Unveiling Tst3, a Multi-Target Gating Modifier Scorpion α Toxin from Tityus stigmurus Venom of Northeast Brazil: Evaluation and Comparison with Well-Studied Ts3 Toxin of Tityus serrulatus.

Studies on the interaction sites of peptide toxins and ion channels typically involve site-directed mutations in toxins. However, natural mutant toxins exist among them, offering insights into how the evolutionary process has conserved crucial sequences for activities and molecular target selection. In this study, we present a comparative investigation using electrophysiological approaches and computational analysis between two alpha toxins from evolutionarily close scorpion species of the genus Tityus, namely, Tst3 and Ts3 from T. stigmurus and T. serrulatus, respectively. These toxins exhibit three natural substitutions near the C-terminal region, which is directly involved in the interaction between alpha toxins and Nav channels. Additionally, we characterized the activity of the Tst3 toxin on Nav1.1-Nav1.7 channels. The three natural changes between the toxins did not alter sensitivity to Nav1.4, maintaining similar intensities regarding their ability to alter opening probabilities, delay fast inactivation, and induce persistent currents. Computational analysis demonstrated a preference for the down conformation of VSD4 and a shift in the conformational equilibrium towards this state. This illustrates that the sequence of these toxins retained the necessary information, even with alterations in the interaction site region. Through electrophysiological and computational analyses, screening of the Tst3 toxin on sodium isoform revealed its classification as a classic α-NaTx with a broad spectrum of activity. It effectively delays fast inactivation across all tested isoforms. Structural analysis of molecular energetics at the interface of the VSD4-Tst3 complex further confirmed this effect.

The binding and mechanism of a positive allosteric modulator of Kv3 channels.

Small-molecule modulators of diverse voltage-gated K+ (Kv) channels may help treat a wide range of neurological disorders. However, developing effective modulators requires understanding of their mechanism of action. We apply an orthogonal approach to elucidate the mechanism of action of an imidazolidinedione derivative (AUT5), a highly selective positive allosteric modulator of Kv3.1 and Kv3.2 channels. AUT5 modulation involves positive cooperativity and preferential stabilization of the open state. The cryo-EM structure of the Kv3.1/AUT5 complex at a resolution of 2.5 Å reveals four equivalent AUT5 binding sites at the extracellular inter-subunit interface between the voltage-sensing and pore domains of the channel's tetrameric assembly. Furthermore, we show that the unique extracellular turret regions of Kv3.1 and Kv3.2 essentially govern the selective positive modulation by AUT5. High-resolution apo and bound structures of Kv3.1 demonstrate how AUT5 binding promotes turret rearrangements and interactions with the voltage-sensing domain to favor the open conformation.

α-Alkylidene δ-lactones inhibit quorum sensing phenotypes in Chromobacterium strain CV026 showing interaction with the CviR receptor.

Disruption of bacterial quorum sensing (QS) is presented as a promising strategy to overcome clinically relevant and phytopathogenic bacteria. This work presents α-alkylidene δ-lactones as new chemical scaffolds that inhibit the biosynthesis of violacein in the biosensor strain Chromobacterium CV026. Three molecules displayed higher than 50% violacein reduction when tested at concentrations lower than 625 µM. The most active α-alkylidene δ-lactone inhibited the hydrolysis of chitin concomitantly with the inhibition of violacein production in CV026, suggesting the disruption of its QS machinery. Further, RT-qPCR and competition experiments showed this molecule to be a transcriptional inhibitor of the QS-regulated operon vioABCDE. Docking calculations suggested a good correlation between binding affinity energies and inhibition effects, with all molecules positioned within the CviR autoinducer-binding domain (AIBD). The most active lactone yielded the best binding affinity energy, most probably due to its unprecedented binding with the AIBD. Our results show α-alkylidene δ-lactones as promising chemical scaffolds for the development of new QS inhibitors affecting LuxR/LuxI-systems.

Allosteric Modulation of Membrane Proteins by Small Low-Affinity Ligands.

Membrane proteins may respond to a variety of ligands under an applied external stimulus. These ligands include small low-affinity molecules that account for functional effects in the mM range. Understanding the modulation of protein function by low-affinity ligands requires characterizing their atomic-level interactions under dilution, challenging the current resolution of theoretical and experimental routines. Part of the problem derives from the fact that small low-affinity ligands may interact with multiple sites of a membrane protein in a highly degenerate manner to a degree that it is better conceived as a partition phenomenon, hard to track at the molecular interface of the protein. Looking for new developments in the field, we rely on the classic two-state Boltzmann model to devise a novel theoretical description of the allosteric modulation mechanism of membrane proteins in the presence of small low-affinity ligands and external stimuli. Free energy stability of the partition process and its energetic influence on the protein coupling with the external stimulus are quantified. The outcome is a simple formulation that allows the description of the equilibrium shifts of the protein in terms of the grand-canonical partition function of the ligand at dilute concentrations. The model's predictions of the spatial distribution and response probability shift across a variety of ligand concentrations, and thermodynamic conjugates can be directly compared to macroscopic measurements, making it especially useful to interpret experimental data at the atomic level. Illustration and discussion of the theory is shown in the context of general anesthetics and voltage-gated channels for which structural data are available.

Concentration-dependent thermodynamic analysis of the partition process of small ligands into proteins.

In the category of functional low-affinity interactions, small ligands may interact with multiple protein sites in a highly degenerate manner. Better conceived as a partition phenomenon at the molecular interface of proteins, such low-affinity interactions appear to be hidden to our current experimental resolution making their structural and functional characterization difficult in the low concentration regime of physiological processes. Characterization of the partition phenomenon under higher chemical forces could be a relevant strategy to tackle the problem provided the results can be scaled back to the low concentration range. Far from being trivial, such scaling demands a concentration-dependent understanding of self-interactions of the ligands, structural perturbations of the protein, among other molecular effects. Accordingly, we elaborate a novel and detailed concentration-dependent thermodynamic analysis of the partition process of small ligands aiming at characterizing the stability and structure of the dilute phenomenon from high concentrations. In analogy to an "aggregate" binding constant of a small molecule over multiple sites of a protein receptor, the model defines the stability of the process as a macroscopic equilibrium constant for the partition number of ligands that can be used to analyze biochemical and functional data of two-component systems driven by low-affinity interactions. Acquisition of such modeling-based structural information is expected to be highly welcome by revealing more traceable protein-binding spots for non-specific ligands.

Trivial and nontrivial error sources account for misidentification of protein partners in mutual information approaches.

The problem of finding the correct set of partners for a given pair of interacting protein families based on multi-sequence alignments (MSAs) has received great attention over the years. Recently, the native contacts of two interacting proteins were shown to store the strongest mutual information (MI) signal to discriminate MSA concatenations with the largest fraction of correct pairings. Although that signal might be of practical relevance in the search for an effective heuristic to solve the problem, the number of MSA concatenations with near-native MI is large, imposing severe limitations. Here, a Genetic Algorithm that explores possible MSA concatenations according to a MI maximization criteria is shown to find degenerate solutions with two error sources, arising from mismatches among (i) similar and (ii) non-similar sequences. If mistakes made among similar sequences are disregarded, type-(i) solutions are found to resolve correct pairings at best true positive (TP) rates of 70%-far above the very same estimates in type-(ii) solutions. A machine learning classification algorithm helps to show further that differences between optimized solutions based on TP rates are not artificial and may have biological meaning associated with the three-dimensional distribution of the MI signal. Type-(i) solutions may therefore correspond to reliable results for predictive purposes, found here to be more likely obtained via MI maximization across protein systems having a minimum critical number of amino acid contacts on their interaction surfaces (N > 200).

Donepezil Inhibits Acetylcholinesterase via Multiple Binding Modes at Room Temperature.

Donepezil is a second generation acetylcholinesterase (AChE) inhibitor for treatment of Alzheimer's disease (AD). AChE is important for neurotransmission at neuromuscular junctions and cholinergic brain synapses by hydrolyzing acetylcholine into acetate and choline. In vitro data support that donepezil is a reversible, mixed competitive and noncompetitive inhibitor of AChE. The experimental fact then suggests a more complex binding mechanism beyond the molecular view in X-ray models resolved at cryogenic temperatures that show a unique binding mode of donepezil in the active site of the enzyme. Aiming at clarifying the mechanism behind that mixed competitive and noncompetitive nature of the inhibitor, we have applied molecular dynamics (MD) simulations and docking and free-energy calculations to investigate microscopic details and energetics of donepezil association for conditions of substrate-free and -bound states of the enzyme. Liquid-phase MD simulation at room temperature shows AChE transits between "open" and "closed" conformations to control accessibility to the active site and ligand binding. As shown by docking and free-energy calculations, association of donepezil involves its reversible axial displacement and reorientation in the active site of the enzyme, assisted by water molecules. Donepezil binds equally well the main-door anionic binding site PAS, the acyl pocket, and the catalytic site CAS by respectively adopting outward-inward-inward orientations regardless of substrate occupancy-the overall stability of that reaction process depends however on co-occupancy of the enzyme being preferential for its substrate-free state. All together, our findings support a physiologically relevant mechanism of AChE inhibition by donepezil involving multistable interactions modes at the molecular origin of the inhibitor's activity.

Nucleosome binding peptide presents laudable biophysical and in vivo effects.

Chromatin state is highly dependent on the nucleosome binding proteins. Herein, we used a multipronged approach employing biophysical and in vivo experiments to characterize the effects of Nucleosome Binding Peptides (NBPeps) on nucleosome and cell activity. We performed a series of structure-based calculations on the nucleosome surface interaction with GMIP1 (a novel NBPep generated in silico), and HMGN2 (nucleosome binding motif of HMGN2), which contains sites that bind DNA and the acid patch, and also LANA and H4pep (nucleosome binding motif of H4 histone tail) that only bind to the acidic patch. Biochemical assays shows that H4pep, but not HMGN2, GMIP1 and LANA, is highly specific for targeting the nucleosome, with important effects on the final nucleosome structure and robust in vivo effects. These findings suggest that NBPeps might have important therapeutic implications and relevance as tools for chromatin investigation.

Coevolutive, evolutive and stochastic information in protein-protein interactions.

Here, we investigate the contributions of coevolutive, evolutive and stochastic information in determining protein-protein interactions (PPIs) based on primary sequences of two interacting protein families A and B. Specifically, under the assumption that coevolutive information is imprinted on the interacting amino acids of two proteins in contrast to other (evolutive and stochastic) sources spread over their sequences, we dissect those contributions in terms of compensatory mutations at physically-coupled and uncoupled amino acids of A and B. We find that physically-coupled amino-acids at short range distances store the largest per-contact mutual information content, with a significant fraction of that content resulting from coevolutive sources alone. The information stored in coupled amino acids is shown further to discriminate multi-sequence alignments (MSAs) with the largest expectation fraction of PPI matches - a conclusion that holds against various definitions of intermolecular contacts and binding modes. When compared to the informational content resulting from evolution at long-range interactions, the mutual information in physically-coupled amino-acids is the strongest signal to distinguish PPIs derived from cospeciation and likely, the unique indication in case of molecular coevolution in independent genomes as the evolutive information must vanish for uncorrelated proteins.

Atomistic Model for Simulations of the Sedative Hypnotic Drug 2,2,2-Trichloroethanol.

2,2,2-Trichloroethanol (TCE) is the active form of the sedative hypnotic drug chloral hydrate, one of the oldest sleep medications in the market. Understanding of TCE's action mechanisms to its many targets, particularly within the ion channel family, could benefit from the state-of-the-art computational molecular studies. In this direction, we employed de novo modeling aided by the force field toolkit to develop CHARMM36-compatible TCE parameters. The classical potential energy function was calibrated targeting molecular conformations, local interactions with water molecules, and liquid bulk properties. Reference data comes from both tabulated thermodynamic properties and ab initio calculations at the MP2 level. TCE solvation free energy calculations in water and oil reproduce a lipophilic, yet nonhydrophobic, behavior. Indeed, the potential mean force profile for TCE partition through the phospholipid bilayer reveals the sedative's preference for the interfacial region. The calculated partition coefficient also matches experimental measures. Further validation of the proposed parameters is supported by the model's ability to recapitulate quenching experiments demonstrating TCE binding to bovine serum albumin.

Binding of the general anesthetic sevoflurane to ion channels.

The direct-site hypothesis assumes general anesthetics bind ion channels to impact protein equilibrium and function, inducing anesthesia. Despite advancements in the field, a first principle all-atom demonstration of this structure-function premise is still missing. We focus on the clinically used sevoflurane interaction to anesthetic-sensitive Kv1.2 mammalian channel to resolve if sevoflurane binds protein's well-characterized open and closed structures in a conformation-dependent manner to shift channel equilibrium. We employ an innovative approach relying on extensive docking calculations and free-energy perturbation of all potential binding sites revealed by the latter, and find sevoflurane binds open and closed structures at multiple sites under complex saturation and concentration effects. Results point to a non-trivial interplay of site and conformation-dependent modes of action involving distinct binding sites that increase channel open-probability at diluted ligand concentrations. Given the challenge in exploring more complex processes potentially impacting channel-anesthetic interaction, the result is revealing as it demonstrates the process of multiple anesthetic binding events alone may account for open-probability shifts recorded in measurements.

Fat nucleosome: Role of lipids on chromatin.

Structural changes in chromatin regulate gene expression and define phenotypic outcomes. Molecules that bind to the nucleosome, the complex of DNA and histone proteins, are key modulators of chromatin structure. Most recently, the formation of condensed chromatin regions based on phase-separation in the cell, a basic physical mechanism, was proposed. Increased understanding of the mechanisms of interaction between chromatin and lipids suggest that small lipid molecules, such as cholesterol and short-chain fatty acids, can regulate important nuclear functions. New biophysical data has suggested that cholesterol interacts with nucleosome through multiple binding sites and affects chromatin structure in vitro. Regardless of the mechanism of how lipids bind to chromatin, there is currently little awareness that lipids may be stored in chromatin and influence its state. Focusing on lipids that bind to nuclear receptors, clinically relevant transcription factors, we discuss the potential interactions of the nucleosome with steroid hormones, bile acids and fatty acids, which suggest that other lipid chemotypes may also impact chromatin structure through binding to common sites on the nucleosome. Herein, we review the main impacts of lipids on the nuclear environment, emphasizing its role on chromatin architecture. We postulate that lipids that bind to nucleosomes and affect chromatin states are likely to be worth investigating as tools to modify disease phenotypes at a molecular level.

Concentration-Dependent Binding of Small Ligands to Multiple Saturable Sites in Membrane Proteins.

Membrane proteins are primary targets for most therapeutic indications in cancer and neurological diseases, binding over 50% of all known small molecule drugs. Understanding how such ligands impact membrane proteins requires knowledge on the molecular structure of ligand binding, a reasoning that has driven relentless efforts in drug discovery and translational research. Binding of small ligands appears however highly complex involving interaction to multiple transmembrane protein sites featuring single or multiple occupancy states. Within this scenario, looking for new developments in the field, we investigate the concentration-dependent binding of ligands to multiple saturable sites in membrane proteins. The study relying on docking and free-energy perturbation provides us with an extensive description of the probability density of protein-ligand states that allows for computation of thermodynamic properties of interest. It also provides one- and three-dimensional spatial descriptions for the ligand density across the protein-membrane system which can be of interest for structural purposes. Illustration and discussion of the results are shown for binding of the general anesthetic sevoflurane against Kv1.2, a mammalian ion channel for which experimental data are available.

Biophysical studies of cholesterol effects on chromatin.

Changes in chromatin structure regulate gene expression and genome maintenance. Molecules that bind to the nucleosome, the complex of DNA and histone proteins, are key modulators of chromatin structure. Previous work indicated that cholesterol, a ubiquitous cellular lipid, may bind to chromatin in vivo, suggesting a potential function for lipids in modulating chromatin architecture. However, the molecular mechanisms of cholesterol's action on chromatin structure have remained unclear. Here, we explored the biophysical impact of cholesterol on nucleosome and chromatin fibers reconstituted in vitro and characterized in silico the cholesterol binding to the nucleosome. Our findings support that cholesterol assists 10 and 30 nm chromatin formation and induces folding of long chromatin fibers as a result of direct interaction of the cholesterol to six nucleosomal binding sites.

Mechanistic Insights into the Modulation of Voltage-Gated Ion Channels by Inhalational Anesthetics.

General anesthesia is a relatively safe medical procedure, which for nearly 170 years has allowed life saving surgical interventions in animals and people. However, the molecular mechanism of general anesthesia continues to be a matter of importance and debate. A favored hypothesis proposes that general anesthesia results from direct multisite interactions with multiple and diverse ion channels in the brain. Neurotransmitter-gated ion channels and two-pore K+ channels are key players in the mechanism of anesthesia; however, new studies have also implicated voltage-gated ion channels. Recent biophysical and structural studies of Na+ and K+ channels strongly suggest that halogenated inhalational general anesthetics interact with gates and pore regions of these ion channels to modulate function. Here, we review these studies and provide a perspective to stimulate further advances.

Positive Allosteric Modulation of Kv Channels by Sevoflurane: Insights into the Structural Basis of Inhaled Anesthetic Action.

Inhalational general anesthesia results from the poorly understood interactions of haloethers with multiple protein targets, which prominently includes ion channels in the nervous system. Previously, we reported that the commonly used inhaled anesthetic sevoflurane potentiates the activity of voltage-gated K+ (Kv) channels, specifically, several mammalian Kv1 channels and the Drosophila K-Shaw2 channel. Also, previous work suggested that the S4-S5 linker of K-Shaw2 plays a role in the inhibition of this Kv channel by n-alcohols and inhaled anesthetics. Here, we hypothesized that the S4-S5 linker is also a determinant of the potentiation of Kv1.2 and K-Shaw2 by sevoflurane. Following functional expression of these Kv channels in Xenopus oocytes, we found that converse mutations in Kv1.2 (G329T) and K-Shaw2 (T330G) dramatically enhance and inhibit the potentiation of the corresponding conductances by sevoflurane, respectively. Additionally, Kv1.2-G329T impairs voltage-dependent gating, which suggests that Kv1.2 modulation by sevoflurane is tied to gating in a state-dependent manner. Toward creating a minimal Kv1.2 structural model displaying the putative sevoflurane binding sites, we also found that the positive modulations of Kv1.2 and Kv1.2-G329T by sevoflurane and other general anesthetics are T1-independent. In contrast, the positive sevoflurane modulation of K-Shaw2 is T1-dependent. In silico docking and molecular dynamics-based free-energy calculations suggest that sevoflurane occupies distinct sites near the S4-S5 linker, the pore domain and around the external selectivity filter. We conclude that the positive allosteric modulation of the Kv channels by sevoflurane involves separable processes and multiple sites within regions intimately involved in channel gating.

Electric fingerprint of voltage sensor domains.

A dynamic transmembrane voltage field has been suggested as an intrinsic element in voltage sensor (VS) domains. Here, the dynamic field contribution to the VS energetics was analyzed via electrostatic calculations applied to a number of atomistic structures made available recently. We find that the field is largely static along with the molecular motions of the domain, and more importantly, it is minimally modified across VS variants. This finding implies that sensor domains transfer approximately the same amount of gating charges when moving the electrically charged S4 helix between fixed microscopic configurations. Remarkably, the result means that the observed operational diversity of the domain, including the extension, rate, and voltage dependence of the S4 motion, as dictated by the free energy landscape theory, must be rationalized in terms of dominant variations of its chemical free energy.

Pore waters regulate ion permeation in a calcium release-activated calcium channel.

The recent crystal structure of Orai, the pore unit of a calcium release-activated calcium (CRAC) channel, is used as the starting point for molecular dynamics and free-energy calculations designed to probe this channel's conduction properties. In free molecular dynamics simulations, cations localize preferentially at the extracellular channel entrance near the ring of Glu residues identified in the crystal structure, whereas anions localize in the basic intracellular half of the pore. To begin to understand ion permeation, the potential of mean force (PMF) was calculated for displacing a single Na(+) ion along the pore of the CRAC channel. The computed PMF indicates that the central hydrophobic region provides the major hindrance for ion diffusion along the permeation pathway, thereby illustrating the nonconducting nature of the crystal structure conformation. Strikingly, further PMF calculations demonstrate that the mutation V174A decreases the free energy barrier for conduction, rendering the channel effectively open. This seemingly dramatic effect of mutating a nonpolar residue for a smaller nonpolar residue in the pore hydrophobic region suggests an important role for the latter in conduction. Indeed, our computations show that even without significant channel-gating motions, a subtle change in the number of pore waters is sufficient to reshape the local electrostatic field and modulate the energetics of conduction, a result that rationalizes recent experimental findings. The present work suggests the activation mechanism for the wild-type CRAC channel is likely regulated by the number of pore waters and hence pore hydration governs the conductance.

Conduction in a biological sodium selective channel.

The crystal structure of NavAb, a bacterial voltage gated Na(+) channel, exhibits a selectivity filter (SF) wider than that of K(+) channels. This new structure provides the opportunity to explore the mechanism of conduction and help rationalize its selectivity for sodium. Recent molecular dynamics (MD) simulations of single- and two-ion permeation processes have revealed that a partially hydrated Na(+) permeates the channel by exploring three SF binding sites while being loosely coupled to other ions and/or water molecules; a finding that differs significantly from the behavior of K(+) selective channels. Herein, we present results derived from a combination of metadynamics and voltage-biased MD simulations that throws more light on the nature of the Na(+) conduction mechanism. Conduction under 0 mV bias explores several distinct pathways involving the binding of two ions to three possible SF sites. While these pathways are very similar to those observed in the presence of a negative potential (inward conduction), a completely different mechanism operates for outward conduction at positive potentials.