Calcium regulates acid-sensing ion channel 3 activation by competing with protons in the channel pore and at an allosteric binding site.

The extracellular Ca2+ concentration changes locally under certain physiological and pathological conditions. Such variations affect the function of ion channels of the nervous system and consequently also neuronal signalling. We investigated here the mechanisms by which Ca2+ controls the activity of acid-sensing ion channel (ASIC) 3. ASICs are neuronal, H+-gated Na+ channels involved in several physiological and pathological processes, including the expression of fear, learning, pain sensation and neurodegeneration after ischaemic stroke. It was previously shown that Ca2+ negatively modulates the ASIC pH dependence. While protons are default activators of ASIC3, this channel can also be activated at pH7.4 by the removal of the extracellular Ca2+. Two previous studies concluded that low pH opens ASIC3 by displacing Ca2+ ions that block the channel pore at physiological pH. We show here that an acidic residue, distant from the pore, together with pore residues, controls the modulation of ASIC3 by Ca2+. Our study identifies a new regulatory site in ASIC3 and demonstrates that ASIC3 activation involves an allosteric mechanism together with Ca2+ unbinding from the channel pore. We provide a molecular analysis of a regulatory mechanism found in many ion channels.

The Voltage-Dependent Deactivation of the KvAP Channel Involves the Breakage of Its S4 Helix.

Voltage-gated potassium channels (Kv) allow ion permeation upon changes of the membrane electrostatic potential (Vm). Each subunit of these tetrameric channels is composed of six transmembrane helices, of which the anti-parallel helix bundle S1-S4 constitutes the voltage-sensor domain (VSD) and S5-S6 forms the pore domain. Here, using 82 molecular dynamics (MD) simulations involving 266 replicated VSDs, we report novel responses of the archaebacterial potassium channel KvAP to membrane polarization. We show that the S4 α-helix, which is straight in the experimental crystal structure solved under depolarized conditions (Vm ∼ 0), breaks into two segments when the cell membrane is hyperpolarized (Vm << 0), and reversibly forms a single straight helix following depolarization (Vm = 0). The outermost segment of S4 translates along the normal to the membrane, bringing new perspective to previously paradoxical accessibility experiments that were initially thought to imply the displacement of the whole VSD across the membrane. The novel model is applied through steered and unbiased MD simulations to the recently solved whole structure of KvAP. The simulations show that the resting state involves a re-orientation of the S5 α-helix by ∼ 5-6 degrees in respect to the normal of the bilayer, which could result in the constriction and closure of the selectivity filter. Our findings support the idea that the breakage of S4 under (hyper)polarization is a general feature of Kv channels with a non-swapped topology.

Divergent Cl- and H+ pathways underlie transport coupling and gating in CLC exchangers and channels.

The CLC family comprises H+-coupled exchangers and Cl- channels, and mutations causing their dysfunction lead to genetic disorders. The CLC exchangers, unlike canonical 'ping-pong' antiporters, simultaneously bind and translocate substrates through partially congruent pathways. How ions of opposite charge bypass each other while moving through a shared pathway remains unknown. Here, we use MD simulations, biochemical and electrophysiological measurements to identify two conserved phenylalanine residues that form an aromatic pathway whose dynamic rearrangements enable H+ movement outside the Cl- pore. These residues are important for H+ transport and voltage-dependent gating in the CLC exchangers. The aromatic pathway residues are evolutionarily conserved in CLC channels where their electrostatic properties and conformational flexibility determine gating. We propose that Cl- and H+ move through physically distinct and evolutionarily conserved routes through the CLC channels and transporters and suggest a unifying mechanism that describes the gating mechanism of both CLC subtypes.

Mechanism of activation at the selectivity filter of the KcsA K+ channel.

Potassium channels are opened by ligands and/or membrane potential. In voltage-gated K+ channels and the prokaryotic KcsA channel, conduction is believed to result from opening of an intracellular constriction that prevents ion entry into the pore. On the other hand, numerous ligand-gated K+ channels lack such gate, suggesting that they may be activated by a change within the selectivity filter, a narrow region at the extracellular side of the pore. Using molecular dynamics simulations and electrophysiology measurements, we show that ligand-induced conformational changes in the KcsA channel removes steric restraints at the selectivity filter, thus resulting in structural fluctuations, reduced K+ affinity, and increased ion permeation. Such activation of the selectivity filter may be a universal gating mechanism within K+ channels. The occlusion of the pore at the level of the intracellular gate appears to be secondary.

Phylogenetic, structural, and functional characterization of AMT3;1, an ammonium transporter induced by mycorrhization among model grasses.

In the arbuscular mycorrhizal (AM) symbiosis, plants satisfy part of their nitrogen (N) requirement through the AM pathway. In sorghum, the ammonium transporters (AMT) AMT3;1, and to a lesser extent AMT4, are induced in cells containing developing arbuscules. Here, we have characterized orthologs of AMT3;1 and AMT4 in four other grasses in addition to sorghum. AMT3;1 and AMT4 orthologous genes are induced in AM roots, suggesting that in the common ancestor of these five plant species, both AMT3;1 and AMT4 were already present and upregulated upon AM colonization. An artificial microRNA approach was successfully used to downregulate either AMT3;1 or AMT4 in rice. Mycorrhizal root colonization and hyphal length density of knockdown plants were not affected at that time, indicating that the manipulation did not modify the establishment of the AM symbiosis and the interaction between both partners. However, expression of the fungal phosphate transporter FmPT was significantly reduced in knockdown plants, indicating a reduction of the nutrient fluxes from the AM fungus to the plant. The AMT3;1 knockdown plants (but not the AMT4 knockdown plants) were significantly less stimulated in growth by AM fungal colonization, and uptake of both 15N and 33P from the AM fungal network was reduced. This confirms that N and phosphorus nutrition through the mycorrhizal pathway are closely linked. But most importantly, it indicates that AMT3;1 is the prime plant transporter involved in the mycorrhizal ammonium transfer and that its function during uptake of N cannot be performed by AMT4.

Unidirectional Transport Mechanism in an ATP Dependent Exporter.

ATP-binding cassette (ABC) transporters use the energy of ATP binding and hydrolysis to move a large variety of compounds across biological membranes. P-glycoprotein, involved in multidrug resistance, is the most investigated eukaryotic family member. Although a large number of biochemical and structural approaches have provided important information, the conformational dynamics underlying the coupling between ATP binding/hydrolysis and allocrite transport remains elusive. To tackle this issue, we performed molecular dynamic simulations for different nucleotide occupancy states of Sav1866, a prokaryotic P-glycoprotein homologue. The simulations reveal an outward-closed conformation of the transmembrane domain that is stabilized by the binding of two ATP molecules. The hydrolysis of a single ATP leads the X-loop, a key motif of the ATP binding cassette, to interfere with the transmembrane domain and favor its outward-open conformation. Our findings provide a structural basis for the unidirectionality of transport in ABC exporters and suggest a ratio of one ATP hydrolyzed per transport cycle.

Conformational dynamics and role of the acidic pocket in ASIC pH-dependent gating.

Acid-sensing ion channels (ASICs) are proton-activated Na+ channels expressed in the nervous system, where they are involved in learning, fear behaviors, neurodegeneration, and pain sensation. In this work, we study the role in pH sensing of two regions of the ectodomain enriched in acidic residues: the acidic pocket, which faces the outside of the protein and is the binding site of several animal toxins, and the palm, a central channel domain. Using voltage clamp fluorometry, we find that the acidic pocket undergoes conformational changes during both activation and desensitization. Concurrently, we find that, although proton sensing in the acidic pocket is not required for channel function, it does contribute to both activation and desensitization. Furthermore, protonation-mimicking mutations of acidic residues in the palm induce a dramatic acceleration of desensitization followed by the appearance of a sustained current. In summary, this work describes the roles of potential pH sensors in two extracellular domains, and it proposes a model of acidification-induced conformational changes occurring in the acidic pocket of ASIC1a.

Gating energetics of a voltage-dependent K+ channel pore domain.

We used targeted molecular dynamics, informed by experimentally determined inter-atomic distances defining the pore region of open and closed states of the KvAP voltage-gated potassium channel, to generate a gating pathway of the pore domain in the absence of the voltage-sensing domains. We then performed umbrella sampling simulations along this pathway to calculate a potential of mean force that describes the free energy landscape connecting the closed and open conformations of the pore domain. The resulting energetic landscape displays three minima, corresponding to stable open, closed, and intermediate conformations with roughly similar stabilities. We found that the extent of hydration of the interior of the pore domain could influence the free energy landscape for pore opening/closing. © 2017 Wiley Periodicals, Inc.

Catch-bond mechanism of the bacterial adhesin FimH.

Ligand-receptor interactions that are reinforced by mechanical stress, so-called catch-bonds, play a major role in cell-cell adhesion. They critically contribute to widespread urinary tract infections by pathogenic Escherichia coli strains. These pathogens attach to host epithelia via the adhesin FimH, a two-domain protein at the tip of type I pili recognizing terminal mannoses on epithelial glycoproteins. Here we establish peptide-complemented FimH as a model system for fimbrial FimH function. We reveal a three-state mechanism of FimH catch-bond formation based on crystal structures of all states, kinetic analysis of ligand interaction and molecular dynamics simulations. In the absence of tensile force, the FimH pilin domain allosterically accelerates spontaneous ligand dissociation from the FimH lectin domain by 100,000-fold, resulting in weak affinity. Separation of the FimH domains under stress abolishes allosteric interplay and increases the affinity of the lectin domain. Cell tracking demonstrates that rapid ligand dissociation from FimH supports motility of piliated E. coli on mannosylated surfaces in the absence of shear force.

A Rigorous and Efficient Method To Reweight Very Large Conformational Ensembles Using Average Experimental Data and To Determine Their Relative Information Content.

Flexible polypeptides such as unfolded proteins may access an astronomical number of conformations. The most advanced simulations of such states usually comprise tens of thousands of individual structures. In principle, a comparison of parameters predicted from such ensembles to experimental data provides a measure of their quality. In practice, analyses that go beyond the comparison of unbiased average data have been impossible to carry out on the entirety of such very large ensembles and have, therefore, been restricted to much smaller subensembles and/or nondeterministic algorithms. Here, we show that such very large ensembles, on the order of 10(4) to 10(5) conformations, can be analyzed in full by a maximum entropy fit to experimental average data. Maximizing the entropy of the population weights of individual conformations under experimental χ(2) constraints is a convex optimization problem, which can be solved in a very efficient and robust manner to a unique global solution even for very large ensembles. Since the population weights can be determined reliably, the reweighted full ensemble presents the best model of the combined information from simulation and experiment. Furthermore, since the reduction of entropy due to the experimental constraints is well-defined, its value provides a robust measure of the information content of the experimental data relative to the simulated ensemble and an indication for the density of the sampling of conformational space. The method is applied to the reweighting of a 35,000 frame molecular dynamics trajectory of the nonapeptide EGAAWAASS by extensive NMR (3)J coupling and RDC data. The analysis shows that RDCs provide significantly more information than (3)J couplings and that a discontinuity in the RDC pattern at the central tryptophan is caused by a cluster of helical conformations. Reweighting factors are moderate and consistent with errors in MD force fields of less than 3kT. The required reweighting is larger for an ensemble derived from a statistical coil model, consistent with its coarser nature. We call the method COPER, for convex optimization for ensemble reweighting. Similar advantages of large-scale efficiency and robustness can be obtained for other ensemble analysis methods with convex targets and constraints, such as constrained χ(2) minimization and the maximum occurrence method.

Allocrite Sensing and Binding by the Breast Cancer Resistance Protein (ABCG2) and P-Glycoprotein (ABCB1).

The ATP binding cassette (ABC) transporters ABCG2 and ABCB1 perform ATP hydrolysis-dependent efflux of structurally highly diverse compounds, collectively called allocrites. Whereas much is known about allocrite-ABCB1 interactions, the chemical nature and strength of ABCG2-allocrite interactions have not yet been assessed. We quantified and characterized interactions of allocrite with ABCG2 and ABCB1 using a set of 39 diverse compounds. We also investigated potential allocrite binding sites based on available transporter structures and structural models. We demonstrate that ABCG2 binds its allocrites from the lipid membrane, despite their hydrophilicity. Hence, binding of allocrite to both transporters is a two-step process, starting with a lipid-water partitioning step, driven mainly by hydrophobic interactions, followed by a transporter binding step in the lipid membrane. We show that binding of allocrite to both transporters increases with the number of hydrogen bond acceptors in allocrites. Scrutinizing the transporter translocation pathways revealed ample hydrogen bond donors for allocrite binding. Importantly, the hydrogen bond donor strength is, on average, higher in ABCG2 than in ABCB1, which explains the higher measured affinity of allocrite for ABCG2. π-π stacking and π-cation interactions play additional roles in binding of allocrite to ABCG2 and ABCB1. With this analysis, we demonstrate that these membrane-mediated weak electrostatic interactions between transporters and allocrites allow for transporter promiscuity toward allocrites. The different sensitivities of the transporters to allocrites' charge and amphiphilicity provide transporter specificity. In addition, we show that the different hydrogen bond donor strengths in the two transporters allow for affinity tuning.

Mechanism of NH4(+) Recruitment and NH3 Transport in Rh Proteins.

In human cells, membrane proteins of the rhesus (Rh) family excrete ammonium and play a role in pH regulation. Based on high-resolution structures, Rh proteins are generally understood to act as NH3 channels. Given that cell membranes are permeable to gases like NH3, the role of such proteins remains a paradox. Using molecular and quantum mechanical calculations, we show that a crystallographically identified site in the RhCG pore actually recruits NH4(+), which is found in higher concentration and binds with higher affinity than NH3, increasing the efficiency of the transport mechanism. A proton is transferred from NH4(+) to a signature histidine (the only moiety thermodynamically likely to accept a proton) followed by the diffusion of NH3 down the pore. The excess proton is circulated back to the extracellular vestibule through a hydrogen bond network, which involves a highly conserved and functionally important aspartic acid, resulting in the net transport of NH3.

Backbone hydration determines the folding signature of amino acid residues.

The relation between the sequence of a protein and its three-dimensional structure remains largely unknown. A lasting dream is to elucidate the side-chain-dependent driving forces that govern the folding process. Different structural data suggest that aromatic amino acids play a particular role in the stabilization of protein structures. To better understand the underlying mechanism, we studied peptides of the sequence EGAAXAASS (X = Gly, Ile, Tyr, Trp) through comparison of molecular dynamics (MD) trajectories and NMR residual dipolar coupling (RDC) measurements. The RDC data for aromatic substitutions provide evidence for a kink in the peptide backbone. Analysis of the MD simulations shows that the formation of internal hydrogen bonds underlying a helical turn is key to reproduce the experimental RDC values. The simulations further reveal that the driving force leading to such helical-turn conformations arises from the lack of hydration of the peptide chain on either side of the bulky aromatic side chain, which can potentially act as a nucleation point initiating the folding process.

A PDI-catalyzed thiol-disulfide switch regulates the production of hydrogen peroxide by human Ero1.

Oxidative folding in the endoplasmic reticulum (ER) involves ER oxidoreductin 1 (Ero1)-mediated disulfide formation in protein disulfide isomerase (PDI). In this process, Ero1 consumes oxygen (O2) and releases hydrogen peroxide (H2O2), but none of the published Ero1 crystal structures reveal any potential pathway for entry and exit of these reactants. We report that additional mutation of the Cys(208)-Cys(241) disulfide in hyperactive Ero1α (Ero1α-C104A/C131A) potentiates H2O2 production, ER oxidation, and cell toxicity. This disulfide clamps two helices that seal the flavin cofactor where O2 is reduced to H2O2. Through its carboxyterminal active site, PDI unlocks this seal by forming a Cys(208)/Cys(241)-dependent mixed-disulfide complex with Ero1α. The H2O2-detoxifying glutathione peroxidase 8 also binds to the Cys(208)/Cys(241) loop region. Supported by O2 diffusion simulations, these data describe the first enzymatically controlled O2 access into a flavoprotein active site, provide molecular-level understanding of Ero1α regulation and H2O2 production/detoxification, and establish the deleterious consequences of constitutive Ero1 activity.

Initial steps of inactivation at the K+ channel selectivity filter.

K(+) efflux through K(+) channels can be controlled by C-type inactivation, which is thought to arise from a conformational change near the channel's selectivity filter. Inactivation is modulated by ion binding near the selectivity filter; however, the molecular forces that initiate inactivation remain unclear. We probe these driving forces by electrophysiology and molecular simulation of MthK, a prototypical K(+) channel. Either Mg(2+) or Ca(2+) can reduce K(+) efflux through MthK channels. However, Ca(2+), but not Mg(2+), can enhance entry to the inactivated state. Molecular simulations illustrate that, in the MthK pore, Ca(2+) ions can partially dehydrate, enabling selective accessibility of Ca(2+) to a site at the entry to the selectivity filter. Ca(2+) binding at the site interacts with K(+) ions in the selectivity filter, facilitating a conformational change within the filter and subsequent inactivation. These results support an ionic mechanism that precedes changes in channel conformation to initiate inactivation.

Functional asymmetry within the Sec61p translocon.

The Sec61 translocon forms a pore to translocate polypeptide sequences across the membrane and offers a lateral gate for membrane integration of hydrophobic (H) segments. A central constriction of six apolar residues has been shown to form a seal, but also to determine the hydrophobicity threshold for membrane integration: Mutation of these residues in yeast Sec61p to glycines, serines, aspartates, or lysines lowered the hydrophobicity required for integration; mutation to alanines increased it. Whereas four leucines distributed in an oligo-alanine H segment were sufficient for 50% integration, we now find four leucines in the N-terminal half of the H segment to produce significantly more integration than in the C-terminal half, suggesting functional asymmetry within the translocon. Scanning a cluster of three leucines through an oligo-alanine H segment showed high integration levels, except around the position matching that of the hydrophobic constriction in the pore where integration was strongly reduced. Both asymmetry and the position effect of H-segment integration disappeared upon mutation of the constriction residues to glycines or serines, demonstrating that hydrophobicity at this position within the translocon is responsible for the phenomenon. Asymmetry was largely retained, however, when constriction residues were replaced by alanines. These results reflect on the integration mechanism of transmembrane domains and show that membrane insertion of H segments strongly depends not only on their intrinsic hydrophobicity but also on the local conditions in the translocon interior. Thus, the contribution of hydrophobic residues in the H segment is not simply additive and displays cooperativeness depending on their relative position.

Different hydration patterns in the pores of AmtB and RhCG could determine their transport mechanisms.

The ammonium transporters of the Amt/Rh family facilitate the diffusion of ammonium across cellular membranes. Functional data suggest that Amt proteins, notably found in plants, transport the ammonium ion (NH4(+)), whereas human Rhesus (Rh) proteins transport ammonia (NH3). Comparison between the X-ray structures of the prokaryotic AmtB, assumed to be representative of Amt proteins, and the human RhCG reveals important differences at the level of their pore. Despite these important functional and structural differences between Amt and Rh proteins, studies of the AmtB transporter have led to the suggestion that proteins of both subfamilies work according to the same mechanism and transport ammonia. We performed molecular dynamics simulations of the AmtB and RhCG proteins under different water and ammonia occupancy states of their pore. Free energy calculations suggest that the probability of finding NH3 molecules in the pore of AmtB is negligible in comparison to finding water. The presence of water in the pore of AmtB could support the transport of proton. The pore lumen of RhCG is found to be more hydrophobic due to the presence of a phenylalanine conserved among Rh proteins. Simulations of RhCG also reveal that the signature histidine dyad is occasionally exposed to the extracellular bulk, which is never observed in AmtB. These different hydration patterns are consistent with the idea that Amt and Rh proteins are not functionally equivalent and that permeation takes place according to two distinct mechanisms.

Molecular determinants of desensitization in an ENaC/degenerin channel.

Epithelial Na(+) channel (ENaC)/degenerin family members are involved in mechanosensation, blood pressure control, pain sensation, and the expression of fear. Several of these channel types display a form of desensitization that allows the channel to limit Na(+) influx during prolonged stimulation. We used site-directed mutagenesis and chemical modification, functional analysis, and molecular dynamics simulations to investigate the role of the lower palm domain of the acid-sensing ion channel 1, a member of the ENaC/degenerin family. The lower palm domains of this trimeric channel are arranged around a central vestibule, at ∼20 Šabove the plasma membrane and are covalently linked to the transmembrane channel parts. We show that the lower palm domains approach one another during desensitization. Residues in the palm co-determine the pH dependence of desensitization, its kinetics, and the stability of the desensitized state. Mutations of palm residues impair desensitization by preventing the closing movement of the palm. Overexpression of desensitization-impaired channel mutants in central neurons allowed--in contrast to overexpression of wild type--a sustained signaling response to rapid pH fluctuations. We identify and describe here the function of an important regulatory domain that most likely has a conserved role in ENaC/degenerin channels.

Self-Learning Adaptive Umbrella Sampling Method for the Determination of Free Energy Landscapes in Multiple Dimensions.

The potential of mean force describing conformational changes of biomolecules is a central quantity that determines the function of biomolecular systems. Calculating an energy landscape of a process that depends on three or more reaction coordinates might require a lot of computational power, making some of multidimensional calculations practically impossible. Here, we present an efficient automatized umbrella sampling strategy for calculating multidimensional potential of mean force. The method progressively learns by itself, through a feedback mechanism, which regions of a multidimensional space are worth exploring and automatically generates a set of umbrella sampling windows that is adapted to the system. The self-learning adaptive umbrella sampling method is first explained with illustrative examples based on simplified reduced model systems, and then applied to two non-trivial situations: the conformational equilibrium of the pentapeptide Met-enkephalin in solution and ion permeation in the KcsA potassium channel. With this method, it is demonstrated that a significant smaller number of umbrella windows needs to be employed to characterize the free energy landscape over the most relevant regions without any loss in accuracy.

A limited 4 Å radial displacement of the S4-S5 linker is sufficient for internal gate closing in Kv channels.

Voltage-gated ion channels are responsible for the generation of action potentials in our nervous system. Conformational rearrangements in their voltage sensor domains in response to changes of the membrane potential control pore opening and thus ion conduction. Crystal structures of the open channel in combination with a wealth of biophysical data and molecular dynamics simulations led to a consensus on the voltage sensor movement. However, the coupling between voltage sensor movement and pore opening, the electromechanical coupling, occurs at the cytosolic face of the channel, from where no structural information is available yet. In particular, the question how far the cytosolic pore gate has to close to prevent ion conduction remains controversial. In cells, spectroscopic methods are hindered because labeling of internal sites remains difficult, whereas liposomes or detergent solutions containing purified ion channels lack voltage control. Here, to overcome these problems, we controlled the state of the channel by varying the lipid environment. This way, we directly measured the position of the S4-S5 linker in both the open and the closed state of a prokaryotic Kv channel (KvAP) in a lipid environment using Lanthanide-based resonance energy transfer. We were able to reconstruct the movement of the covalent link between the voltage sensor and the pore domain and used this information as restraints for molecular dynamics simulations of the closed state structure. We found that a small decrease of the pore radius of about 3-4 Å is sufficient to prevent ion permeation through the pore.