Divalent Cation Binding Research Articles

Cobalt ion interaction with TMEM16A calcium-activated chloride channel: Inhibition and potentiation.

TMEM16A, a Ca2+-sensitive Cl- channel, plays key roles in many physiological functions related to Cl- transport across lipid membranes. Activation of this channel is mediated via binding intracellular Ca2+ to the channel with a relatively high apparent affinity, roughly in the sub-μM to low μM concentration range. Recently available high-resolution structures of TMEM16 molecules reveal that the high-affinity Ca2+ activation sites are formed by several acidic amino acids, using their negatively charged sidechain carboxylates to coordinate the bound Ca2+. In this study, we examine the interaction of TMEM16A with a divalent cation, Co2+, which by itself cannot activate current in TMEM16A. This divalent cation, however, has two effects when applied intracellularly. It inhibits the Ca2+-induced TMEM16A current by competing with Ca2+ for the aforementioned high-affinity activation sites. In addition, Co2+ also potentiates the Ca2+-induced current with a low affinity. This potentiation effect requires high concentration (mM) of Co2+, similar to our previous findings that high concentrations (mM) of intracellular Ca2+ ([Ca2+]i) can induce more TMEM16A current after the Ca2+-activation sites are saturated by tens of μM [Ca2+]i. The degrees of potentiation by Co2+ and Ca2+ also roughly correlate with each other. Interestingly, mutating a pore residue of TMEM16A, Y589, alters the degree of potentiation in that the smaller the sidechain of the replaced residue, the larger the potentiation induced by divalent cations. We suggest that the Co2+ potentiation and the Ca2+ potentiation share a similar mechanism by increasing Cl- flux through the channel pore, perhaps due to an increase of positive pore potential after the binding of divalent cations to phospholipids in the pore. A smaller sidechain of a pore residue may allow the pore to accommodate more phospholipids, thus enhancing the current potentiation caused by high concentrations of divalent cations.

Regulation of Leishmaniamajor PAS domain-containing phosphoglycerate kinase by cofactor Mg2+ ion at neutral pH.

Recently, we described the PAS domain-containing phosphoglycerate kinase (PGK) from Leishmaniamajor (LmPAS-PGK) that shows acidic pH (5.5)-dependent optimum catalytic activity. The PAS domain of LmPAS-PGK is expected to regulate PGK activity during catalysis, but the mechanism of regulation by PAS domain at the molecular level is uncharacterized. In this work, we have utilized the full-length, PAS domain-deleted, and mutant enzymes to measure the enzymatic activity in the presence of divalent cation at various pH values. Catalytic activity measurement indicates that Mg2+ binding through PAS domain inhibits the PGK activity at pH 7.5, and this inhibition is withdrawn at pH 5.5. To identify the Mg2+ binding residues of the PAS domain, we exploited a systematic mutational analysis of all (four) His residues in the PAS domain for potential divalent cation binding. Replacement of His-57 with alanine resulted in depression in the presence of Mg2+ at pH 7.5, but H71A, H89A, and H111A showed similar characteristics with respect to the wild-type protein. Fluorescence and isothermal titration calorimetry studies revealed that H57 is responsible for Mg2+ binding in the absence of substrates. Thus, the protonated form of His57 at acidic pH 5.5 destabilizes the Mg2+ binding in the PAS domain, which is an essential requirement in the wild-type LmPAS-PGK for a conformational alteration in the sensor domain that, sequentially, activates the PGK domain, resulting in the synthesis of higher amounts of ATP.

Free-energy landscapes of membrane co-translocational protein unfolding

Protein post-translational translocation is found at the plasma membrane of prokaryotes and protein import into organellae. Translocon structures are becoming available, however the dynamics of proteins during membrane translocation remain largely obscure. Here we study, at the single-molecule level, the folding landscape of a model protein while forced to translocate a transmembrane pore. We use a DNA tag to drive the protein into the α-hemolysin pore under a quantifiable force produced by an applied electric potential. Using a voltage-quench approach we find that the protein fluctuates between the native state and an intermediate in the translocation process at estimated forces as low as 1.9 pN. The fluctuation kinetics provide the free energy landscape as a function of force. We show that our stable, ≈15 kBT, substrate can be unfolded and translocated with physiological membrane potentials and that selective divalent cation binding may have a profound effect on the translocation kinetics.

Comparing Nonbonded Metal Ion Models in the Divalent Cation Binding Protein PsaA.

Divalent metal cations are essential for many biological processes; however, accurately modeling divalent metal ions has proved a significant challenge for molecular dynamics force fields. Here we show that the choice of ion model influences the observed dynamics in PsaA, a metal binding protein from Streptococcus pneumoniae. We conduct extensive unbiased simulations and free energy calculations of PsaA bound to its cognate ligand Mn2+ and inhibitory ligand Zn2+ using three nonbonded ion models: a 12-6 model, a 12-6-4 model, and a multisite model. The observed coordination geometries and metal binding dynamics are sensitive to the choice of ion model, with the most dramatic differences observed in free energy calculations of ion release. We show that the conformational ensemble of Mn-bound PsaA is more similar to the crystallographic metal bound open state. This work extends the current model of PsaA metal binding and provides a framework for the rationalization of experimentally determined metal binding behavior. Our findings support the use of the 12-6-4 ion model for further simulations of divalent cation binding proteins.

Effects of Cation Binding to the Intracellular Vestibule of TMEM16 Ion Transport Pathways

TMEM16A and TMEM16F are prototype molecules for the two functional categories of TMEM16 family members, respectively, Ca2+-activated chloride channels and phospholipid scramblases. Upon binding of sub-µM to low µM intracellular Ca2+, TMEM16A conducts anionic currents while TMEM16F is permeable to both cations and anions. In this study, we found another divalent cation, Co2+, while unable to induce current in TMEM16A, inhibits the Ca2+-induced current via competing with Ca2+ for the high-affinity (sub-µM to low µM) channel activation sites. In addition, Co2+ also potentiates the TMEM16A current induced by Ca2+. All alkaline earth divalent cations, including Ca2+ itself, can generate this current potentiation with low affinities—at least hundreds of µM to mM concentrations are required for observing the effect. On the other hand, divalent cations at mM concentrations generate very little potentiation in TMEM16F, likely because the changes in local anion and cation concentrations caused by divalent cation bindings cancel out each other. Nonetheless, divalent cations change the cation/anion selectivity for the TMEM16F current conduction. Manipulating membrane phospholipids alters this low-affinity effect of divalent cations. Recent studies have shown that membrane phospholipids participate in various gating and permeation functions of TMEM16 molecules. Our results suggest that intracellular divalent cations bind to membrane phospholipids with a low affinity to alter the electrostatic potential near or in the ion-transport pathways of TMEM16 molecules. Monovalent cations and polyamine molecules also exert similar effects, although with different apparent affinities. The physiological significance of the TMEM16 current modulation by altering membrane surface potential will be discussed.

Binding of divalent cations to acetate: molecular simulations guided by Raman spectroscopy.

In spite of the biological importance of the binding of Zn2+, Ca2+, and Mg2+ to the carboxylate group, cation-acetate binding affinities and binding modes remain actively debated. Here, we report the first use of Raman multivariate curve resolution (Raman-MCR) vibrational spectroscopy to obtain self-consistent free and bound metal acetate spectra and one-to-one binding constants, without the need to invoke any a priori assumptions regarding the shapes of the corresponding vibrational bands. The experimental results, combined with classical molecular dynamics simulations with a force field effectively accounting for electronic polarization via charge scaling and ab initio simulations, indicate that the measured binding constants pertain to direct (as opposed to water separated) ion pairing. The resulting binding constants do not scale with cation size, as the binding constant to Zn2+ is significantly larger than that to either Mg2+ or Ca2+, although Zn2+ and Mg2+ have similar radii that are about 25% smaller than Ca2+. Remaining uncertainties in the metal acetate binding free energies are linked to fundamental ambiguities associated with identifying the range of structures pertaining to non-covalently bound species.

Array-based Investigation of Amino Acids Responsible for Regulation of Transamidase and Kinase Activities of Transglutaminase 2

Transglutaminase 2 (TGase2) is involved in a variety of cellular processes and diseases via its transamidase and kinase activities, which are regulated by conformational changes induced by the binding of nucleotides and divalent cations. However, due to the lack of an appropriate assay system, the function of critical amino acid residues in the regulation of both activities is unclear. Thus, we designed site-directed TGase2 mutants that were then used in protein arrays to investigate the effects of the mutations on the regulation of TGase2 transamidase and kinase activities. We found that the Lys444Ala mutation, but not the Arg580Lys and Lys663Ala mutations, completely inhibited the transamidase activity. Additionally, the mutations at Lys444 and Lys663 inhibited the kinase activity by 27% and 48%, respectively, but the mutations at Cys277 and Arg580 had no effect. Furthermore, a kinetic analysis of the transamidation reaction revealed that the Lys663Ala mutation increased the affinity of TGase2 for the substrate fibrinogen. Thus, this array-based approach would be helpful for investigation of amino acids responsible for regulation of the TGase2 transamidase and kinase activities and the pathogenesis of TGase2- mediated diseases.

Human Serine Racemase: Key Residues/Active Site Motifs and Their Relation to Enzyme Function.

Serine racemase (SR) is the first racemase enzyme to be identified in human biology and converts L-serine to D-serine, an important neuronal signaling molecule that serves as a co-agonist of the NMDA (N-methyl-D-aspartate) receptor. This overview describes key molecular features of the enzyme, focusing on the side chains and binding motifs that control PLP (pyridoxal phosphate) cofactor binding as well as activity modulation through the binding of both divalent cations and ATP, the latter showing allosteric modulation. Discussed are catalytically important residues in the active site including K56 and S84—the si- and re-face bases, respectively,—and R135, a residue that appears to play a critical role in the binding of both negatively charged alternative substrates and inhibitors. The interesting bifurcated mechanism followed by this enzyme whereby substrate L-serine can be channeled either into D-serine (racemization pathway) or into pyruvate (β-elimination pathway) is discussed extensively, as are studies that focus on a key loop region (the so-called “triple serine loop”), the modification of which can be used to invert the normal in vitro preference of this enzyme for the latter pathway over the former. The possible cross-talk between the PLP enzymes hSR and hCBS (human cystathionine β-synthase) is discussed, as the former produces D-serine and the latter produces H2S, both of which stimulate the NMDAR and both of which have been implicated in neuronal infarction pursuant to ischemic stroke. Efforts to gain a more complete mechanistic understanding of these PLP enzymes are expected to provide valuable insights for the development of specific small molecule modulators of these enzymes as tools to study their roles in neuronal signaling and in modulation of NMDAR function.

Predicting Site-Binding Modes of Ions and Water to Nucleic Acids Using Molecular Solvation Theory.

Site binding of ions and water shapes nucleic acids folding, dynamics, and biological function, complementing the more diffuse, nonspecific "territorial" ion binding. Unlike territorial binding, prediction of site-specific binding to nucleic acids remains an unsolved challenge in computational biophysics. This work presents a new toolset based on the 3D-RISM molecular solvation theory and topological analysis that predicts cation and water site binding to nucleic acids. 3D-RISM is shown to accurately capture alkali cations and water binding to the central channel, transversal loops, and grooves of the Oxytricha nova's telomeres' G-quadruplex ( Oxy-GQ), in agreement with high-resolution crystallographic data. To improve the computed cation occupancy along the Oxy-GQ central channel, it was necessary to refine and validate new cation-oxygen parameters using structural and thermodynamic data available for crown ethers and ion channels. This single set of parameters that describes both localized and delocalized binding to various biological systems is used to gain insight into cation occupancy along the Oxy-GQ channel under various salt conditions. The paper concludes with prospects for extending the method to predict divalent cation binding to nucleic acids. This work advances the forefront of theoretical methods able to provide predictive insight into ion atmosphere effects on nucleic acids function.

Voltage-dependent dynamics of the BK channel cytosolic gating ring are coupled to the membrane-embedded voltage sensor.

In humans, large conductance voltage- and calcium-dependent potassium (BK) channels are regulated allosterically by transmembrane voltage and intracellular Ca2+. Divalent cation binding sites reside within the gating ring formed by two Regulator of Conductance of Potassium (RCK) domains per subunit. Using patch-clamp fluorometry, we show that Ca2+ binding to the RCK1 domain triggers gating ring rearrangements that depend on transmembrane voltage. Because the gating ring is outside the electric field, this voltage sensitivity must originate from coupling to the voltage-dependent channel opening, the voltage sensor or both. Here we demonstrate that alterations of the voltage sensor, either by mutagenesis or regulation by auxiliary subunits, are paralleled by changes in the voltage dependence of the gating ring movements, whereas modifications of the relative open probability are not. These results strongly suggest that conformational changes of RCK1 domains are specifically coupled to the voltage sensor function during allosteric modulation of BK channels.

A conserved cation binding site in the DNA binding domain of forkhead box transcription factors regulates DNA binding by FOXP2

FOXP2 is a transcriptional repressor involved in development of the human brain and is the first gene product to be linked to the evolution of human speech. FOXP2 belongs to the FOX superfamily of proteins that share a common winged helix DNA binding domain – the forkhead domain. A divalent cation (Mg2+ or Ca2+) has been identified bound to a group of highly conserved residues in a number of FOX forkhead domain crystal structures. This work aims to investigate the role of the conserved divalent cation binding site by studying both the structure and DNA-binding function of the FOXP2 forkhead domain when in the presence and absence of either cation (Mg2+or Ca2+). The presence of the cations does not significantly alter the structure of the apo-FOXP2 forkhead domain. However, when in the presence of a cognate oligonucleotide sequence, differences are observed upon addition of divalent cation. These differences occur both in the structure and in the thermodynamic DNA binding signature of the FOXP2 forkhead domain. The incorporation of molecular dynamics simulations together with the experimental data provides us with sufficient insight so as to propose a possible role for divalent cations in the regulation of DNA binding to FOX transcription factors.

Divalent cation and chloride ion sites of chicken acid sensing ion channel 1a elucidated by x-ray crystallography

Acid sensing ion channels (ASICs) are proton-gated ion channels that are members of the degenerin/epithelial sodium channel superfamily and are expressed throughout central and peripheral nervous systems. ASICs have been implicated in multiple physiological processes and are subject to numerous forms of endogenous and exogenous regulation that include modulation by Ca2+ and Cl- ions. However, the mapping of ion binding sites as well as a structure-based understanding of the mechanisms underlying ionic modulation of ASICs have remained elusive. Here we present ion binding sites of chicken ASIC1a in resting and desensitized states at high and low pH, respectively, determined by anomalous diffraction x-ray crystallography. The acidic pocket serves as a nexus for divalent cation binding at both low and high pH, while we observe divalent cation binding within the central vestibule on the resting channel at high pH only. Moreover, neutralization of residues positioned to coordinate divalent cations via individual and combined Glu to Gln substitutions reduced, but did not extinguish, modulation of proton-dependent gating by Ca2+. Additionally, we demonstrate that anion binding at the canonical thumb domain site is state-dependent and present a previously undetected anion site at the mouth of the extracellular fenestrations on the resting channel. Our results map anion and cation sites on ASICs across multiple functional states, informing possible mechanisms of modulation and providing a blueprint for the design of therapeutics targeting ASICs.

Cezomycin Is Activated by CalC to Its Ester Form for Further Biosynthesis Steps in the Production of Calcimycin in Streptomyces chartreusis NRRL 3882.

Calcimycin, N-demethyl calcimycin, and cezomycin are polyether divalent cation ionophore secondary metabolites produced by Streptomyces chartreusis A thorough understanding of the organization of their encoding genes, biosynthetic pathway(s), and cation specificities is vitally important for their efficient future production and therapeutic use. So far, this has been lacking, as has information concerning any biosynthetic relationships that may exist between calcimycin and cezomycin. In this study, we observed that when a Cal- (calB1 mutant) derivative of a calcimycin-producing strain of S. chartreusis (NRRL 3882) was grown on cezomycin, calcimycin production was restored. This suggested that calcimycin synthesis may have resulted from postsynthetic modification of cezomycin rather than from a de novo process through a novel and independent biosynthetic mechanism. Systematic screening of a number of Cal-S. chartreusis mutants lacking the ability to convert cezomycin to calcimycin allowed the identification of a gene, provisionally named calC, which was involved in the conversion step. Molecular cloning and heterologous expression of the CalC protein along with its purification to homogeneity and negative-staining electron microscopy allowed the determination of its apparent molecular weight, oligomeric forms in solution, and activity. These experiments allowed us to confirm that the protein possessed ATP pyrophosphatase activity and was capable of ligating coenzyme A (CoA) with cezomycin but not 3-hydroxyanthranilic acid. The CalC protein's apparent Km and kcat for cezomycin were observed to be 190 μM and 3.98 min-1, respectively, and it possessed the oligomeric form in solution. Our results unequivocally show that cezomycin is postsynthetically modified to calcimycin by the CalC protein through its activation of cezomycin to a CoA ester form.IMPORTANCE Calcimycin is a secondary metabolite divalent cation-ionophore that has been studied in the context of human health. However, detail is lacking with respect to both calcimycin's biosynthesis and its biochemical/biophysical properties as well as information regarding its, and its analogues', divalent cation binding specificities and other activities. Such knowledge would be useful in understanding how calcimycin and related compounds may be effective in modifying the calcium channel ion flux and might be useful in influencing the homeostasis of magnesium and manganese ions for the cure or control of human and bacterial infectious diseases. The results presented here unequivocally show that CalC protein is essential for the production of calcimycin, which is essentially a derivative of cezomycin, and allow us to propose a biosynthetic mechanism for calcimycin's production.

Utilizing homology of Wound Inducible Transcript 3.0 (WIT3) as a stepping stone to investigate the function of Suppressor of IKKepsilon (SIKE)

Wound inducible transcript 3.0 (WIT3.0) is a novel cytoskeleton protein that regulates fibroblast migration and initiates rapid wound closure. WIT3 shares ~50% sequence identity with an innate immune protein of unknown function, Suppressor of IKKepsilon (SIKE), that also associates with cytoskeletal proteins. We hypothesize that SIKE and WIT3 will have similar biochemical and biophysical characteristics that contribute to a shared function. To assess these similarities, we undertook characterization of WIT3 structure utilizing circular dichroism, fluorescence‐based thermal shift assays, ligand binding studies using an ANS reporter, and crosslinking with BS3. Using PHYRE2, a WIT3 model was predicted that consisted of an alpha helical coil with 30% disordered regions. The secondary structure of WIT3 was assessed via circular dichroism. Wavelength spectra is consistent with an alpha helical protein and thermal melt data showed a linear unfolding pattern with Tm of 37°C, suggesting a minimally stable protein. As part of the PHYRE2 model, 3DLigandSite proposed a zinc binding site. To assess an interaction between WIT3 and divalent cations, WIT3‐ANS titration and fluorescence‐based thermal shift assays were completed +/‐ Mg, Mn, Ca, Ni and Zn. An interaction between WIT3 and Zn was confirmed where, in the presence of zinc, WIT3's affinity for ANS was 2 fold higher than in the absence of zinc. The initial comparison suggests that WIT3 and SIKE share similar helical structure and similar FTS melt curve topography indicating exposed hydrophobic residues prior to denaturation, but different structural stability and a potential divalent cation binding site on WIT3. Together, these data suggest that WIT3 and SIKE share several biochemical characteristics that may allow these two proteins to have complimentary function.Support or Funding InformationStudies were funded in part by USD SURE.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Biophysical characterization of Suppressor of IKKepsilon structure, stability, and metal binding properties

Suppressor of IKKepsilon (SIKE) is phosphorylated in the signaling cascade that initiates the anti‐viral innate immune response mediated by Toll‐like receptor 3. Although shown to be associated with cytoskeletal proteins, the function of SIKE in association with the cytoskeleton or as part of the innate immune response is not yet known. To gain insight into SIKE's function, structural and biophysical characterization were undertaken. Fluorescence‐based thermal shift assays (FTSA) indicated that the SIKE structure displayed hydrophobic residues prior to thermal denaturation. With increasing temperature, additional hydrophobic character was displayed giving rise to unique FTS curves showing three transitions, initial binding of fluor, unfolding, and quenching of fluorescence, presumably by protein aggregation. From modeling software, a divalent cation binding site was predicted. Using FTSA, the addition of zinc and calcium appear to alter the thermal stability of SIKE. Using ANS as a reporter group or direct tryptophan fluorescence of SIKE, the binding affinity of SIKE to various divalent cations, zinc, magnesium, manganese and calcium, was assessed. Fluorescence quenching experiments with neutral and charged quenchers were used to probe the local environment of the lone Trp residue of SIKE. The quenching coefficient for the Trp in SIKE determined from acrylamide studies was significantly lower than that of free Trp in solution indicating that the residue is fairly protected. Together, these studies provide biophysical parameters with which to interrogate and refine our predicted SIKE structure that is being employed in interpreting how SIKE interacts with cytoskeletal proteins to mediate host defense mechanisms.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Binding of Divalent Cations to Insulin: Capillary Electrophoresis and Molecular Simulations.

In the present study, we characterize the binding of divalent cations to insulin in aqueous salt solutions by means of capillary electrophoresis and molecular dynamics simulations. The results show a strong pH dependence. At low pH, at which all the carboxylate groups are protonated and the protein has an overall positive charge, all the cations exhibit only weak and rather unspecific interactions with insulin. In contrast, at close to neutral pH, when all the carboxylate groups are deprotonated and negatively charged, the charge-neutralizing effect of magnesium, calcium, and zinc, in particular, on the electrophoretic mobility of insulin is significant. This is also reflected in the results of molecular dynamics simulations showing accumulation of cations at the protein surface, which becomes smaller in magnitude upon effective inclusion of electronic polarization via charge rescaling.

Allosteric inhibitor remotely modulates the conformation of the orthestric pockets in mutant IDH2/R140Q

Neomorphic mutation R140Q in the metabolic enzyme isocitrate dehydrogenase 2 (IDH2) is found to be a driver mutation in cancers. Recent studies revealed that allosteric inhibitors could selectively inhibit IDH2/R140Q and induce differentiation of TF-1 erythroleukemia and primary human AML cells. However, the allosteric inhibition mechanism is not very clear. Here, we report the results from computational studies that AGI-6780 binds tightly with the divalent cation binding helices at the homodimer interface and prevents the transition of IDH2/R140Q homodimer to a closed conformation that is required for catalysis, resulting in the decrease of the binding free energy of NADPHs. If the allosteric inhibitor is removed, the original open catalytic center of IDH2/R140Q will gradually reorganize to a quasi-closed conformation and the enzymatic activity might recover. Unlike IDH2/R140Q, AGI-6780 locks one monomer of the wild-type IDH2 in an inactive open conformation and the other in a half-closed conformation, which can be used to explain the selectivity of AGI-6780. Our results suggest that conformational changes are the primary contributors to the inhibitory potency of the allosteric inhibitor. Our study will also facilitate the understanding of the inhibitory and selective mechanisms of AG-221 (a promising allosteric inhibitor that has been approved by FDA) for mutant IDH2.

Threading the biophysics of mammalian Slo1 channels onto structures of an invertebrate Slo1 channel.

For those interested in the machinery of ion channel gating, the Ca2+ and voltage-activated BK K+ channel provides a compelling topic for investigation, by virtue of its dual allosteric regulation by both voltage and intracellular Ca2+ and because its large-single channel conductance facilitates detailed kinetic analysis. Over the years, biophysical analyses have illuminated details of the allosteric regulation of BK channels and revealed insights into the mechanism of BK gating, e.g., inner cavity size and accessibility and voltage sensor-pore coupling. Now the publication of two structures of an Aplysia californica BK channel-one liganded and one metal free-promises to reinvigorate functional studies and interpretation of biophysical results. The new structures confirm some of the previous functional inferences but also suggest new perspectives regarding cooperativity between Ca2+-binding sites and the relationship between voltage- and Ca2+-dependent gating. Here we consider the extent to which the two structures explain previous functional data on pore-domain properties, voltage-sensor motions, and divalent cation binding and activation of the channel.

Behavior of Spherical Poly(2-acrylamido-2-methylpropanesulfonate) Polyelectrolyte Brushes on Silica Nanoparticles up to Extreme Salinity with Weak Divalent Cation Binding at Ambient and High Temperature

The colloidal stability of nanoparticles (NPs) stabilized by grafted polyelectrolyte (PE) brushes in concentrated divalent ion solutions, at either ambient or high temperature, is of interest in a wide variety of applications including medicine, personal care products, oil and gas recovery, reservoir imaging, and environmental remediation. Previous attempts to determine the length of PE brushes at these conditions have been limited by lack of colloidal stability particularly when divalent ions form complexes with the charges on the brushes. We find that brushes of highly acidic strong PE poly(2-acrylamido-2-methylpropanesulfonate, AMPS) end-grafted to silica NPs provide colloidal stability at salinities up to 4.5 M CaCl2 or NaCl. Thus, the brush behavior could be studied with dynamic light scattering (DLS) and the electrophoretic mobility by phase analysis light scattering (PALS) from the salt-free condition to the extreme salinities of 4.5 M. In monovalent NaCl solutions, the highly extended poly(AMPS) b...

All atom NMDA receptor transmembrane domain model development and simulations in lipid bilayers and water.

N-methyl-d-aspartate receptors (NMDARs) are members of the ionotropic glutamate receptor family that mediate excitatory synaptic transmission in the central nervous system. The channels of NMDARs are permeable to Ca2+ but blocked by Mg2+, distinctive properties that underlie essential brain processes such as induction of synaptic plasticity. However, due to limited structural information about the NMDAR transmembrane ion channel forming domain, the mechanism of divalent cation permeation and block is understood poorly. In this paper we developed an atomistic model of the transmembrane domain (TMD) of NMDARs composed of GluN1 and GluN2A subunits (GluN1/2A receptors). The model was generated using (a) a homology model based on the structure of the NaK channel and a partially resolved structure of an AMPA receptor (AMPAR), and (b) a partially resolved X-ray structure of GluN1/2B NMDARs. Refinement and extensive Molecular Dynamics (MD) simulations of the NMDAR TMD model were performed in explicit lipid bilayer membrane and water. Targeted MD with simulated annealing was introduced to promote structure refinement. Putative positions of the Mg2+ and Ca2+ ions in the ion channel divalent cation binding site are proposed. Differences in the structural and dynamic behavior of the channel protein in the presence of Mg2+ or Ca2+ are analyzed. NMDAR protein conformational flexibility was similar with no ion bound to the divalent cation binding site and with Ca2+ bound, whereas Mg2+ binding reduced protein fluctuations. While bound at the binding site both ions retained their preferred ligand coordination numbers: 6 for Mg2+, and 7–8 for Ca2+. Four asparagine side chain oxygens, a back-bone oxygen, and a water molecule participated in binding a Mg2+ ion. The Ca2+ ion first coordination shell ligands typically included four to five side-chain oxygen atoms of the binding site asparagine residues, two water molecules and zero to two backbone oxygens of the GluN2B subunits. These results demonstrate the importance of high-resolution channel structures for elucidation of mechanisms of NMDAR permeation and block.