavin McKinley, Alejandra Fernandez-Cid, Shubhashish M M Mukhopadhyay, Nicola A Burgess-Brown, Lucie Delemotte, Manuel Covarrubias, Katharina L Dürr

Kv3 channels have distinctive gating kinetics tailored for rapid repolarization in fast-spiking neurons. Malfunction of this process due to genetic variants in the KCNC1 gene causes severe epileptic disorders, yet the structural determinants for the unusual gating properties remain elusive. Here, we present cryo-electron microscopy structures of the human Kv3.1a channel, revealing a unique arrangement of the cytoplasmic tetramerization domain T1 which facilitates interactions with C-terminal axonal targeting motif and key components of the gating machinery. Additional interactions between S1/S2 linker and turret domain strengthen the interface between voltage sensor and pore domain. Supported by molecular dynamics simulations, electrophysiological and mutational analyses, we identify several residues in the S4/S5 linker which influence the gating kinetics and an electrostatic interaction between acidic residues in α6 of T1 and R449 in the pore-flanking S6T helices. These findings provide insights into gating control and disease mechanisms and may guide strategies for the design of pharmaceutical drugs targeting Kv3 channels.

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Published 1 June 2022 in eLife (doi 10.7554/eLife.77672):

Subtype-specific responses of hKv7.4 and hKv7.5 channels to polyunsaturated fatty acids reveal an unconventional modulatory site and mechanism

Damon JA Frampton, Koushik Choudhury, Johan Nikesjö, Lucie Delemotte, Sara I Liin

The KV7.4 and KV7.5 subtypes of voltage-gated potassium channels play a role in important physiological processes such as sound amplification in the cochlea and adjusting vascular smooth muscle tone. Therefore, the mechanisms that regulate KV7.4 and KV7.5 channel function are of interest. Here, we study the effect of polyunsaturated fatty acids (PUFAs) on human KV7.4 and KV7.5 channels expressed in Xenopus oocytes. We report that PUFAs facilitate activation of hKV7.5 by shifting the V50 of the conductance versus voltage (G(V)) curve toward more negative voltages. This response depends on the head group charge, as an uncharged PUFA analogue has no effect and a positively charged PUFA analogue induces positive V50 shifts. In contrast, PUFAs inhibit activation of hKV7.4 by shifting V50 toward more positive voltages. No effect on V50 of hKV7.4 is observed by an uncharged or a positively charged PUFA analogue. Thus, the hKV7.5 channel’s response to PUFAs is analogous to the one previously observed in hKV7.1–7.3 channels, whereas the hKV7.4 channel response is opposite, revealing subtype-specific responses to PUFAs. We identify a unique inner PUFA interaction site in the voltage-sensing domain of hKV7.4 underlying the PUFA response, revealing an unconventional mechanism of modulation of hKV7.4 by PUFAs.

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Published 6 April 2022 in Journal of Fluid Mechanics (doi 10.1017/jfm.2022.219):

Nanoscale sheared droplet: volume-of-fluid, phase-field and no-slip molecular dynamics

Uǧis Lācis, Michele Pellegrino, Johan Sundin, Gustav Amberg, Stéphane Zaleski, Berk Hess, Shervin Bagheri

The motion of the three-phase contact line between two immiscible fluids and a solid surface arises in a variety of wetting phenomena and technological applications. One challenge in continuum theory is the effective representation of molecular motion close to the contact line. Here, we characterize the molecular processes of the moving contact line to assess the accuracy of two different continuum two-phase models. Specifically, molecular dynamics simulations of a two-dimensional droplet between two moving plates are used to create reference data for different capillary numbers and contact angles. We use a simple-point-charge/extended water model. This model provides a very small slip and a more realistic representation of the molecular physics than Lennard-Jones models. The Cahn–Hilliard phase-field model and the volume-of-fluid model are calibrated against the drop displacement from molecular dynamics reference data. It is shown that the calibrated continuum models can accurately capture droplet displacement and droplet break-up for different capillary numbers and contact angles. However, we also observe differences between continuum and atomistic simulations in describing the transient and unsteady droplet behaviour, in particular, close to dynamical wetting transitions. The molecular dynamics of the sheared droplet provide insight into the line friction experienced by the advancing and receding contact lines. The presented results will serve as a stepping stone towards developing accurate continuum models for nanoscale hydrodynamics.

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Published 23 February 2022 in eLife (doi 10.7554/eLife.74773):

Identification of electroporation sites in the complex lipid organization of the plasma membrane

Lea Rems, Xinru Tang, Fangwei Zhao, Sergio Pérez-Conesa, Ilaria Testa, Lucie Delemotte

The plasma membrane of a biological cell is a complex assembly of lipids and membrane proteins, which tightly regulate transmembrane transport. When a cell is exposed to strong electric field, the membrane integrity becomes transiently disrupted by formation of transmembrane pores. This phenomenon termed electroporation is already utilized in many rapidly developing applications in medicine including gene therapy, cancer treatment, and treatment of cardiac arrhythmias. However, the molecular mechanisms of electroporation are not yet sufficiently well understood; in particular, it is unclear where exactly pores form in the complex organization of the plasma membrane. In this study, we combine coarse-grained molecular dynamics simulations, machine learning methods, and Bayesian survival analysis to identify how formation of pores depends on the local lipid organization. We show that pores do not form homogeneously across the membrane, but colocalize with domains that have specific features, the most important being high density of polyunsaturated lipids. We further show that knowing the lipid organization is sufficient to reliably predict poration sites with machine learning. Additionally, by analysing poration kinetics with Bayesian survival analysis we show that poration does not depend solely on local lipid arrangement, but also on membrane mechanical properties and the polarity of the electric field. Finally, we discuss how the combination of atomistic and coarse-grained molecular dynamics simulations, machine learning methods, and Bayesian survival analysis can guide the design of future experiments and help us to develop an accurate description of plasma membrane electroporation on the whole-cell level. Achieving this will allow us to shift the optimization of electroporation applications from blind trial-and-error approaches to mechanistic-driven design.

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Published 4 January 2022 in Biophysical Journal (doi 10.1016/j.bpj.2021.12.010):

An open state of a voltage-gated sodium channel involving a π-helix and conserved pore-facing asparagine

Koushik Choudhury, Marina A Kasimova, Sarah McComas, Rebecca J Howard, Lucie Delemotte

Voltage-gated sodium (Nav) channels play critical roles in propagating action potentials and otherwise manipulating ionic gradients in excitable cells. These channels open in response to membrane depolarization, selectively permeating sodium ions until rapidly inactivating. Structural characterization of the gating cycle in this channel family has proved challenging, particularly due to the transient nature of the open state. A structure from the bacterium Magnetococcus marinus Nav (NavMs) was initially proposed to be open, based on its pore diameter and voltage-sensor conformation. However, the functional annotation of this model, and the structural details of the open state, remain disputed. In this work, we used molecular modeling and simulations to test possible open-state models of NavMs. The full-length experimental structure, termed here the α-model, was consistently dehydrated at the activation gate, indicating an inability to conduct ions. Based on a spontaneous transition observed in extended simulations, and sequence/structure comparison to other Nav channels, we built an alternative π-model featuring a helix transition and the rotation of a conserved asparagine residue into the activation gate. Pore hydration, ion permeation, and state-dependent drug binding in this model were consistent with an open functional state. This work thus offers both a functional annotation of the full-length NavMs structure and a detailed model for a stable Nav open state, with potential conservation in diverse ion-channel families.

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Published December 2021 in Journal of Biological Chemistry (doi 10.1016/j.jbc.2021.101355):

A missense mutation converts the Na⁺,K⁺-ATPase into an ion channel and causes therapy-resistant epilepsy

Sofia Ygberg, Evgeny E Akkuratov*, Rebecca J Howard*, Fulya Taylan, Daniel C Jans*, Dhani R Mahato, Adriana Katz, Paula F Kinoshita, Benjamin Portal, Inger Nennesmo, Maria Lindskog, Steven JD Karlish, Magnus Andersson*, Anna Lindstrand, Hjalmar Brismar*, Anita Aperia*

*Current and former members of the SciLifeLab biophysics community

The ion pump Na⁺,K⁺-ATPase is a critical determinant of neuronal excitability; however, its role in the etiology of diseases of the central nervous system (CNS) is largely unknown. We describe here the molecular phenotype of a Trp931Arg mutation of the Na⁺,K⁺-ATPase catalytic α1 subunit in an infant diagnosed with therapy-resistant lethal epilepsy. In addition to the pathological CNS phenotype, we also detected renal wasting of Mg²⁺. We found that membrane expression of the mutant α1 protein was low, and ion pumping activity was lost. Arginine insertion into membrane proteins can generate water-filled pores in the plasma membrane, and our molecular dynamic (MD) simulations of the principle states of Na⁺,K⁺-ATPase transport demonstrated massive water inflow into mutant α1 and destabilization of the ion-binding sites. MD simulations also indicated that a water pathway was created between the mutant arginine residue and the cytoplasm, and analysis of oocytes expressing mutant α1 detected a nonspecific cation current. Finally, neurons expressing mutant α1 were observed to be depolarized compared with neurons expressing wild-type protein, compatible with a lowered threshold for epileptic seizures. The results imply that Na⁺,K⁺-ATPase should be considered a neuronal locus minoris resistentia in diseases associated with epilepsy and with loss of plasma membrane integrity.

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Published 15 November 2021 in Journal of Chemical Information & Modeling (doi 10.1021/acs.jcim.1c00826):

Allosteric effect of nanobody binding on ligand-specific active states of the β2 adrenergic receptor

Yue Chen, Oliver Fleetwood, Sergio Pérez-Conesa, Lucie Delemotte

Nanobody binding stabilizes G-protein-coupled receptors (GPCR) in a fully active state and modulates their affinity for bound ligands. However, the atomic-level basis for this allosteric regulation remains elusive. Here, we investigate the conformational changes induced by the binding of a nanobody (Nb80) on the active-like β2 adrenergic receptor (β2AR) via enhanced sampling molecular dynamics simulations. Dimensionality reduction analysis shows that Nb80 stabilizes structural features of the β2AR with an ∼14 Å outward movement of transmembrane helix 6 and a close proximity of transmembrane (TM) helices 5 and 7, and favors the fully active-like conformation of the receptor, independent of ligand binding, in contrast to the conditions under which no intracellular binding partner is bound, in which case the receptor is only stabilized in an intermediate-active state. This activation is supported by the residues located at hotspots located on TMs 5, 6, and 7, as shown by supervised machine learning methods. Besides, ligand-specific subtle differences in the conformations assumed by intracellular loop 2 and extracellular loop 2 are captured from the trajectories of various ligand-bound receptors in the presence of Nb80. Dynamic network analysis further reveals that Nb80 binding triggers tighter and stronger local communication networks between the Nb80 and the ligand-binding sites, primarily involving residues around ICL2 and the intracellular end of TM3, TM5, TM6, as well as ECL2, ECL3, and the extracellular ends of TM6 and TM7. In particular, we identify unique allosteric signal transmission mechanisms between the Nb80-binding site and the extracellular domains in conformations modulated by a full agonist, BI167107, and a G-protein-biased partial agonist, salmeterol, involving mainly TM1 and TM2, and TM5, respectively. Altogether, our results provide insights into the effect of intracellular binding partners on the GPCR activation mechanism, which should be taken into account in structure-based drug discovery.

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Published 15 October 2021 in eLife (doi 10.7554/eLife.68369):

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

Cathrine Bergh, Stephanie A Heusser, Rebecca Howard, Erik Lindahl

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

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Published 14 September 2021 in Proceedings of the National Academy of Sciences of the USA (doi 10.1073/pnas.2108006118):

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

Marie Lycksell, Urška Rovšnik, Cathrine Bergh, Nicolai T. Johansen, Anne Martel, Lionel Porcar, Lise Arleth, Rebecca J Howard, Erik Lindahl

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.

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Published online August 31, 2021 in Journal of Investigative Dermatology (doi https://doi.org/10.1016/j.jid.2021.06.037):

The Skin’s Barrier: A Cryo-EM Based Overview of its Architecture and Stepwise Formation

Lars Norlén, Magnus Lundborg, Christian Wennberg, Ali Narangifard, Bertil Daneholt

A major role of the skin is to serve as a barrier toward the environment. The skin’s permeability barrier consists of a lipid structure positioned in the stratum corneum. Recent progress in high-resolution cryo-electron microscopy (cryo-EM) has allowed for elucidation of the architecture of the skin’s barrier and its stepwise formation process representing the final stage of epidermal differentiation. In this review, we present an overview of the skin’s barrier structure and its formation process, as evidenced by cryo-EM.

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Published 17 August 2021 in Proceedings of the National Academy of Sciences of the USA (doi 10.1073/pnas.2025320118):

Functional cross-talk between phosphorylation and disease-causing mutations in the cardiac sodium channel Na_v1.5

Iacopo Galleano, Hendrik Harms, Koushik Choudhury, Keith Khoo, Lucie Delemotte, Stephan A Pless

The voltage-gated sodium channel Na_v1.5 initiates the cardiac action potential. Alterations of its activation and inactivation properties due to mutations can cause severe, life-threatening arrhythmias. Yet despite intensive research efforts, many functional aspects of this cardiac channel remain poorly understood. For instance, Na_v1.5 undergoes extensive posttranslational modification in vivo, but the functional significance of these modifications is largely unexplored, especially under pathological conditions. This is because most conventional approaches are unable to insert metabolically stable posttranslational modification mimics, thus preventing a precise elucidation of the contribution by these modifications to channel function. Here, we overcome this limitation by using protein semisynthesis of Na_v1.5 in live cells and carry out complementary molecular dynamics simulations. We introduce metabolically stable phosphorylation mimics on both wild-type (WT) and two pathogenic long-QT mutant channel backgrounds and decipher functional and pharmacological effects with unique precision. We elucidate the mechanism by which phosphorylation of Y1495 impairs steady-state inactivation in WT Na_v1.5. Surprisingly, we find that while the Q1476R patient mutation does not affect inactivation on its own, it enhances the impairment of steady-state inactivation caused by phosphorylation of Y1495 through enhanced unbinding of the inactivation particle. We also show that both phosphorylation and patient mutations can impact Na_v1.5 sensitivity toward the clinically used antiarrhythmic drugs quinidine and ranolazine, but not flecainide. The data highlight that functional effects of Na_v1.5 phosphorylation can be dramatically amplified by patient mutations. Our work is thus likely to have implications for the interpretation of mutational phenotypes and the design of future drug regimens.

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Available online 1 July 2021 in Life Science Alliance (doi 10.26508/lsa.202101011):

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

Urška Rovšnik, Yuxuan Zhuang, Björn O Forsberg, Marta Carroni, Linnea Yvonnesdotter, Rebecca J Howard, Erik Lindahl 

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 side chain rearrangements at the subunit/domain interface, particularly involving functionally important residues in the β1–β2 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 allow us to define distinct protonation and activation steps in pH-stimulated conformational cycling in GLIC, including interfacial rearrangements largely conserved in the pentameric channel family.

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Available online 3 July 2021 in the Journal of Molecular Biology (doi 10.1016/j.jmb.2021.167128):

Elephants in the dark: insights and incongruities in pentameric ligand-gated ion channel models

Rebecca J Howard

The superfamily of pentameric ligand-gated ion channels (pLGICs) comprises key players in electrochemical signal transduction across evolution, including historic model systems for receptor allostery and targets for drug development. Accordingly, structural studies of these channels have steadily increased, and now approach 250 depositions in the protein data bank. This review contextualizes currently available structures in the pLGIC family, focusing on morphology, ligand binding, and gating in three model subfamilies: the prokaryotic channel GLIC, the cation-selective nicotinic acetylcholine receptor, and the anion-selective glycine receptor. Common themes include the challenging process of capturing and annotating channels in distinct functional states; partially conserved gating mechanisms, including remodeling at the extracellular/transmembrane-domain interface; and diversity beyond the protein level, arising from posttranslational modifications, ligands, lipids, and signaling partners. Interpreting pLGIC structures can be compared to describing an elephant in the dark, relying on touch alone to comprehend the many parts of a monumental beast: each structure represents a snapshot in time under specific experimental conditions, which must be understood and integrated with further structure, function, and simulations data to build a comprehensive model, and understand how one channel may fundamentally differ from another.

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Available online 19 June 2021 in the Journal of Biological Chemistry (doi 10.1016/j.jbc.2021.100899):

Regulation of a pentameric ligand-gated ion channel by a semi-conserved cationic-lipid binding site

Akshay Sridhar*, Sarah CR Lummis*, Diletta Pasini, Aujan Mehregan, Marijke Brams, Kumiko Kambara, Daniel Bertrand, Erik Lindahl, Rebecca J Howard, Chris Ulens

Pentameric ligand-gated ion channels (pLGICs) are crucial mediators of electrochemical signal transduction in various organisms from bacteria to humans. Lipids play an important role in regulating pLGIC function, yet the structural bases for specific pLGIC-lipid interactions remain poorly understood. The bacterial channel ELIC recapitulates several properties of eukaryotic pLGICs, including activation by the neurotransmitter GABA, and binding and modulation by lipids, offering a simplified model system for structure-function relationship studies. In this study, functional effects of non-canonical amino acid substitution of a potential lipid-interacting residue (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-π binding. Polar contacts from other regions of the protein, particularly M3 residue Q264, further support lipid binding via headgroup ester linkages. Aromatic residues were identified at analogous sites in a handful of eukaryotic family members, including the human GABA_A receptor ε subunit, suggesting conservation of relevant interactions in other evolutionary branches. Further mutagenesis experiments indicated that mutations at this site in ε-containing GABA_A receptors can change the apparent affinity of the agonist response to GABA, suggesting a potential role of this site in channel gating. In conclusion, this work details type-specific lipid interactions, which adds to our growing understanding of how lipids modulate pLGICs.

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*equal contributions