A molecular switch controls the impact of cholesterol on a Kir channel.

SignificanceCholesterol is one of the main components found in plasma membranes and is involved in lipid-dependent signaling enabled by integral membrane proteins such as inwardly rectifying potassium (Kir) channels. Similar to other ion channels, most of the Kir channels are down-regulated by cholesterol. One of the very few notable exceptions is Kir3.4, which is up-regulated by this important lipid. Here, we discovered and characterized a molecular switch that controls the impact (up-regulation vs. down-regulation) of cholesterol on Kir3.4. Our results provide a detailed molecular mechanism of tunable cholesterol regulation of a potassium channel.

A second S4 movement opens hyperpolarization-activated HCN channels.

Rhythmic activity in pacemaker cells, as in the sino-atrial node in the heart, depends on the activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. As in depolarization-activated K+ channels, the fourth transmembrane segment S4 functions as the voltage sensor in hyperpolarization-activated HCN channels. But how the inward movement of S4 in HCN channels at hyperpolarized voltages couples to channel opening is not understood. Using voltage clamp fluorometry, we found here that S4 in HCN channels moves in two steps in response to hyperpolarizations and that the second S4 step correlates with gate opening. We found a mutation in sea urchin HCN channels that separate the two S4 steps in voltage dependence. The E356A mutation in S4 shifts the main S4 movement to positive voltages, but channel opening remains at negative voltages. In addition, E356A reveals a second S4 movement at negative voltages that correlates with gate opening. Cysteine accessibility and molecular models suggest that the second S4 movement opens up an intracellular crevice between S4 and S5 that would allow radial movement of the intracellular ends of S5 and S6 to open HCN channels.

Selectivity filter modalities and rapid inactivation of the hERG1 channel.

The human ether-á-go-go-related gene (hERG1) channel conducts small outward K+ currents that are critical for cardiomyocyte membrane repolarization. The gain-of-function mutation N629D at the outer mouth of the selectivity filter (SF) disrupts inactivation and K+-selective transport in hERG1, leading to arrhythmogenic phenotypes associated with long-QT syndrome. Here, we combined computational electrophysiology with Markov state model analysis to investigate how SF-level gating modalities control selective cation transport in wild-type (WT) and mutant (N629D) hERG1 variants. Starting from the recently reported cryogenic electron microscopy (cryo-EM) open-state channel structure, multiple microseconds-long molecular-dynamics (MD) trajectories were generated using different cation configurations at the filter, voltages, electrolyte concentrations, and force-field parameters. Most of the K+ permeation events observed in hERG1-WT simulations occurred at microsecond timescales, influenced by the spontaneous dehydration/rehydration dynamics at the filter. The SF region displayed conductive, constricted, occluded, and dilated states, in qualitative agreement with the well-documented flickering conductance of hERG1. In line with mutagenesis studies, these gating modalities resulted from dynamic interaction networks involving residues from the SF, outer-mouth vestibule, P-helices, and S5-P segments. We found that N629D mutation significantly stabilizes the SF in a state that is permeable to both K+ and Na+, which is reminiscent of the SF in the nonselective bacterial NaK channel. Increasing the external K+ concentration induced "WT-like" SF dynamics in N629D, in qualitative agreement with the recovery of flickering currents in experiments. Overall, our findings provide an understanding of the molecular mechanisms controlling selective transport in K+ channels with a nonconventional SF sequence.

The voltage-sensing domain of a hERG1 mutant is a cation-selective channel.

A cationic leak current known as an "omega current" may arise from mutations of the first charged residue in the S4 of the voltage sensor domains of sodium and potassium voltage-gated channels. The voltage-sensing domains (VSDs) in these mutated channels act as pores allowing nonspecific passage of cations, such as Li+, K+, Cs+, and guanidinium. Interestingly, no omega currents have been previously detected in the nonswapped voltage-gated potassium channels such as the human-ether-a-go-go-related (hERG1), hyperpolarization-activated cyclic nucleotide-gated, and ether-a-go-go channels. In this work, we discovered a novel omega current by mutating the first charged residue of the S4 of the hERG1, K525 to serine. To characterize this omega current, we used various probes, including the hERG1 pore domain blocker, dofetilide, to show that the omega current does not require cation flux via the canonical pore domain. In addition, the omega flux does not cross the conventional selectivity filter. We also show that the mutated channel (K525S hERG1) conducts guanidinium. These data are indicative of the formation of an omega current channel within the VSD. Using molecular dynamics simulations with replica-exchange umbrella sampling simulations of the wild-type hERG1 and the K525S hERG1, we explored the molecular underpinnings governing the cation flow in the VSD of the mutant. We also show that the wild-type hERG1 may form water crevices supported by the biophysical surface accessibility data. Overall, our multidisciplinary study demonstrates that the VSD of hERG1 may act as a cation-selective channel wherein a mutation of the first charged residue in the S4 generates an omega current. Our simulation uncovers the atomistic underpinning of this mechanism.

Identification of multiple substrate binding sites in SLC4 transporters in the outward-facing conformation: Insights into the transport mechanism.

Solute carrier family 4 (SLC4) transporters mediate the transmembrane transport of HCO3-, CO32-, and Cl- necessary for pH regulation, transepithelial H+/base transport, and ion homeostasis. Substrate transport with varying stoichiometry and specificity is achieved through an exchange mechanism and/or through coupling of the uptake of anionic substrates to typically co-transported Na+. Recently solved outward-facing structures of two SLC4 members (human anion exchanger 1 [hAE1] and human electrogenic sodium bicarbonate cotransporter 1 [hNBCe1]) with different transport modes (Cl-/HCO3- exchange versus Na+-CO32- symport) revealed highly conserved three-dimensional organization of their transmembrane domains. However, the exact location of the ion binding sites and their protein-ion coordination motifs are still unclear. In the present work, we combined site identification by ligand competitive saturation mapping and extensive molecular dynamics sampling with functional mutagenesis studies which led to the identification of two substrate binding sites (entry and central) in the outward-facing states of hAE1 and hNBCe1. Mutation of residues in the identified binding sites led to impaired transport in both proteins. We also showed that R730 in hAE1 is crucial for anion binding in both entry and central sites, whereas in hNBCe1, a Na+ acts as an anchor for CO32- binding to the central site. Additionally, protonation of the central acidic residues (E681 in hAE1 and D754 in hNBCe1) alters the ion dynamics in the permeation cavity and may contribute to the transport mode differences in SLC4 proteins. These results provide a basis for understanding the functional differences between hAE1 and hNBCe1 and may facilitate potential drug development for diseases such as proximal and distal renal tubular acidosis.

The Single Residue K12 Governs the Exceptional Voltage Sensitivity of Mitochondrial Voltage-Dependent Anion Channel Gating.

The voltage-dependent anion channel (VDAC) is a ß-barrel channel of the mitochondrial outer membrane (MOM) that passively transports ions, metabolites, polypeptides, and single-stranded DNA. VDAC responds to a transmembrane potential by "gating," i.e. transitioning to one of a variety of low-conducting states of unknown structure. The gated state results in nearly complete suppression of multivalent mitochondrial metabolite (such as ATP and ADP) transport, while enhancing calcium transport. Voltage gating is a universal property of ß-barrel channels, but VDAC gating is anomalously sensitive to transmembrane potential. Here, we show that a single residue in the pore interior, K12, is responsible for most of VDAC's voltage sensitivity. Using the analysis of over 40 µs of atomistic molecular dynamics (MD) simulations, we explore correlations between motions of charged residues inside the VDAC pore and geometric deformations of the ß-barrel. Residue K12 is bistable; its motions between two widely separated positions along the pore axis enhance the fluctuations of the ß-barrel and augment the likelihood of gating. Single channel electrophysiology of various K12 mutants reveals a dramatic reduction of the voltage-induced gating transitions. The crystal structure of the K12E mutant at a resolution of 2.6 Å indicates a similar architecture of the K12E mutant to the wild type; however, 60 µs of atomistic MD simulations using the K12E mutant show restricted motion of residue 12, due to enhanced connectivity with neighboring residues, and diminished amplitude of barrel motions. We conclude that ß-barrel fluctuations, governed particularly by residue K12, drive VDAC gating transitions.

Defective cell adhesion function of solute transporter, SLC4A11, in endothelial corneal dystrophies.

Corneal endothelial cell (CEnC) loss is often associated with blinding endothelial corneal dystrophies: dominantly inherited, common (5%) Fuchs endothelial corneal dystrophy (FECD) and recessive, rare congenital hereditary endothelial dystrophy (CHED). Mutations of SLC4A11, an abundant corneal solute transporter, cause CHED and some cases of FECD. The link between defective SLC4A11 solute transport function and CEnC loss is, however, unclear. Cell adhesion assays using SLC4A11-transfected HEK293 cells and primary human CEnC revealed that SLC4A11 promotes adhesion to components of Descemet's membrane (DM), the basement membrane layer to which CEnC bind. An antibody against SLC4A11 extracellular loop 3 (EL3) suppressed cell adhesion, identifying EL3 as the DM-binding site. Earlier studies showed that some SLC4A11 mutations cause FECD and CHED by impairing solute transport activity or cell surface trafficking. Without affecting these functions, FECD-causing mutations in SLC4A11-EL3 compromised cell adhesion capacity. In an energy-minimized SLC4A11-EL3 three-dimensional model, these mutations cluster and are buried within the EL3 structure. A GST fusion protein of SLC4A11-EL3 interacts with principal DM protein, COL8A2, as identified by mass spectrometry. Engineered SLC4A11-EL3-containing protein, STIC (SLC4A11-EL3 Transmembrane-GPA Integrated Chimera), promotes cell adhesion in transfected HEK293 cells and primary human CEnC, confirming the cell adhesion role of EL3. Taken together, the data suggest that SLC4A11 directly binds DM to serve as a cell adhesion molecule (CAM). These data further suggest that cell adhesion defects contribute to FECD and CHED pathology. Observations with STIC point toward a new therapeutic direction in these diseases: replacement of lost cell adhesion capacity.

A Computational Pipeline to Predict Cardiotoxicity: From the Atom to the Rhythm.

RATIONALE: Drug-induced proarrhythmia is so tightly associated with prolongation of the QT interval that QT prolongation is an accepted surrogate marker for arrhythmia. But QT interval is too sensitive a marker and not selective, resulting in many useful drugs eliminated in drug discovery. OBJECTIVE: To predict the impact of a drug from the drug chemistry on the cardiac rhythm. METHODS AND RESULTS: In a new linkage, we connected atomistic scale information to protein, cell, and tissue scales by predicting drug-binding affinities and rates from simulation of ion channel and drug structure interactions and then used these values to model drug effects on the hERG channel. Model components were integrated into predictive models at the cell and tissue scales to expose fundamental arrhythmia vulnerability mechanisms and complex interactions underlying emergent behaviors. Human clinical data were used for model framework validation and showed excellent agreement, demonstrating feasibility of a new approach for cardiotoxicity prediction. CONCLUSIONS: We present a multiscale model framework to predict electrotoxicity in the heart from the atom to the rhythm. Novel mechanistic insights emerged at all scales of the system, from the specific nature of proarrhythmic drug interaction with the hERG channel, to the fundamental cellular and tissue-level arrhythmia mechanisms. Applications of machine learning indicate necessary and sufficient parameters that predict arrhythmia vulnerability. We expect that the model framework may be expanded to make an impact in drug discovery, drug safety screening for a variety of compounds and targets, and in a variety of regulatory processes.

Refinement of a cryo-EM structure of hERG: Bridging structure and function.

The human-ether-a-go-go-related gene (hERG) encodes the voltage-gated potassium channel (KCNH2 or Kv11.1, commonly known as hERG). This channel plays a pivotal role in the stability of phase 3 repolarization of the cardiac action potential. Although a high-resolution cryo-EM structure is available for its depolarized (open) state, the structure surprisingly did not feature many functionally important interactions established by previous biochemical and electrophysiology experiments. Using molecular dynamics flexible fitting (MDFF), we refined the structure and recovered the missing functionally relevant salt bridges in hERG in its depolarized state. We also performed electrophysiology experiments to confirm the functional relevance of a novel salt bridge predicted by our refinement protocol. Our work shows how refinement of a high-resolution cryo-EM structure helps to bridge the existing gap between the structure and function in the voltage-sensing domain (VSD) of hERG.

Molecular determinants of pro-arrhythmia proclivity of d- and l-sotalol via a multi-scale modeling pipeline.

Drug isomers may differ in their proarrhythmia risk. An interesting example is the drug sotalol, an antiarrhythmic drug comprising d- and l- enantiomers that both block the hERG cardiac potassium channel and confer differing degrees of proarrhythmic risk. We developed a multi-scale in silico pipeline focusing on hERG channel - drug interactions and used it to probe and predict the mechanisms of pro-arrhythmia risks of the two enantiomers of sotalol. Molecular dynamics (MD) simulations predicted comparable hERG channel binding affinities for d- and l-sotalol, which were validated with electrophysiology experiments. MD derived thermodynamic and kinetic parameters were used to build multi-scale functional computational models of cardiac electrophysiology at the cell and tissue scales. Functional models were used to predict inactivated state binding affinities to recapitulate electrocardiogram (ECG) QT interval prolongation observed in clinical data. Our study demonstrates how modeling and simulation can be applied to predict drug effects from the atom to the rhythm for dl-sotalol and also increased proarrhythmia proclivity of d- vs. l-sotalol when accounting for stereospecific beta-adrenergic receptor blocking.

Toward Reducing hERG Affinities for DAT Inhibitors with a Combined Machine Learning and Molecular Modeling Approach.

Psychostimulant drugs, such as cocaine, inhibit dopamine reuptake via blockading the dopamine transporter (DAT), which is the primary mechanism underpinning their abuse. Atypical DAT inhibitors are dissimilar to cocaine and can block cocaine- or methamphetamine-induced behaviors, supporting their development as part of a treatment regimen for psychostimulant use disorders. When developing these atypical DAT inhibitors as medications, it is necessary to avoid off-target binding that can produce unwanted side effects or toxicities. In particular, the blockade of a potassium channel, human ether-a-go-go (hERG), can lead to potentially lethal ventricular tachycardia. In this study, we established a counter screening platform for DAT and against hERG binding by combining machine learning-based quantitative structure-activity relationship (QSAR) modeling, experimental validation, and molecular modeling and simulations. Our results show that the available data are adequate to establish robust QSAR models, as validated by chemical synthesis and pharmacological evaluation of a validation set of DAT inhibitors. Furthermore, the QSAR models based on subsets of the data according to experimental approaches used have predictive power as well, which opens the door to target specific functional states of a protein. Complementarily, our molecular modeling and simulations identified the structural elements responsible for a pair of DAT inhibitors having opposite binding affinity trends at DAT and hERG, which can be leveraged for rational optimization of lead atypical DAT inhibitors with desired pharmacological properties.

Emerging Diversity in Lipid-Protein Interactions.

Membrane lipids interact with proteins in a variety of ways, ranging from providing a stable membrane environment for proteins to being embedded in to detailed roles in complicated and well-regulated protein functions. Experimental and computational advances are converging in a rapidly expanding research area of lipid-protein interactions. Experimentally, the database of high-resolution membrane protein structures is growing, as are capabilities to identify the complex lipid composition of different membranes, to probe the challenging time and length scales of lipid-protein interactions, and to link lipid-protein interactions to protein function in a variety of proteins. Computationally, more accurate membrane models and more powerful computers now enable a detailed look at lipid-protein interactions and increasing overlap with experimental observations for validation and joint interpretation of simulation and experiment. Here we review papers that use computational approaches to study detailed lipid-protein interactions, together with brief experimental and physiological contexts, aiming at comprehensive coverage of simulation papers in the last five years. Overall, a complex picture of lipid-protein interactions emerges, through a range of mechanisms including modulation of the physical properties of the lipid environment, detailed chemical interactions between lipids and proteins, and key functional roles of very specific lipids binding to well-defined binding sites on proteins. Computationally, despite important limitations, molecular dynamics simulations with current computer power and theoretical models are now in an excellent position to answer detailed questions about lipid-protein interactions.

VDAC Gating Thermodynamics, but Not Gating Kinetics, Are Virtually Temperature Independent.

The voltage-dependent anion channel (VDAC) is the most abundant protein in the mitochondrial outer membrane and an archetypical ß-barrel channel. Here, we study the effects of temperature on VDAC channels reconstituted in planar lipid membranes at the single- and multichannel levels within the 20°C to 40°C range. The temperature dependence of conductance measured on a single channel in 1 M KCl shows an increase characterized by a 10°C temperature coefficient Q10 = 1.22 ± 0.02, which exceeds that of the bathing electrolyte solution conductivity, Q10 = 1.17 ± 0.01. The rates of voltage-induced channel transition between the open and closed states measured on multichannel membranes also show statistically significant increases, with temperatures that are consistent with activation energy barriers of ∼10 ± 3 kcal/mol. At the same time, the gating thermodynamics, as characterized by the gating charge and voltage of equipartitioning, does not display any measurable temperature dependence. The two parameters stay within 3.2 ± 0.2 elementary charges and 30 ± 2 mV, respectively. Thus, whereas the channel kinetics, specifically its conductance and rates of gating response to voltage steps, demonstrates a clear increase with temperature, the conformational voltage-dependent equilibria are virtually insensitive to temperature. These results, which may be a general feature of ß-barrel channel gating, suggest either an entropy-driven gating mechanism or a role for enthalpy-entropy compensation.

SLC4A11 function: evidence for H+(OH-) and NH3-H+ transport.

Whether SLC4A11 transports ammonia and its potential mode of ammonia transport (NH4+, NH3, or NH3-2H+ transport have been proposed) are controversial. In the absence of ammonia, whether SLC4A11 mediates significant conductive H+(OH-) transport is also controversial. The present study was performed to determine the mechanism of human SLC4A11 ammonia transport and whether the transporter mediates conductive H+(OH-) transport in the absence of ammonia. We quantitated H+ flux by monitoring changes in intracellular pH (pHi) and measured whole cell currents in patch-clamp studies of HEK293 cells expressing the transporter in the absence and presence of NH4Cl. Our results demonstrate that SLC4A11 mediated conductive H+(OH-) transport that was stimulated by raising the extracellular pH (pHe). Ammonia-induced HEK293 whole cell currents were also stimulated by an increase in pHe. In studies using increasing NH4Cl concentrations with equal NH4+ extracellular and intracellular concentrations, the shift in the reversal potential (Erev) due to the addition of ammonia was compatible with NH3-H+ transport competing with H+(OH-) rather than NH3-nH+ (n ≥ 2) transport. The increase in equivalent H+(OH-) flux observed in the presence of a transcellular H+ gradient was also compatible with SLC4A11-mediated NH3-H+ flux. The NH3 versus Erev data fit a theoretical model suggesting that NH3-H+ and H+(OH-) competitively interact with the transporter. Studies of mutant SLC4A11 constructs in the putative SLC4A11 ion coordination site showed that both H+(OH-) transport and ammonia-induced whole cell currents were blocked suggesting that the H+(OH-) and NH3-H+ transport processes share common features involving the SLC4A11 transport mechanism.

Assessing hERG1 Blockade from Bayesian Machine-Learning-Optimized Site Identification by Ligand Competitive Saturation Simulations.

Drug-induced cardiotoxicity is a potentially lethal and yet one of the most common side effects with the drugs in clinical use. Most of the drug-induced cardiotoxicity is associated with an off-target pharmacological blockade of K+ currents carried out by the cardiac Human-Ether-a-go-go-Related (hERG1) potassium channel. There is a compulsory preclinical stage safety assessment for the hERG1 blockade for all classes of drugs, which adds substantially to the cost of drug development. The availability of a high-resolution cryogenic electron microscopy (cryo-EM) structure for the channel in its open/depolarized state solved in 2017 enabled the application of molecular modeling for rapid assessment of drug blockade by molecular docking and simulation techniques. More importantly, if successful, in silico methods may allow a path to lead-compound salvaging by mapping out key block determinants. Here, we report the blind application of the site identification by the ligand competitive saturation (SILCS) protocol to map out druggable/regulatory hotspots in the hERG1 channel available for blockers and activators. The SILCS simulations use small solutes representative of common functional groups to sample the chemical space for the entire protein and its environment using all-atom simulations. The resulting chemical maps, FragMaps, explicitly account for receptor flexibility, protein-fragment interactions, and fragment desolvation penalty allowing for rapid ranking of potential ligands as blockers or nonblockers of hERG1. To illustrate the power of the approach, SILCS was applied to a test set of 55 blockers with diverse chemical scaffolds and pIC50 values measured under uniform conditions. The original SILCS model was based on the all-atom modeling of the hERG1 channel in an explicit lipid bilayer and was further augmented with a Bayesian-optimization/machine-learning (BML) stage employing an independent literature-derived training set of 163 molecules. BML approach was used to determine weighting factors for the FragMaps contributions to the scoring function. pIC50 predictions from the combined SILCS/BML approach to the 55 blockers showed a Pearson correlation (PC) coefficient of >0.535 relative to the experimental data. SILCS/BML model was shown to yield substantially improved performance as compared to commonly used rigid and flexible molecular docking methods for a well-established cohort of hERG1 blockers, where no correlation with experimental data was recorded. SILCS/BML results also suggest that a proper weighting of protonation states of common blockers present at physiological pH is essential for accurate predictions of blocker potency. The precalculated and optimized SILCS FragMaps can now be used for the rapid screening of small molecules for their cardiotoxic potential as well as for exploring alternative binding pockets in the hERG1 channel with applications to the rational design of activators.

Mapping of Ion and Substrate Binding Sites in Human Sodium Iodide Symporter (hNIS).

The human sodium iodide symporter (hNIS) is a theranostic reporter gene which concentrates several clinically approved SPECT and PET radiotracers and plays an essential role for the synthesis of thyroid hormones as an iodide transporter in the thyroid gland. Development of hNIS mutants which could enhance translocation of the desired imaging ions is currently underway. Unfortunately, it is hindered by lack of understanding of the 3D organization of hNIS and its relation to anion transport. There are no known crystal structures of hNIS in any of its conformational states. Homology modeling can be very effective in such situations; however, the low sequence identity between hNIS and relevant secondary transporters with available experimental structures makes the choice of a template and the generation of 3D models nontrivial. Here, we report a combined application of homology modeling and molecular dynamics refining of the hNIS structure in its semioccluded state. The modeling was based on templates from the LeuT-fold protein family and was done with emphasis on the refinement of the substrate-ion binding pocket. The consensus model developed in this work is compared to available biophysical and biochemical experimental data for a number of different LeuT-fold proteins. Some functionally important residues contributing to the formation of putative binding sites and permeation pathways for the cotransported Na+ ions and I- substrate were identified. The model predictions were experimentally tested by generation of mutant versions of hNIS and measurement of relative (to WT hNIS) 125I- uptake of 35 hNIS variants.

Benchmarking polarizable and non-polarizable force fields for Ca2+-peptides against a comprehensive QM dataset.

Explicit description of atomic polarizability is critical for the accurate treatment of inter-molecular interactions by force fields (FFs) in molecular dynamics (MD) simulations aiming to investigate complex electrostatic environments such as metal-binding sites of metalloproteins. Several models exist to describe key monovalent and divalent cations interacting with proteins. Many of these models have been developed from ion-amino-acid interactions and/or aqueous-phase data on cation solvation. The transferability of these models to cation-protein interactions remains uncertain. Herein, we assess the accuracy of existing FFs by their abilities to reproduce hierarchies of thousands of Ca2+-dipeptide interaction energies based on density-functional theory calculations. We find that the Drude polarizable FF, prior to any parameterization, better approximates the QM interaction energies than any of the non-polarizable FFs. Nevertheless, it required improvement in order to address polarization catastrophes where, at short Ca2+-carboxylate distances, the Drude particle of oxygen overlaps with the divalent cation. To ameliorate this, we identified those conformational properties that produced the poorest prediction of interaction energies to reduce the parameter space for optimization. We then optimized the selected cation-peptide parameters using Boltzmann-weighted fitting and evaluated the resulting parameters in MD simulations of the N-lobe of calmodulin. We also parameterized and evaluated the CTPOL FF, which incorporates charge-transfer and polarization effects in additive FFs. This work shows how QM-driven parameter development, followed by testing in condensed-phase simulations, may yield FFs that can accurately capture the structure and dynamics of ion-protein interactions.

Structural features and lipid binding domain of tubulin on biomimetic mitochondrial membranes.

Dimeric tubulin, an abundant water-soluble cytosolic protein known primarily for its role in the cytoskeleton, is routinely found to be associated with mitochondrial outer membranes, although the structure and physiological role of mitochondria-bound tubulin are still unknown. There is also no consensus on whether tubulin is a peripheral membrane protein or is integrated into the outer mitochondrial membrane. Here the results of five independent techniques-surface plasmon resonance, electrochemical impedance spectroscopy, bilayer overtone analysis, neutron reflectometry, and molecular dynamics simulations-suggest that α-tubulin's amphipathic helix H10 is responsible for peripheral binding of dimeric tubulin to biomimetic "mitochondrial" membranes in a manner that differentiates between the two primary lipid headgroups found in mitochondrial membranes, phosphatidylethanolamine and phosphatidylcholine. The identification of the tubulin dimer orientation and membrane-binding domain represents an essential step toward our understanding of the complex mechanisms by which tubulin interacts with integral proteins of the mitochondrial outer membrane and is important for the structure-inspired design of tubulin-targeting agents.

The Pore-Lipid Interface: Role of Amino-Acid Determinants of Lipophilic Access by Ivabradine to the hERG1 Pore Domain.

Abnormal cardiac electrical activity is a common side effect caused by unintended block of the promiscuous drug target human ether-à-go-go-related gene (hERG1), the pore-forming domain of the delayed rectifier K+ channel in the heart. hERG1 block leads to a prolongation of the QT interval, a phase of the cardiac cycle that underlies myocyte repolarization detectable on the electrocardiogram. Even newly released drugs such as heart-rate lowering agent ivabradine block the rapid delayed rectifier current IKr, prolong action potential duration, and induce potentially lethal arrhythmia known as torsades de pointes. In this study, we describe a critical drug-binding pocket located at the lateral pore surface facing the cellular membrane. Mutations of the conserved M651 residue alter ivabradine-induced block but not by the common hERG1 blocker dofetilide. As revealed by molecular dynamics simulations, binding of ivabradine to a lipophilic pore access site is coupled to a state-dependent reorientation of aromatic residues F557 and F656 in the S5 and S6 helices. We show that the M651 mutation impedes state-dependent dynamics of F557 and F656 aromatic cassettes at the protein-lipid interface, which has a potential to disrupt drug-induced block of the channel. This fundamentally new mechanism coupling the channel dynamics and small-molecule access from the membrane into the hERG1 intracavitary site provides a simple rationale for the well established state-dependence of drug blockade. SIGNIFICANCE STATEMENT: The drug interference with the function of the cardiac hERG channels represents one of the major sources of drug-induced heart disturbances. We found a novel and a critical drug-binding pocket adjacent to a lipid-facing surface of the hERG1 channel, which furthers our molecular understanding of drug-induced QT syndrome.

Nonequilibrium path-ensemble averages for symmetric protocols.

According to the nonequilibrium work relations, path-ensembles generated by irreversible processes in which a system is driven out of equilibrium according to a predetermined protocol may be used to compute equilibrium free energy differences and expectation values. Estimation has previously been improved by considering data collected from the reverse process, which starts in equilibrium in the final thermodynamic state of the forward process and is driven according to the time-reversed protocol. Here, we develop a theoretically rigorous statistical estimator for nonequilibrium path-ensemble averages specialized for symmetric protocols, in which forward and reverse processes are identical. The estimator is tested with a number of model systems: a symmetric 1D potential, an asymmetric 1D potential, the unfolding of deca-alanine, separating a host-guest system, and translocating a potassium ion through a gramicidin A ion channel. When reconstructing free energies using data from symmetric protocols, the new estimator outperforms existing rigorous unidirectional and bidirectional estimators, converging more quickly and resulting in a smaller error. However, in most cases, using the bidirectional estimator with data from a forward and reverse pair of asymmetric protocols outperforms the corresponding symmetric protocol and estimator with the same amount of simulation time. Hence, the new estimator is only recommended when the bidirectional estimator is not feasible or is expected to perform poorly. The symmetric estimator shows similar performance to a unidirectional protocol of half the length and twice the number of trajectories.