Hydrophobic Gates Research Articles

SWEET family transporters act as water conducting carrier proteins in plants.

Dedicated water channels are involved in the facilitated diffusion of water molecules across the cell membrane in plants. Transporter proteins are also known to transport water molecules along with substrates, however the molecular mechanism of water permeation is not well understood in plant transporters. Here, we show plant sugar transporters from the SWEET (Sugar Will Eventually be Exported Transporter) family act as water-conducting carrier proteins via a variety of passive and active mechanisms that allow diffusion of water molecules from one side of the membrane to the other. This study provides a molecular perspective on how plant membrane transporters act as water carrier proteins, a topic that has not been extensively explored in literature. Water permeation in membrane transporters could occur via four distinct mechanisms which form our hypothesis for water transport in SWEETs. These hypothesis are tested using molecular dynamics simulations of the outward-facing, occluded, and inward-facing state of AtSWEET1 to identify the water permeation pathways and the flux associated with them. The hydrophobic gates at the center of the transport tunnel act as a barrier that restricts water permeation. We have performed in silico single and double mutations of the hydrophobic gate residues to examine the changes in the water conductivity. Surprisingly, the double mutant allows the water permeation to the intracellular half of the membrane and forms a continuous water channel. These computational results are validated by experimentally examining the transport of hydrogen peroxide molecules by the AtSWEET family of transporters. We have also shown that the transport of hydrogen peroxide follows the similar mechanism as water transport in AtSWEET1. Finally, we conclude that similar water-conduction states are also present in other SWEET transporters due to the high sequence and structure conservation exhibited by this transporter family.

Deciphering the mannose transfer mechanism of mycobacterial PimE by molecular dynamics simulations.

Phosphatidyl-myo-inositol mannosides (PIMs), Lipomannan (LM), and Lipoarabinomannan (LAM) are essential components of the cell envelopes of mycobacteria. At the beginning of the biosynthesis of these compounds, phosphatidylinositol (PI) is mannosylated and acylated by various enzymes to produce Ac1/2PIM4, which is used to synthesize either Ac1/2PIM6 or LM/LAM. The protein PimE, a membrane-bound glycosyltransferase (GT-C), catalyzes the addition of a mannose group to Ac1PIM4 to produce Ac1PIM5, using polyprenolphosphate mannose (PPM) as the mannose donor. PimE-deleted Mycobacterium smegmatis (Msmeg) showed structural deformity and increased antibiotic and copper sensitivity. Despite knowing that the mutation D58A caused inactivity in Msmeg, how PimE catalyzes the transfer of mannose from PPM to Ac1/2PIM4 remains unknown. In this study, analyzing the AlphaFold structure of PimE revealed the presence of a tunnel through the D58 residue with two differently charged gates. Molecular docking suggested PPM binds to the hydrophobic tunnel gate, whereas Ac1PIM4 binds to the positively charged tunnel gate. Molecular dynamics (MD) simulations further demonstrated the critical roles of the residues N55, F87, L89, Y163, Q165, K197, L198, R251, F277, W324, H326, and I375 in binding PPM and Ac1PIM4. The mutation D58A caused a faster release of PPM from the catalytic tunnel, explaining the loss of PimE activity. Along with a hypothetical mechanism of mannose transfer by PimE, we also observe the presence of tunnels through a negatively charged aspartate or glutamate with two differently-charged gates among most GT-C enzymes. Common hydrophobic gates of GT-C enzymes probably harbor sugar donors, whereas, differently-charged tunnel gates accommodate various sugar-acceptors.

Structural basis of promiscuous substrate transport by Organic Cation Transporter 1

Organic Cation Transporter 1 (OCT1) plays a crucial role in hepatic metabolism by mediating the uptake of a range of metabolites and drugs. Genetic variations can alter the efficacy and safety of compounds transported by OCT1, such as those used for cardiovascular, oncological, and psychological indications. Despite its importance in drug pharmacokinetics, the substrate selectivity and underlying structural mechanisms of OCT1 remain poorly understood. Here, we present cryo-EM structures of full-length human OCT1 in the inward-open conformation, both ligand-free and drug-bound, indicating the basis for its broad substrate recognition. Comparison of our structures with those of outward-open OCTs provides molecular insight into the alternating access mechanism of OCTs. We observe that hydrophobic gates stabilize the inward-facing conformation, whereas charge neutralization in the binding pocket facilitates the release of cationic substrates. These findings provide a framework for understanding the structural basis of the promiscuity of drug binding and substrate translocation in OCT1.

Microscopic Characterization of the Chloride Permeation Pathway in the Human Excitatory Amino Acid Transporter 1 (EAAT1).

Excitatory amino acid transporters (EAATs) are glutamate transporters that belong to the solute carrier 1A (SLC1A) family. They couple glutamate transport to the cotransport of three sodium (Na+) ions and one proton (H+) and the counter-transport of one potassium (K+) ion. In addition to this coupled transport, binding of cotransported species to EAATs activates a thermodynamically uncoupled chloride (Cl-) conductance. Structures of SLC1A family members have revealed that these transporters use a twisting elevator mechanism of transport, where a mobile transport domain carries substrate and coupled ions across the membrane, while a static scaffold domain anchors the transporter in the membrane. We recently demonstrated that the uncoupled Cl- conductance is activated by the formation of an aqueous pore at the domain interface during the transport cycle in archaeal GltPh. However, a pathway for the uncoupled Cl- conductance has not been reported for the EAATs, and it is unclear if such a pathway is conserved. Here, we employ all-atom molecular dynamics (MD) simulations combined with enhanced sampling, free-energy calculations, and experimental mutagenesis to approximate large-scale conformational changes during the transport process and identified a Cl--conducting conformation in human EAAT1 (hEAAT1). Sampling the large-scale structural transitions in hEAAT1 allowed us to capture an intermediate conformation formed during the transport cycle with a continuous aqueous pore at the domain interface. The free-energy calculations performed for the conduction of Cl- and Na+ ions through the captured conformation highlight the presence of two hydrophobic gates that control low-barrier movement of Cl- through the aqueous pathway. Overall, our findings provide insights into the mechanism by which a human neurotransmitter transporter supports functional duality of active transport and passive Cl- permeation and confirm the commonality of this mechanism in different members of the SLC1A family.

Occlusion of the human serotonin transporter is mediated by serotonin-induced conformational changes in the bundle domain

The human serotonin transporter (hSERT) terminates neurotransmission by removing serotonin (5HT) from the synaptic cleft, an essential process for proper functioning of serotonergic neurons. Structures of the hSERT have revealed its molecular architecture in four conformations, including the outward-open and occluded states, and show the transporter’s engagement with co-transported ions and the binding mode of inhibitors. In this study, we investigated the molecular mechanism by which the hSERT occludes and sequesters the substrate 5HT. This first step of substrate uptake into cells is a structural change consisting of the transition from the outward-open to the occluded state. Inhibitors such as the antidepressants citalopram, fluoxetine, and sertraline inhibit this step of the transport cycle. Using molecular dynamics simulations, we reached a fully occluded state, in which the transporter-bound 5HT becomes fully shielded from both sides of the membrane by two closed hydrophobic gates. Analysis of 5HT-triggered occlusion showed that bound 5HT serves as an essential trigger for transporter occlusion. Moreover, simulations revealed a complex sequence of steps and showed that movements of bundle domain helices are only partially correlated. 5HT-triggered occlusion is initially dominated by movements of transmembrane helix 1b, while in the final step, only transmembrane helix 6a moves and relaxes an intermediate change in its secondary structure.

Antagonistic Mechanism of α-Conotoxin BuIA toward the Human α3β2 Nicotinic Acetylcholine Receptor

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that are abundantly expressed in the central and peripheral nervous systems, playing an important role in mediating neurotransmitter release and inter-synaptic signaling. Dysfunctional nAChRs are associated with neurological disorders, and studying the structure and function of nAChRs is essential for development of drugs or strategies for treatment of related diseases. α-Conotoxins are selective antagonists of the nAChR and are an important class of drug leads. So far, the antagonistic mechanism of α-conotoxins toward the nAChRs is still unclear. In this study, we built an α3β2 nAChR homology model and investigated its conformational transition mechanism upon binding with a highly potent inhibitor, α-conotoxin BuIA, through μs molecular dynamic simulations and site-directed mutagenesis studies. The results suggested that the α3β2 nAChR underwent global conformational transitions and was stabilized into a closed state with three hydrophobic gates present in the transmembrane domain by BuIA. Finally, the probable antagonistic mechanism of BuIA was proposed. Overall, the closed-state model of the α3β2 nAChR bound with BuIA is not only essential for understanding the antagonistic mechanism of α-conotoxins but also particularly valuable for development of therapeutic inhibitors in future.

Glutamate transporters have a chloride channel with two hydrophobic gates.

Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, therefore its precise control is vital for maintaining normal brain function and preventing excitotoxicity1. Removal of extracellular glutamate is achieved by plasma membrane-bound transporters, which couple glutamate transport to sodium, potassium and pH gradients using an elevator mechanism2–5. Glutamate transporters also conduct chloride ions via a channel-like process that is thermodynamically uncoupled from transport6–8. However, the molecular mechanisms that allow these dual-function transporters to carry out two seemingly contradictory roles are unknown. Here we report the cryo-electron microscopy structure of a glutamate transporter homologue in an open-channel state, revealing an aqueous cavity that is formed during the transport cycle. By studying functional properties combined with molecular dynamics simulations, we show that this cavity is an aqueous-accessible chloride permeation pathway gated by two hydrophobic regions and is conserved across mammalian and archaeal glutamate transporters. Our findings provide insight into the mechanism by which glutamate transporters support their dual function and add a crucial piece of information to aid mapping of the complete transport cycle shared by the SLC1A transporter family.

Modelling Water Behaviour in Hydrophobic Gates of Ion Channels

Ion channels are a key class of membrane proteins as they enable ions and water to pass into and out of cells. Some of these ion channels possess a hydrophobic gate, which enables the channel to switch between wetted and de-wetted states without sterically occluding the channel. The transition between wetting and de-wetting can be determined as a function of both the radius and hydrophobicity of the channel lining. Molecular dynamics simulations are a powerful tool for exploring this parameter space. However, due to the complexity of modelling bulk water interactions and the translation of these to nano-confined environments, the level of theory chosen for modelling the water is an important consideration. We present a study of how different techniques for modelling water affect simulations of hydrophobic gating in selected ion channels.

A Novel Chloride Conducting Conformation in Human Glutamate Transporters

Excitatory amino acid transporters (EAATs), which belong to the solute carrier 1A (SLC1A) family, couple glutamate transport to the co-transport of three sodium (Na+) ions and one proton (H+) and the counter-transport of one potassium (K+) ion. In addition to this coupled transport, binding of substrate and Na+ ions to EAATs activates a reversible and thermodynamically uncoupled chloride (Cl-) conductance. Mutations that alter this Cl- conductance have been linked to episodic ataxia type 6 (EA6). Structures of human EAATs and its homologues have highlighted that they share a similar trimeric structure and perform an elevator-like mechanism for the substrate transport, but a clear pathway for the uncoupled Cl- conductance has not been observed in the available structures. Therefore, the molecular determinants required for its activation remain unclear. Here, we present an extensive computational study, including microsecond-long molecular dynamics (MD) simulations and free-energy calculations, closely coupled to cryo-EM structure determination, electrophysiology, and mutagenesis to characterize a clear pathway for the uncoupled Cl- conductance in human and bacterial glutamate transporters. We have designed a modeling protocol which incorporates driven simulations combined with experimentally-derived restraints to capture the Cl- conducting conformation in human EAAT1. The free-energy calculations performed for the conduction of Cl- and Na+ ions through the captured conformation, highlight the presence of two hydrophobic gates which control the selective movement of Cl- through an aqueous-accessible permeation pathway. The computationally captured pathway was further validated by the cryo-EM structure of the Cl- conducting conformation in archaeal glutamate transporter (GlTph), mutagenesis, and functional studies. Overall, our findings provide insights into the mechanism by which glutamate transporters support their dual function and add a crucial piece of information to aid mapping of the complete transport cycle shared by the SLC1A transporter family.

Glutamate Transporters (EAATS) Contain a Conserved Chloride Channel with Two Hydrophobic Gates

Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, therefore its precise control is vital for maintaining normal brain function and preventing excitotoxicity. Removal of extracellular glutamate is achieved by plasma membrane-bound transporters known as Excitatory Amino Acid Transporters (EAATs) which belong to the solute carrier 1A (SLC1A) family. The EAATs couple glutamate transport to sodium, potassium and pH gradients using an elevator transport mechanism. Glutamate transporters also conduct chloride ions via a channel-like process that is thermodynamically uncoupled from transport. However, the molecular mechanisms that allow these dual-function transporters to carry out two seemingly contradictory roles are unknown. Here we report the cryo-electron microscopy structure of a glutamate transporter homologue (GltPh) in an open-channel state, revealing an aqueous cavity that is formed at the interface of the transport and scaffold domains during the transport cycle. Using functional studies and molecular dynamics simulations, we show that this cavity is an aqueous-accessible chloride permeation pathway gated by two hydrophobic regions and is conserved across mammalian and archaeal glutamate transporters. Our findings reveal the mechanism by which glutamate transporters support their dual functions and provide a crucial piece of information to aid mapping of the complete transport cycle shared by the SLC1A transporter family.

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.

Computational Study of the Ion and Water Permeation and Transport Mechanisms of the SARS-CoV-2 Pentameric E Protein Channel.

Coronavirus disease 2019 (COVID-19) is caused by a novel coronavirus (SARS-CoV-2) and represents the causative agent of a potentially fatal disease that is a public health emergency of international concern. Coronaviruses, including SARS-CoV-2, encode an envelope (E) protein, which is a small, hydrophobic membrane protein; the E protein of SARS-CoV-2 shares a high level of homology with severe acute respiratory syndrome coronavirus (SARS-CoV). In this study, we provide insights into the function of the SARS-CoV-2 E protein channel and the ion and water permeation mechanisms using a combination of in silico methods. Based on our results, the pentameric E protein promotes the penetration of cation ions through the channel. An analysis of the potential mean force (PMF), pore radius and diffusion coefficient reveals that Leu10 and Phe19 are the hydrophobic gates of the channel. In addition, the pore exhibits a clear wetting/dewetting transition with cation selectivity under transmembrane voltage, indicating that it is a hydrophobic voltage-dependent channel. Overall, these results provide structure-based insights and molecular dynamic information that are needed to understand the regulatory mechanisms of ion permeability in the pentameric SARS-CoV-2 E protein channel.

Finding the gates

Structural Biology Hundreds of ion channel structures are available thanks in large part to recent advances in electron microscopy. Insights into channel gating have come from structures of the same channel in different conformational states, supported by biophysical approaches. Gating can occur in hydrophobic regions of the pore even when the diameter is larger than that of a water molecule. Rao et al. simulate the water distribution in nearly 200 representative ion channel structures. Using a machine learning approach, they classify the hydrophobicity–pore radius landscape into two regions, depending on whether water is likely to be excluded or not. This enables them to predict the presence of hydrophobic gates by analysis of the hydrophobicity and pore radius of a single set of coordinates without molecular dynamic simulation of the structure. Proc. Natl. Acad. Sci. U.S.A. 116 , 13989 (2019).

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.

Hydrophobic pore gates regulate ion permeation in polycystic kidney disease 2 and 2L1 channels

PKD2 and PKD1 genes are mutated in human autosomal dominant polycystic kidney disease. PKD2 can form either a homomeric cation channel or a heteromeric complex with the PKD1 receptor, presumed to respond to ligand(s) and/or mechanical stimuli. Here, we identify a two-residue hydrophobic gate in PKD2L1, and a single-residue hydrophobic gate in PKD2. We find that a PKD2 gain-of-function gate mutant effectively rescues PKD2 knockdown-induced phenotypes in embryonic zebrafish. The structure of a PKD2 activating mutant F604P by cryo-electron microscopy reveals a π- to α-helix transition within the pore-lining helix S6 that leads to repositioning of the gate residue and channel activation. Overall the results identify hydrophobic gates and a gating mechanism of PKD2 and PKD2L1.

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 β-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.

Molecular simulation studies of hydrophobic gating in nanopores and ion channels.

Gating in channels and nanopores plays a key role in regulating flow of ions across membranes. Molecular simulations provide a 'computational microscope' which enables us to examine the physical nature of gating mechanisms at the level of the single channel molecule. Water enclosed within the confines of a nanoscale pore may exhibit unexpected behaviour. In particular, if the molecular surfaces lining the pore are hydrophobic this promotes de-wetting of the pore. De-wetting is observed as stochastic liquid-vapour transitions within a pore, and may lead to functional closure of a pore to the flow of ions and/or water. Such behaviour was first observed in simulations of simple model nanopores and referred to as 'hydrophobic gating'. Simulations of both the nicotinic acetylcholine receptor and of TWIK-1 potassium channels (the latter alongside experimental studies) suggest hydrophobic gating may occur in some biological ion channels. Current studies are focused on designing hydrophobic gates into biomimetic nanopores.

Designing Hydrophobic Gates into Biomimetic Nanopores

The use of nanopores is fast being a major scientific tool in molecular analysis and detection due to their ability to detect polynucleotides, proteins and small molecules. Biomimetic modelling of pores allows for a specific function to be incorporated into the molecular structure of the nanopore, based on amino acid motifs found in existing protein structures.An initial beta barrel model was built computationally, based on the transmembrane domain of 14 stranded beta-barrel pore, alpha-hemolysin. Hydrophobic and hydrophilic residues were built in a specific arrangement within the structure to replicate an hourglass shape cavity with a central constriction. From this, pore conductions were observed via Molecular Dynamics (MD) and selected models were transformed into hybrid pores in which the location of hydrophobic residues differed to give constricting regions surrounded by hydrophilic residues. From All Atom MD simulations, a hydrophobic gating mechanism has been established within these toy models with intermittent water currents through the pore giving an insight into possible biomimetic motifs which could be biochemically integrated into the wild type protein.

Bubbles, Gating, and Anesthetics in Ion Channels

We suggest that bubbles are the bistable hydrophobic gates responsible for the on-off transitions of single channel currents. In this view, many types of channels gate by the same physical mechanism—dewetting by capillary evaporation—but different types of channels use different sensors to modulate hydrophobic properties of the channel wall and thereby trigger and control bubbles and gating. Spontaneous emptying of channels has been seen in many simulations. Because of the physics involved, such phase transitions are inherently sensitive, unstable threshold phenomena that are difficult to simulate reproducibly and thus convincingly. We present a thermodynamic analysis of a bubble gate using morphometric density functional theory of classical (not quantum) mechanics. Thermodynamic analysis of phase transitions is generally more reproducible and less sensitive to details than simulations. Anesthetic actions of inert gases—and their interactions with hydrostatic pressure (e.g., nitrogen narcosis)—can be easily understood by actions on bubbles. A general theory of gas anesthesia may involve bubbles in channels. Only experiments can show whether, or when, or which channels actually use bubbles as hydrophobic gates: direct observation of bubbles in channels is needed. Existing experiments show thin gas layers on hydrophobic surfaces in water and suggest that bubbles nearly exist in bulk water.