Charged Binding Sites Research Articles

Small Molecule Modulator of the mTORC2 Pathway Discovered from a DEL Library Designed to Bind to Pleckstrin Homology Domains.

Pleckstrin homology (PH) domains are structural motifs critical for cellular processes, such as signal transduction and cytoskeletal organization. Due to their involvement in various diseases, PH domains are promising therapeutic targets, yet their highly charged and hydrophobic binding sites are not ideal for traditional small drugs. In this study, we designed a DNA-encoded library (DEL) mimicking phospholipids to identify novel modulators targeting PH domains with uncharted chemical properties. Screening against several PH domains led to the discovery of 2DII, a small molecule that selectively binds to mSin1PH. This compound can modulate mTORC2 activity by impairing mTORC2's membrane interactions, resulting in reduced AKT1 phosphorylation. A micromapping via Dexter energy transfer based on 2DII bearing an iridium catalyst (2DII-Ir), along with a biotin-diazirine small molecule was used for target identification by proteomics, which confirmed mSin1 as the primary intracellular target of 2DII, demonstrating its potential for selective mTORC2 pathway modulation. These findings introduce a novel strategy for targeting PH domains and provide a foundation for the development of therapeutic interventions that modulate PH-domain-dependent signaling pathways.

PH-Dependent Assembly and Stability of Toll-Like Receptor 3/dsRNA Signaling Complex: Insights from Constant pH Molecular Dynamics and Metadynamics Simulations.

The pH-dependent assembly of Toll-like receptors (TLRs), which triggers a threshold-like response, is a key principle in immune signaling. While crystallography has revealed the intricate structure of these assembly complexes, the mechanisms underlying their pH dependency remain unclear. Herein, constant pH simulations and metadynamics are employed to investigate the pH-dependent assembly and stability of the TLR3/dsRNA signaling complex. The findings demonstrate that system pH regulates complex assembly and stability by modulating the protonation and charge states of histidines. Histidines in TLR3 act as pH-dependent, positively charged binding sites that capture negatively charged dsRNA. Additionally, these histidines form a [H682⁺]-[E626⁻] dipole, facilitating the assembly of two TLR3 molecules into an antisymmetric dimer through dipole-dipole interactions. Surprisingly, TLR3 can shift the pKa values of key histidines from their model pKa of 6.5, increasing protonation likelihood and enhancing ligand binding. Notably, the aromatic residue Phe84, located within the dsRNA binding site [His39⁺-His60⁺-Phe84-His108⁺], alters the pKa of His60 through cation-π interactions with its protonated state. This study offers new insights into the molecular mechanisms underlying pH-dependent immune signaling via higher-order assemblies and suggests potential applications for histidine in self-assembling biomaterials.

Membrane potential stimulates ADP import and ATP export by the mitochondrial ADP/ATP carrier due to its positively charged binding site.

The mitochondrial adenosine 5'-diphosphate (ADP)/adenosine 5'-triphosphate (ATP) carrier imports ADP into the mitochondrion and exports ATP to the cell. Here, we demonstrate that 3.3 positive charges are translocated with the negatively charged substrate in each transport step. They can be assigned to three positively charged residues of the central substrate-binding site and two asparagine/arginine pairs. In this way, the membrane potential stimulates not only the ATP4- export step, as a net -0.7 charge is transported, but also the ADP3- import step, as a net +0.3 charge is transported with the electric field. These positive charge movements also inhibit the import of ATP and export of ADP in the presence of a membrane potential, allowing these nucleotides to be maintained at high concentrations in the cytosol and mitochondrial matrix to drive the hydrolysis and synthesis of ATP, respectively. Thus, this is the mechanism by which the membrane potential drives adenine nucleotide exchange with high directional fluxes to fuel the cellular processes.

Unravelling the metal uptake process in mosses: Comparison of aquatic and terrestrial species as air pollution biomonitors

Transplanted mosses have been widely shown to be excellent tools for biomonitoring air pollution; however, it is not clear how the functional groups present on their surfaces affect the uptake of metal cations. In the present study, we examined differences in trace metal accumulation in two terrestrial and one aquatic moss species, and investigated whether the differences depended on their physico-chemical characteristics. In the laboratory, we determined C, N and H contents in their tissues and obtained the ATR-FTIR spectra (to identify the presence of functional groups). We also conducted surface acid-base titrations and metal adsorption assays with Cd, Cu and Pb. In the field, we exposed transplants of each species near different air-polluting industries, and determined the mosses enrichment of Al, Cd, Co, Cr, Cu, Fe, Ni, Pb and V. Laboratory results demonstrated higher metal uptake capacity in the terrestrial mosses Sphagnum palustre and Pseudoscleropodium purum, compared to that in the aquatic moss Fontinalis antipyretica, which can be attributed to a greater abundance of acidic functional groups (i.e. negatively charged binding sites) on the surface of the terrestrial mosses. The affinity of moss for certain elements depends on the abundance and nature of surface functional groups. Accordingly, the metal concentrations generally reached higher levels in S. palustre transplants compared to the other species, except for the uptake of Hg, which was higher in F. antipyretica. However, the findings also suggest an interaction between the type of environment (terrestrial or aquatic) and the moss characteristics that may influence the abovementioned trend. Thus, irrespective of the physico-chemical characteristics, metal uptake varied depending on the environment of origin of the mosses “i.e. atmospheric or aquatic”. In other words, the findings suggest that species that accumulate more metals in terrestrial environments will accumulate lower amounts of metals in aquatic environments and vice versa.

Inhibition of AdeB, AceI, and AmvA Efflux Pumps Restores Chlorhexidine and Benzalkonium Susceptibility in Acinetobacter baumannii ATCC 19606.

The management of infections caused by Acinetobacter baumannii is hindered by its intrinsic tolerance to a wide variety of biocides. The aim of the study was to analyze the role of different A. baumannii efflux pumps (EPs) in tolerance to chlorhexidine (CHX) and benzalkonium (BZK) and identify non-toxic compounds, which can restore susceptibility to CHX and BZK in A. baumannii. A. baumannii ATCC 19606 strain was tolerant to both CHX and BZK with MIC and MBC value of 32 mg/L. CHX subMIC concentrations increased the expression of adeB and adeJ (RND superfamily), aceI (PACE family) and amvA (MFS superfamily) EP genes. The values of CHX MIC and MBC decreased by eightfold in ΔadeB and twofold in ΔamvA or ΔaceI mutants, respectively, while not affected in ΔadeJ mutant; EPs double and triple deletion mutants showed an additive effect on CHX MIC. CHX susceptibility was restored in double and triple deletion mutants with inactivation of adeB gene. BZK MIC was decreased by fourfold in ΔadeB mutant, and twofold in ΔamvA and ΔaceI mutants, respectively; EPs double and triple deletion mutants showed an additive effect on BZK MIC. BZK susceptibility was recovered in ΔadeB ΔaceI ΔadeJ and ΔamvA ΔadeB ΔadeJ triple mutants. The structural comparison of AdeB and AdeJ protomers showed a more negatively charged entrance binding site and F-loop in AdeB, which may favor the transport of CHX. The carbonyl cyanide m-chlorophenylhydrazine protonophore (CCCP) EP inhibitor reduced dose-dependently CHX MIC in A. baumannii ATCC 19606 and in ΔadeJ, ΔaceI, or ΔamvA mutants, but not in ΔadeB mutant. Either piperine (PIP) or resveratrol (RV) at non-toxic concentrations inhibited CHX MIC in A. baumannii ATCC 19606 parental strain and EPs gene deletion mutants, and CHX-induced EP gene expression. Also, RV inhibited BZK MIC and EP genes expression in A. baumannii ATCC 19606 parental strain and EPs mutants. These results demonstrate that tolerance to CHX and BZK in A. baumannii is mediated by the activation of AdeB, AceI and AmvA EPs, AdeB playing a major role. Importantly, inhibition of EP genes expression by RV restores CHX and BZK susceptibility in A. baumannii.

Straightforward Incorporation of Multiple Ligand Types into Molecular Dynamics Simulations for Efficient Binding Site Detection and Characterization.

Binding site identification and characterization is an important initial step in structure-based drug design. To account for the effects of protein flexibility and solvation, several cosolvent molecular dynamics (MD) simulation methods that incorporate small organic molecules into the protein's solvent box to probe for binding sites have been developed. However, most of these methods are highly inefficient, as they allow for the use of only one probe type at a time, which means that multiple sets of simulations have to be performed to map different types of binding sites. The high probe concentrations used in some of these methods also necessitate the use of artificial repulsive forces to prevent the probes from aggregating. Here, we present multiple-ligand-mapping MD (mLMMD), a method that incorporates multiple types of probes for simultaneous and efficient mapping of different types of binding sites without the need for introduction of artificial forces that may cause unintended mapping artifacts. We validate the method on a diverse set of 10 proteins and show that the mLMMD probes are able to reliably identify hydrophobic, hydrogen-bonding, charged, and cryptic binding sites in all of the test cases. Our results also highlight the potential utility of mLMMD for virtual screening and rational drug design.

Abstract LB-A20: True small molecule STING agonist generates systemic and potent anti-tumor immunity required for effective cancer immunotherapy

Abstract STING (Stimulator of Interferon Genes) is a master regulator of type I interferons and a key mediator of innate immunity. Activation of STING provides two critical anti-tumor responses – (1) The “spark” for initiating a robust innate immune response and (2) Priming and activation of a potent T cell mediated anti-tumor adaptive response. By serving as a bridge between the two arms of the immune system, activation of STING has the potential to transform the tumor microenvironment from immunologically “cold” to “hot” (inflamed), making otherwise resistant tumors respond to checkpoint blockade as well as other T cell targeting immunotherapies. Many preclinical and clinical drug discovery programs targeting STING activation are based on modifications of the naturally occurring cyclic dinucleotide (CDN) agonists. However, the CDN molecules suffer from poor drug-like properties and consequently cannot be delivered systemically. Thus, the benefits of STING activation by these first generation CDNs will be limited to only a subset of tumors that are accessible to intratumoral injection. The large, polar and charged binding site as well as the mechanism of activation of STING present unique drug discovery challenges to designing a small molecule that can induce the optimal, active conformation of the STING dimer while maintaining the physical-chemical properties required for systemic exposure. Using our proprietary physics-driven discovery engine with our integrative chemistry/biophysics/biology approach we have developed novel small molecule agonists of STING that exhibit selective and potent activity across all known isoforms of the human protein. Our small molecule agonist rapidly activates the STING/TBK1/IRF3 pathway (phosphorylation and translocation) and induces the expression of type I IFN in cells in a STING-dependent manner. Treatment of primary immune cells from healthy human donors results in the activation of dendritic cells and upregulation of costimulatory markers. Drug characteristics and STING pharmacology allow for unique dosing paradigm in vivo with robust induction of type I IFN after intravenous (i.v.) administration. A single i.v. dose produces potent and durable anti-tumor immunity and complete tumor regression in mice bearing syngeneic tumors. Activation of tumor directed T cell response provides long term protection against tumor re-challenge and effective systemic immunity. Our work highlights the tremendous potential of a small molecule STING agonist as a well-tolerated, systemically delivered cancer immunotherapeutic. Citation Format: Meghana M Kulkarni, Timothy Dwight, Kristen Marino, Bryce K Allen, Mohammed Taimi, Cecilia Bastos, Sujen Lai, Cheryl Koh, Ramya Samant, Bethany Tesar, Huang Huang, Brian Chamberlain, James M Rice, Dazhi Tan, Zhixiong Lin, Sharon Shechter, Stella Li, Sam Sparks, Holly Soutter, Steven Swann, Woody Sherman, Christopher Winter. True small molecule STING agonist generates systemic and potent anti-tumor immunity required for effective cancer immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A20. doi:10.1158/1535-7163.TARG-19-LB-A20

Fate of selenium in soil: A case study in a maize (Zea mays L.) field under two irrigation regimes and fertilized with sodium selenite

Selenium (Se) is a trace element necessary for both human and livestock nutrition. To increase Se human intake, soil Se fertilizations were performed but the fate of the added Se remains unclear. The present research aims to: (1) determine the influence of Se fertilization on the fractionation of Se in soil; (2) assess the influence of water availability on the distribution of soil Se chemical fractions; and (3) monitor the Se content in soil, leachates and plants. To reach these goals, 200 g Se ha−1 was applied to soil as sodium selenite in maize crops under two irrigation regimes, and the Se content in plant, soil chemical fractions and leachates were analyzed. Se application increased the total Se content of the soil, specifically it increased the Se content of the soluble, exchangeable and organic fractions with more pronounced effect in the soils with higher water availability. These differences disappeared over time likely due to the Se loss through volatilization. The hypothesis of Se volatilization is confirmed by the absence of both leachates during the maize growing season and differences among the treatments of Se content in sub-soil samples. Also, although the Se treated plants showed higher Se content than the untreated ones, overall <1% of the added Se was assimilated by plants. Hence, this study demonstrated that the addition of selenite to the soil increased the Se contents of the plants, but the Se does not accumulate in the soil because it is likely lost via volatilization. Further, leaching of Se into groundwater is avoided due to its association with both the soil organic matter and positively charged binding sites of soil, and due to its loss via volatilization. Therefore, soil Se fertilization could increase the nutritional value of plants without consequences on the environment.

Effects of electrostatic interactions on ligand dissociation kinetics.

We study unbinding of multivalent cationic ligands from oppositely charged polymeric binding sites sparsely grafted on a flat neutral substrate. Our molecular dynamics simulations are suggested by single-molecule studies of protein-DNA interactions. We consider univalent salt concentrations spanning roughly a 1000-fold range, together with various concentrations of excess ligands in solution. To reveal the ionic effects on unbinding kinetics of spontaneous and facilitated dissociation mechanisms, we treat electrostatic interactions both at a Debye-Hückel (DH) (or implicit ions, i.e., use of an electrostatic potential with a prescribed decay length) level and by the more precise approach of considering all ionic species explicitly in the simulations. We find that the DH approach systematically overestimates unbinding rates, relative to the calculations where all ion pairs are present explicitly in solution, although many aspects of the two types of calculation are qualitatively similar. For facilitated dissociation (FD) (acceleration of unbinding by free ligands in solution) explicit-ion simulations lead to unbinding at lower free-ligand concentrations. Our simulations predict a variety of FD regimes as a function of free-ligand and ion concentrations; a particularly interesting regime is at intermediate concentrations of ligands where nonelectrostatic binding strength controls FD. We conclude that explicit-ion electrostatic modeling is an essential component to quantitatively tackle problems in molecular ligand dissociation, including nucleic-acid-binding proteins.

Identification of Gold Sensing Peptide by Integrative Proteomics and a Bacterial Two-Component System.

The proteomics strategy was utilized to analyze and identify the gold adsorption proteins from Tepidimonas fonticaldi AT-A2, due to its outstanding performance in gold-binding and recovery. The results showed that three small proteins, including histidine biosynthesis protein (HisIE), iron donor protein (CyaY) and hypothetical protein_65aa, have a higher ability to adsorb gold ions because of the negatively charged domains or metal binding sites. On the other hand, the Salmonella PmrA/PmrB two-component system first replaces the iron (III)-binding motif using the peptide sequence from hypothetical protein_65aa, and this is then used to reveal the sensing and responsiveness to gold metal ions, which is totally different from the performance of traditional gold binding peptide (GBP) on the crystals on the surface of gold (111). We have successfully demonstrated an integrative proteomics and bacterial two-component system to explore the novel GBP. Finally, the heterologous over-expression of GBP by E. coli and the equilibrium of binding capacity for Au(III) have been conducted.

The Sam-Sam interaction between Ship2 and the EphA2 receptor: design and analysis of peptide inhibitors

The lipid phosphatase Ship2 represents a drug discovery target for the treatment of different diseases, including cancer. Its C-terminal sterile alpha motif domain (Ship2-Sam) associates with the Sam domain from the EphA2 receptor (EphA2-Sam). This interaction is expected to mainly induce pro-oncogenic effects in cells therefore, inhibition of the Ship2-Sam/EphA2-Sam complex may represent an innovative route to discover anti-cancer therapeutics. In the present work, we designed and analyzed several peptide sequences encompassing the interaction interface of EphA2-Sam for Ship2-Sam. Peptide conformational analyses and interaction assays with Ship2-Sam conducted through diverse techniques (CD, NMR, SPR and MST), identified a positively charged penta-amino acid native motif in EphA2-Sam, that once repeated three times in tandem, binds Ship2-Sam. NMR experiments show that the peptide targets the negatively charged binding site of Ship2-Sam for EphA2-Sam. Preliminary in vitro cell-based assays indicate that -at 50 µM concentration- it induces necrosis of PC-3 prostate cancer cells with more cytotoxic effect on cancer cells than on normal dermal fibroblasts. This work represents a pioneering study that opens further opportunities for the development of inhibitors of the Ship2-Sam/EphA2-Sam complex for therapeutic applications.

Molecular Interactions of a DNA Modifying Enzyme APOBEC3F Catalytic Domain with a Single-Stranded DNA

The single-stranded DNA (ssDNA) cytidine deaminase APOBEC3F (A3F) deaminates cytosine (C) to uracil (U) and is a known restriction factor of HIV-1. Its C-terminal catalytic domain (CD2) alone is capable of binding single-stranded nucleic acids and is important for deamination. However, little is known about how the CD2 interacts with ssDNA. Here we report a crystal structure of A3F-CD2 in complex with a 10-nucleotide ssDNA composed of poly-thymine, which reveals a novel positively charged nucleic acid binding site distal to the active center that plays a key role in substrate DNA binding and catalytic activity. Lysine and tyrosine residues within this binding site interact with the ssDNA, and mutating these residues dramatically impairs both ssDNA binding and catalytic activity. This binding site is not conserved in APOBEC3G (A3G), which may explain differences in ssDNA-binding characteristics between A3F-CD2 and A3G-CD2. In addition, we observed an alternative Zn-coordination conformation around the active center. These findings reveal the structural relationships between nucleic acid interactions and catalytic activity of A3F.

An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron.

Group II introns and non-LTR retrotransposons encode a phylogenetically related family of highly processive reverse transcriptases (RTs) that are essential for mobility and persistence of these retroelements. Recent crystallographic studies on members of this RT family have revealed that they are structurally distinct from the retroviral RTs that are typically used in biotechnology. However, quantitative, structure-guided analysis of processivity, efficiency, and accuracy of this alternate RT family has been lacking. Here, we characterize the processivity of a group II intron maturase RT from Eubacterium rectale (E.r.), for which high-resolution structural information is available. We find that the E.r. maturase RT (MarathonRT) efficiently copies transcripts at least 10 kb in length and displays superior intrinsic RT processivity compared to commercial enzymes such as Superscript IV (SSIV). The elevated processivity of MarathonRT is at least partly mediated by a loop structure in the finger subdomain that acts as a steric guard (the α-loop). Additionally, we find that a positively charged secondary RNA binding site on the surface of the RT diminishes the primer utilization efficiency of the enzyme, and that reengineering of this surface enhances capabilities of the MarathonRT. Finally, using single-molecule sequencing, we show that the error frequency of MarathonRT is comparable to that of other high-performance RTs, such as SSIV, which were tested in parallel. Our results provide a structural framework for understanding the enhanced processivity of retroelement RTs, and they demonstrate the potential for engineering a powerful new generation of RT tools for application in biotechnology and research.

Novel insights into the regulation of coagulation by factor V isoforms, tissue factor pathway inhibitorα, and protein S

Factor V (FV) is a regulator of both pro- and anticoagulant pathways. It circulates as a single-chain procofactor, which is activated by thrombin or FXa to FVa that serves as cofactor for FXa in prothrombin activation. The cofactor function of FVa is regulated by activated protein C (APC) and protein S. FV can also function as an anticoagulant APC cofactor in the inhibition of FVIIIa in the membrane-bound tenase complex (FIXa/FVIIIa). In recent years, it has become clear that FV also functions in multiple ways in the tissue factor pathway inhibitor (TFPI) anticoagulant pathway. Of particular importance is a FV splice variant (FV-Short) that serves as a carrier and cofactor to TFPIα in the inhibition of FXa. FV-Short is generated through alternative splicing of exon 13 that encodes the large activation B domain. A highly negatively charged binding site for TFPIα is exposed in the C-terminus of the FV-Short B domain, which binds the positively charged C-terminus of TFPIα, thus keeping TFPIα in circulation. The binding of TFPIα to FV-Short is also instrumental in localizing the inhibitor to the surface of negatively charged phospholipids, where TFPIα inhibits FXa in process that is stimulated by protein S. Plasma FV activation intermediates and partially proteolyzed platelet FV similarly bind TFPIα with high affinity and regulate formation of prothrombinase. The novel insights gained into the interaction between FV isoforms, TFPIα, and protein S have opened a new avenue for research about the mechanisms of coagulation regulation and also for future development of therapeutics aimed at modulating coagulation.

Examining Nanoparticle Adsorption on Electrostatically "Patchy" Glycopolymer Brushes Using Real-Time ζ-Potential Measurements.

Biomaterial surfaces can possess chemical, topographical, or electrostatic heterogeneity, which can profoundly influence their performance. By developing experimental models that reliably simulate this nanoscale heterogeneity, we can predict how heterogeneous surfaces are transformed by their interactions with the dynamic physiological environment. In this work, we present a model surface where well-defined glycopolymer brushes are interspersed with positively charged binding sites, giving rise to an interface presenting a mixture of repulsive and adhesive cues to an approaching virus particle. We show that the density of the affinity sites relative to the glycopolymer brushes can be tuned precisely by modifying the chemical vapor deposition (CVD) copolymerization conditions. Further, we examined the effects of binding site density and glycopolymer brush architecture on the adsorption kinetics of virus-like nanoparticles through a novel approach employing time-resolved ζ-potential measurements. Most materials have charge-bearing, dynamic surfaces that are sensitive to electrostatic effects. Hence, adsorption-triggered changes in ζ-potential measurements can be captured in real time to monitor interfacial events. Real-time ζ-potential measurements present an interesting platform to probe the structure and function of chemically and electrostatically heterogeneous polymer interfaces. To validate this electrokinetic method, we examined the effect of neutravidin concentration on its rate of binding to biotinylated surfaces using ζ-potential and compared our results with QCM studies. By applying electrokinetic methods to examine the roles of glycopolymer brush architecture and surface charge of these tunable glycopolymer coatings, we can enhance our understanding of the interactions of viruses with heterogeneous biomaterial interfaces.

Triazolium cyclodextrin click cluster-resin conjugate: an enrichment material for phosphatidylinositol (3,4,5)-triphosphate.

UltraLink was functionalized with a triazolium cyclodextrin click cluster (CCC) which provides a well-oriented, multivalent, positively charged binding site for PtdIns(3,4,5)P3. MALDI TOF MS and LC ESI MS/MS MRM analysis of spiked PtdIns(3,4,5)P3 in lipid extract suggest that triazolium CCC-UltraLink conjugate can be used as an enrichment material for PtdIns(3,4,5)P3.

Deciphering the binding behaviours of BSA using ionic AIE-active fluorescent probes.

The binding behaviours of a transport protein, bovine serum albumin (BSA), in its native, unfolding and refolding states have been probed by monitoring the emission changes of two exogenous AIE-active fluorescent probes, M2 and M3, which are designed to be anionic and cationic, respectively. Due to their AIE properties, both M2 and M3 display emission enhancement when bound to the hydrophobic cavity of BSA. The binding site of M2 and M3 is found to be subdomain IIA. Then, the BSA + M2 and BSA + M3 systems are utilized to fluorescently signal the conformation changes of BSA caused by various external stimuli, including thermally or chemically induced denaturation. The data confirmed the multi-step unfolding process and the existence of a molten-globule intermediate state. The unfolding process consists of the rearrangement of subdomain IIA, the exposure of a negatively charged binding site in domain I that prefers interacting with cationic species, and the transformation of the molten-globule intermediate into the final random coil. The anionic and cationic modifications of the probes enable us to observe that electrostatic interactions play a role in the folding and unfolding of BSA.

Rapid approximate calculation of water binding free energies in the whole hydration domain of (bio)macromolecules.

The evaluation of water binding free energies around solute molecules is important for the thermodynamic characterization of hydration or association processes. Here, a rapid approximate method to estimate water binding free energies around (bio)macromolecules from a single molecular dynamics simulation is presented. The basic idea is that endpoint free-energy calculation methods are applied and the endpoint quantities are monitored on a three-dimensional grid around the solute. Thus, a gridded map of water binding free energies around the solute is obtained, that is, from a single short simulation, a map of favorable and unfavorable water binding sites can be constructed. Among the employed free-energy calculation methods, approaches involving endpoint information pertaining to actual thermodynamic integration calculations or endpoint information as exploited in the linear interaction energy method were examined. The accuracy of the approximate approaches was evaluated on the hydration of a cage-like molecule representing either a nonpolar, polar, or charged water binding site and on α- and β-cyclodextrin molecules. Among the tested approaches, the linear interaction energy method is considered the most viable approach. Applying the linear interaction energy method on the grid around the solute, a semi-quantitative thermodynamic characterization of hydration around the whole solute is obtained. Disadvantages are the approximate nature of the method and a limited flexibility of the solute. © 2016 Wiley Periodicals, Inc.

Tmc1 Point Mutation Affects Ca2+ Sensitivity and Block by Dihydrostreptomycin of the Mechanoelectrical Transducer Current of Mouse Outer Hair Cells.

In the auditory system, the hair cells convert sound-induced mechanical movement of the hair bundles atop these cells into electrical signals through the opening of mechanically gated ion channels at the tips of the bundles. Although the nature of these mechanoelectrical transducer (MET) channels is still unclear, recent studies implicate transmembrane channel-like protein isoform 1 (TMC1) channels in the mammalian cochlea. Using a mutant mouse model (Beethoven) for progressive hearing loss in humans (DFNA36), which harbors a point mutation in the Tmc1 gene, we show that this mutation affects the MET channel pore, reducing its Ca(2+) permeability and its affinity for the permeant blocker dihydrostreptomycin. A number of phenomena that we ascribe to Ca(2+)-dependent adaptation appear stronger, in compensation for the reduced Ca(2+) entry.

Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity

The structure-guided design of chloride-conducting channelrhodopsins has illuminated mechanisms underlying ion selectivity of this remarkable family of light-activated ion channels. The first generation of chloride-conducting channelrhodopsins, guided in part by development of a structure-informed electrostatic model for pore selectivity, included both the introduction of amino acids with positively charged side chains into the ion conduction pathway and the removal of residues hypothesized to support negatively charged binding sites for cations. Engineered channels indeed became chloride selective, reversing near -65 mV and enabling a new kind of optogenetic inhibition; however, these first-generation chloride-conducting channels displayed small photocurrents and were not tested for optogenetic inhibition of behavior. Here we report the validation and further development of the channelrhodopsin pore model via crystal structure-guided engineering of next-generation light-activated chloride channels (iC++) and a bistable variant (SwiChR++) with net photocurrents increased more than 15-fold under physiological conditions, reversal potential further decreased by another ∼ 15 mV, inhibition of spiking faithfully tracking chloride gradients and intrinsic cell properties, strong expression in vivo, and the initial microbial opsin channel-inhibitor-based control of freely moving behavior. We further show that inhibition by light-gated chloride channels is mediated mainly by shunting effects, which exert optogenetic control much more efficiently than the hyperpolarization induced by light-activated chloride pumps. The design and functional features of these next-generation chloride-conducting channelrhodopsins provide both chronic and acute timescale tools for reversible optogenetic inhibition, confirm fundamental predictions of the ion selectivity model, and further elucidate electrostatic and steric structure-function relationships of the light-gated pore.