Mechanism and cellular function of direct membrane binding by the ESCRT and ERES-associated Ca2+-sensor ALG-2.

Apoptosis linked Gene-2 (ALG-2) is a multifunctional intracellular Ca2+ sensor and the archetypal member of the penta-EF hand protein family. ALG-2 functions in the repair of damage to both the plasma and lysosome membranes and in COPII-dependent budding at endoplasmic reticulum exit sites (ERES). In the presence of Ca2+, ALG-2 binds to ESCRT-I and ALIX in membrane repair and to SEC31A at ERES. ALG-2 also binds directly to acidic membranes in the presence of Ca2+ by a combination of electrostatic and hydrophobic interactions. By combining giant unilamellar vesicle-based experiments and molecular dynamics simulations, we show that charge-reversed mutants of ALG-2 at these locations disrupt membrane recruitment. ALG-2 membrane binding mutants have reduced or abrogated ERES localization in response to Thapsigargin-induced Ca2+ release but still localize to lysosomes following lysosomal Ca2+ release. In vitro reconstitution shows that the ALG-2 membrane-binding defect can be rescued by binding to ESCRT-I. These data thus reveal the nature of direct Ca2+-dependent membrane binding and its interplay with Ca2+-dependent protein binding in the cellular functions of ALG-2.

Three-step docking by WIPI2, ATG16L1, and ATG3 delivers LC3 to the phagophore.

The covalent attachment of ubiquitin-like LC3 proteins (microtubule-associated proteins 1A/1B light chain 3) prepares the autophagic membrane for cargo recruitment. We resolve key steps in LC3 lipidation by combining molecular dynamics simulations and experiments in vitro and in cellulo. We show how the E3-like ligaseautophagy-related 12 (ATG12)-ATG5-ATG16L1 in complex with the E2-like conjugase ATG3 docks LC3 onto the membrane in three steps by (i) the phosphatidylinositol 3-phosphate effector protein WD repeat domain phosphoinositide-interacting protein 2 (WIPI2), (ii) helix α2 of ATG16L1, and (iii) a membrane-interacting surface of ATG3. Phosphatidylethanolamine (PE) lipids concentrate in a region around the thioester bond between ATG3 and LC3, highlighting residues with a possible role in the catalytic transfer of LC3 to PE, including two conserved histidines. In a near-complete pathway from the initial membrane recruitment to the LC3 lipidation reaction, the three-step targeting of the ATG12-ATG5-ATG16L1 machinery establishes a high level of regulatory control.

Mechanism and cellular function of direct membrane binding by the ESCRT and ERES-associated Ca2+-sensor ALG-2.

Apoptosis Linked Gene-2 (ALG-2) is a multifunctional intracellular Ca2+ sensor and the archetypal member of the penta-EF hand protein family. ALG-2 functions in the repair of damage to both the plasma and lysosome membranes and in COPII-dependent budding at endoplasmic reticulum exit sites (ERES). In the presence of Ca2+, ALG-2 binds to ESCRT-I and ALIX in membrane repair and to SEC31A at ERES. ALG-2 also binds directly to acidic membranes in the presence of Ca2+ by a combination of electrostatic and hydrophobic interactions. By combining GUV-based experiments and molecular dynamics simulations, we show that charge-reversed mutants of ALG-2 at these locations disrupt membrane recruitment. ALG-2 membrane binding mutants have reduced or abrogated ERES localization in response to Thapsigargin-induced Ca2+ release but still localize to lysosomes following lysosomal Ca2+ release. In vitro reconstitution shows that the ALG-2 membrane-binding defect can be rescued by binding to ESCRT-I. These data thus reveal the nature of direct Ca2+-dependent membrane binding and its interplay with Ca2+-dependent protein binding in the cellular functions of ALG-2.

Membrane curvature sensing and stabilization by the autophagic LC3 lipidation machinery.

How the highly curved phagophore membrane is stabilized during autophagy initiation is a major open question in autophagosome biogenesis. Here, we use in vitro reconstitution on membrane nanotubes and molecular dynamics simulations to investigate how core autophagy proteins in the LC3 (Microtubule-associated proteins 1A/1B light chain 3) lipidation cascade interact with curved membranes, providing insight into their possible roles in regulating membrane shape during autophagosome biogenesis. ATG12(Autophagy-related 12)-ATG5-ATG16L1 was up to 100-fold enriched on highly curved nanotubes relative to flat membranes. At high surface density, ATG12-ATG5-ATG16L1 binding increased the curvature of the nanotubes. While WIPI2 (WD repeat domain phosphoinositide-interacting protein 2) binding directs membrane recruitment, the amphipathic helix α2 of ATG16L1 is responsible for curvature sensitivity. Molecular dynamics simulations revealed that helix α2 of ATG16L1 inserts shallowly into the membrane, explaining its curvature-sensitive binding to the membrane. These observations show how the binding of the ATG12-ATG5-ATG16L1 complex to the early phagophore rim could stabilize membrane curvature and facilitate autophagosome growth.

Gain-of-function mutations in KCNK3 cause a developmental disorder with sleep apnea.

Sleep apnea is a common disorder that represents a global public health burden. KCNK3 encodes TASK-1, a K+ channel implicated in the control of breathing, but its link with sleep apnea remains poorly understood. Here we describe a new developmental disorder with associated sleep apnea (developmental delay with sleep apnea, or DDSA) caused by rare de novo gain-of-function mutations in KCNK3. The mutations cluster around the 'X-gate', a gating motif that controls channel opening, and produce overactive channels that no longer respond to inhibition by G-protein-coupled receptor pathways. However, despite their defective X-gating, these mutant channels can still be inhibited by a range of known TASK channel inhibitors. These results not only highlight an important new role for TASK-1 K+ channels and their link with sleep apnea but also identify possible therapeutic strategies.

Structural basis for cannabinoid-induced potentiation of alpha1-glycine receptors in lipid nanodiscs.

Nociception and motor coordination are critically governed by glycine receptor (GlyR) function at inhibitory synapses. Consequentially, GlyRs are attractive targets in the management of chronic pain and in the treatment of several neurological disorders. High-resolution mechanistic details of GlyR function and its modulation are just emerging. While it has been known that cannabinoids such as Δ9-tetrahydrocannabinol (THC), the principal psychoactive constituent in marijuana, potentiate GlyR in the therapeutically relevant concentration range, the molecular mechanism underlying this effect is still not understood. Here, we present Cryo-EM structures of full-length GlyR reconstituted into lipid nanodisc in complex with THC under varying concentrations of glycine. The GlyR-THC complexes are captured in multiple conformational states that reveal the basis for THC-mediated potentiation, manifested as different extents of opening at the level of the channel pore. Taken together, these structural findings, combined with molecular dynamics simulations and functional analysis, provide insights into the potential THC binding site and the allosteric coupling to the channel pore.

Water Nanoconfined in a Hydrophobic Pore: Molecular Dynamics Simulations of Transmembrane Protein 175 and the Influence of Water Models.

Water molecules within biological ion channels are in a nanoconfined environment and therefore exhibit behaviors which differ from that of bulk water. Here, we investigate the phenomenon of hydrophobic gating, the process by which a nanopore may spontaneously dewet to form a "vapor lock" if the pore is sufficiently hydrophobic and/or narrow. This occurs without steric occlusion of the pore. Using molecular dynamics simulations with both rigid fixed-charge and polarizable (AMOEBA) force fields, we investigate this wetting/dewetting behavior in the transmembrane protein 175 ion channel. We examine how a range of rigid fixed-charge and polarizable water models affect wetting/dewetting in both the wild-type structure and in mutants chosen to cover a range of nanopore radii and pore-lining hydrophobicities. Crucially, we find that the rigid fixed-charge water models lead to similar wetting/dewetting behaviors, but that the polarizable water model resulted in an increased wettability of the hydrophobic gating region of the pore. This has significant implications for molecular simulations of nanoconfined water, as it implies that polarizability may need to be included if we are to gain detailed mechanistic insights into wetting/dewetting processes. These findings are of importance for the design of functionalized biomimetic nanopores (e.g., sensing or desalination) as well as for furthering our understanding of the mechanistic processes underlying biological ion channel function.

Norfluoxetine inhibits TREK-2 K2P channels by multiple mechanisms including state-independent effects on the selectivity filter gate.

The TREK subfamily of two-pore domain K+ (K2P) channels are inhibited by fluoxetine and its metabolite, norfluoxetine (NFx). Although not the principal targets of this antidepressant, TREK channel inhibition by NFx has provided important insights into the conformational changes associated with channel gating and highlighted the role of the selectivity filter in this process. However, despite the availability of TREK-2 crystal structures with NFx bound, the precise mechanisms underlying NFx inhibition remain elusive. NFx has previously been proposed to be a state-dependent inhibitor, but its binding site suggests many possible ways in which this positively charged drug might inhibit channel activity. Here we show that NFx exerts multiple effects on single-channel behavior that influence both the open and closed states of the channel and that the channel can become highly activated by 2-APB while remaining in the down conformation. We also show that the inhibitory effects of NFx are unrelated to its positive charge but can be influenced by agonists which alter filter stability, such as ML335, as well as by an intrinsic voltage-dependent gating process within the filter. NFx therefore not only inhibits channel activity by altering the equilibrium between up and down conformations but also can directly influence filter gating. These results provide further insight into the complex allosteric mechanisms that modulate filter gating in TREK K2P channels and highlight the different ways in which filter gating can be regulated to permit polymodal regulation.

Molecular Simulations of Hydrophobic Gating of Pentameric Ligand Gated Ion Channels: Insights into Water and Ions.

Ion channels are proteins which form gated nanopores in biological membranes. Many channels exhibit hydrophobic gating, whereby functional closure of a pore occurs by local dewetting. The pentameric ligand gated ion channels (pLGICs) provide a biologically important example of hydrophobic gating. Molecular simulation studies comparing additive vs polarizable models indicate predictions of hydrophobic gating are robust to the model employed. However, polarizable models suggest favorable interactions of hydrophobic pore-lining regions with chloride ions, of relevance to both synthetic carriers and channel proteins. Electrowetting of a closed pLGIC hydrophobic gate requires too high a voltage to occur physiologically but may inform designs for switchable nanopores. Global analysis of ∼200 channels yields a simple heuristic for structure-based prediction of (closed) hydrophobic gates. Simulation-based analysis is shown to provide an aid to interpretation of functional states of new channel structures. These studies indicate the importance of understanding the behavior of water and ions within the nanoconfined environment presented by ion channels.

The MscS-like channel YnaI has a gating mechanism based on flexible pore helices.

The mechanosensitive channel of small conductance (MscS) is the prototype of an evolutionarily diversified large family that fine-tunes osmoregulation but is likely to fulfill additional functions. Escherichia coli has six osmoprotective paralogs with different numbers of transmembrane helices. These helices are important for gating and sensing in MscS but the role of the additional helices in the paralogs is not understood. The medium-sized channel YnaI was extracted and delivered in native nanodiscs in closed-like and open-like conformations using the copolymer diisobutylene/maleic acid (DIBMA) for structural studies. Here we show by electron cryomicroscopy that YnaI has an extended sensor paddle that during gating relocates relative to the pore concomitant with bending of a GGxGG motif in the pore helices. YnaI is the only one of the six paralogs that has this GGxGG motif allowing the sensor paddle to move outward. Access to the pore is through a vestibule on the cytosolic side that is fenestrated by side portals. In YnaI, these portals are obstructed by aromatic side chains but are still fully hydrated and thus support conductance. For comparison with large-sized channels, we determined the structure of YbiO, which showed larger portals and a wider pore with no GGxGG motif. Further in silico comparison of MscS, YnaI, and YbiO highlighted differences in the hydrophobicity and wettability of their pores and vestibule interiors. Thus, MscS-like channels of different sizes have a common core architecture but show different gating mechanisms and fine-tuned conductive properties.

Water in Nanopores and Biological Channels: A Molecular Simulation Perspective.

This Review explores the dynamic behavior of water within nanopores and biological channels in lipid bilayer membranes. We focus on molecular simulation studies, alongside selected structural and other experimental investigations. Structures of biological nanopores and channels are reviewed, emphasizing those high-resolution crystal structures, which reveal water molecules within the transmembrane pores, which can be used to aid the interpretation of simulation studies. Different levels of molecular simulations of water within nanopores are described, with a focus on molecular dynamics (MD). In particular, models of water for MD simulations are discussed in detail to provide an evaluation of their use in simulations of water in nanopores. Simulation studies of the behavior of water in idealized models of nanopores have revealed aspects of the organization and dynamics of nanoconfined water, including wetting/dewetting in narrow hydrophobic nanopores. A survey of simulation studies in a range of nonbiological nanopores is presented, including carbon nanotubes, synthetic nanopores, model peptide nanopores, track-etched nanopores in polymer membranes, and hydroxylated and functionalized nanoporous silica. These reveal a complex relationship between pore size/geometry, the nature of the pore lining, and rates of water transport. Wider nanopores with hydrophobic linings favor water flow whereas narrower hydrophobic pores may show dewetting. Simulation studies over the past decade of the behavior of water in a range of biological nanopores are described, including porins and ß-barrel protein nanopores, aquaporins and related polar solute pores, and a number of different classes of ion channels. Water is shown to play a key role in proton transport in biological channels and in hydrophobic gating of ion channels. An overall picture emerges, whereby the behavior of water in a nanopore may be predicted as a function of its hydrophobicity and radius. This informs our understanding of the functions of diverse channel structures and will aid the design of novel nanopores. Thus, our current level of understanding allows for the design of a nanopore which promotes wetting over dewetting or vice versa. However, to design a novel nanopore, which enables fast, selective, and gated flow of water de novo would remain challenging, suggesting a need for further detailed simulations alongside experimental evaluation of more complex nanopore systems.

Mechanisms of activation and desensitization of full-length glycine receptor in lipid nanodiscs.

Glycinergic synapses play a central role in motor control and pain processing in the central nervous system. Glycine receptors (GlyRs) are key players in mediating fast inhibitory neurotransmission at these synapses. While previous high-resolution structures have provided insights into the molecular architecture of GlyR, several mechanistic questions pertaining to channel function are still unanswered. Here, we present Cryo-EM structures of the full-length GlyR protein complex reconstituted into lipid nanodiscs that are captured in the unliganded (closed), glycine-bound (open and desensitized), and allosteric modulator-bound conformations. A comparison of these states reveals global conformational changes underlying GlyR channel gating and modulation. The functional state assignments were validated by molecular dynamics simulations, and the observed permeation events are in agreement with the anion selectivity and conductance of GlyR. These studies provide the structural basis for gating, ion selectivity, and single-channel conductance properties of GlyR in a lipid environment.

Induced Polarization in Molecular Dynamics Simulations of the 5-HT3 Receptor Channel.

Ion channel proteins form water-filled nanoscale pores within lipid bilayers, and their properties are dependent on the complex behavior of water in a nanoconfined environment. Using a simplified model of the pore of the 5-HT3 receptor (5HT3R) which restrains the backbone structure to that of the parent channel protein from which it is derived, we compare additive with polarizable models in describing the behavior of water in nanopores. Molecular dynamics simulations were performed with four conformations of the channel: two closed state structures, an intermediate state, and an open state, each embedded in a phosphatidylcholine bilayer. Water density profiles revealed that for all water models, the closed and intermediate states exhibited strong dewetting within the central hydrophobic gate region of the pore. However, the open state conformation exhibited varying degrees of hydration, ranging from partial wetting for the TIP4P/2005 water model to complete wetting for the polarizable AMOEBA14 model. Water dipole moments calculated using polarizable force fields also revealed that water molecules remaining within dewetted sections of the pore resemble gas phase water. Free energy profiles for Na+ and for Cl- ions within the open state pore revealed more rugged energy landscapes using polarizable force fields, and the hydration number profiles of these ions were also sensitive to induced polarization resulting in a substantive reduction of the number of waters within the first hydration shell of Cl- while it permeates the pore. These results demonstrate that induced polarization can influence the complex behavior of water and ions within nanoscale pores and provides important new insights into their chemical properties.

Characterizing Membrane Association and Periplasmic Transfer of Bacterial Lipoproteins through Molecular Dynamics Simulations.

Escherichia coli lipoprotein precursors at the inner membrane undergo three maturation stages before transport by the Lol system to the outer membrane. Here, we develop a pipeline to simulate the membrane association of bacterial lipoproteins in their four maturation states. This has enabled us to model and simulate 81 of the predicted 114 E. coli lipoproteins and reveal their interactions with the host lipid membrane. As part of this set we characterize the membrane contacts of LolB, the lipoprotein involved in periplasmic translocation. We also consider the means and bioenergetics for lipoprotein localization. Our calculations uncover a preference for LolB over LolA and therefore indicate how a lipoprotein may be favorably transferred from the inner to outer membrane. Finally, we reveal that LolC has a role in membrane destabilization, thereby promoting lipoprotein transfer to LolA.

Publisher Correction: Structure and assembly of calcium homeostasis modulator proteins.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structure and assembly of calcium homeostasis modulator proteins.

The biological membranes of many cell types contain large-pore channels through which a wide variety of ions and metabolites permeate. Examples include connexin, innexin and pannexin, which form gap junctions and/or bona fide cell surface channels. The most recently identified large-pore channels are the calcium homeostasis modulators (CALHMs), through which ions and ATP permeate in a voltage-dependent manner to control neuronal excitability, taste signaling and pathologies of depression and Alzheimer's disease. Despite such critical biological roles, the structures and patterns of their oligomeric assembly remain unclear. Here, we reveal the structures of two CALHMs, chicken CALHM1 and human CALHM2, by single-particle cryo-electron microscopy (cryo-EM), which show novel assembly of the four transmembrane helices into channels of octamers and undecamers, respectively. Furthermore, molecular dynamics simulations suggest that lipids can favorably assemble into a bilayer within the larger CALHM2 pore, but not within CALHM1, demonstrating the potential correlation between pore size, lipid accommodation and channel activity.

A heuristic derived from analysis of the ion channel structural proteome permits the rapid identification of hydrophobic gates.

Ion channel proteins control ionic flux across biological membranes through conformational changes in their transmembrane pores. An exponentially increasing number of channel structures captured in different conformational states are now being determined; however, these newly resolved structures are commonly classified as either open or closed based solely on the physical dimensions of their pore, and it is now known that more accurate annotation of their conductive state requires additional assessment of the effect of pore hydrophobicity. A narrow hydrophobic gate region may disfavor liquid-phase water, leading to local dewetting, which will form an energetic barrier to water and ion permeation without steric occlusion of the pore. Here we quantify the combined influence of radius and hydrophobicity on pore dewetting by applying molecular dynamics simulations and machine learning to nearly 200 ion channel structures. This allows us to propose a simple simulation-free heuristic model that rapidly and accurately predicts the presence of hydrophobic gates. This not only enables the functional annotation of new channel structures as soon as they are determined, but also may facilitate the design of novel nanopores controlled by hydrophobic gates.

CHAP: A Versatile Tool for the Structural and Functional Annotation of Ion Channel Pores.

The control of ion channel permeation requires the modulation of energetic barriers or "gates" within their pores. However, such barriers are often simply identified from the physical dimensions of the pore. Such approaches have worked well in the past, but there is now evidence that the unusual behavior of water within narrow hydrophobic pores can produce an energetic barrier to permeation without requiring steric occlusion of the pathway. Many different ion channels have now been shown to exploit "hydrophobic gating" to regulate ion flow, and it is clear that new tools are required for more accurate functional annotation of the increasing number of ion channel structures becoming available. We have previously shown how molecular dynamics simulations of water can be used as a proxy to predict hydrophobic gates, and we now present a new and highly versatile computational tool, the Channel Annotation Package (CHAP) that implements this methodology.

Cryo-EM reveals two distinct serotonin-bound conformations of full-length 5-HT3A receptor.

The 5-HT3A serotonin receptor1, a cationic pentameric ligand-gated ion channel (pLGIC), is the clinical target for management of nausea and vomiting associated with radiation and chemotherapies2. Upon binding, serotonin induces a global conformational change that encompasses the ligand-binding extracellular domain (ECD), the transmembrane domain (TMD) and the intracellular domain (ICD), the molecular details of which are unclear. Here we present two serotonin-bound structures of the full-length 5-HT3A receptor in distinct conformations at 3.32 Å and 3.89 Å resolution that reveal the mechanism underlying channel activation. In comparison to the apo 5-HT3A receptor, serotonin-bound states underwent a large twisting motion in the ECD and TMD, leading to the opening of a 165 Å permeation pathway. Notably, this motion results in the creation of lateral portals for ion permeation at the interface of the TMD and ICD. Combined with molecular dynamics simulations, these structures provide novel insights into conformational coupling across domains and functional modulation.

Water and hydrophobic gates in ion channels and nanopores.

Ion channel proteins form nanopores in biological membranes which allow the passage of ions and water molecules. Hydrophobic constrictions in such pores can form gates, i.e. energetic barriers to water and ion permeation. Molecular dynamics simulations of water in ion channels may be used to assess whether a hydrophobic gate is closed (i.e. impermeable to ions) or open. If there is an energetic barrier to water permeation then it is likely that a gate will also be impermeable to ions. Simulations of water behaviour have been used to probe hydrophobic gates in two recently reported ion channel structures: BEST1 and TMEM175. In each of these channels a narrow region is formed by three consecutive rings of hydrophobic sidechains and in both cases such analysis demonstrates that the crystal structures correspond to a closed state of the channel. In silico mutations of BEST1 have also been used to explore the effect of changes in the hydrophobicity of the gating constriction, demonstrating that substitution of hydrophobic sidechains with more polar sidechains results in an open gate which allows water permeation. A possible open state of the TMEM175 channel was modelled by the in silico expansion of the hydrophobic gate resulting in the wetting of the pore and free permeation of potassium ions through the channel. Finally, a preliminary study suggests that a hydrophobic gate motif can be transplanted in silico from the BEST1 channel into a simple ß-barrel pore template. Overall, these results suggest that simulations of the behaviour of water in hydrophobic gates can reveal important design principles for the engineering of gates in novel biomimetic nanopores.