Additional Binding Sites Research Articles

The Transcription Factor NMP4 Binds to the AQP5 Promoter and Is a Novel Transcriptional Regulator of the AQP5 Gene.

Aquaporin 5 (AQP5) is a water channel that regulates water transport, cell migration, and proliferation. Therefore, knowledge of its genetic regulation could be relevant to study these mechanisms. The AQP5 promoter region containing the AQP5-1364 A/C single-nucleotide polymorphism (SNP) might be an important regulatory region because the SNP is associated with the etiopathology of several diseases. The aim of this study was to identify a transcription factor that binds to this AQP5 promoter region and to investigate its potential impact upon AQP5 expression. In silico analysis revealed nuclear matrix protein 4 (NMP4) as a putative candidate. Electrophoretic mobility shift assays showed specific binding of NMP4 to the AQP5 promoter region of nt -1370 to nt -1329. Overexpression of NMP4 increased AQP5 promoter activity of the analyzed promoter constructs from nt -469 to nt -1979. Furthermore, an additional NMP4 binding site at position nt -592/nt -602 of the AQP5 promoter was identified. NMP4 overexpression increased AQP5 mRNA expression by 2.5-fold in HEK293 cells. Summarizing, we identified NMP4 as a novel transcriptional regulator of AQP5 expression, which binds to two AQP5 promoter regions. Both regions appear to impact AQP5 expression significantly.

Hill function-based models of transcriptional switches: impact of specific, nonspecific, functional and nonfunctional binding.

We explore minimalist models of transcription in which we take into account that a cis-regulatory sequence is embedded in, and interacts with, a complex genome. The classical Hill equation is the simplest way to represent a transcriptional response. However, it may overlook the fact that a transcription factor (TF) establishes specific and nonspecific nonfunctional interactions with chromatin. Classical papers have shown that nonfunctional binding (not leading to transcription) may influence gene expression. We examine how the presence of additional binding sites for a TF, besides those on the gene(s) of interest, affect the shape and parameters of the transcriptional response. We consider two conditions: at equilibrium and at steady-state. In many cases the TF level is determined by the position of the cell within a spatial or temporal gradient. We show that such gradients can be adjusted by evolutionary selection to compensate for the alteration of the gene transcription response by the presence of nonfunctional binding sites. Finally, we analyse how the transcriptional response is affected by a decrease in TF concentration, as in cases of haploinsufficiency. We show that the nonlinearity of the transcriptional response as a function of [TF] exacerbates the effect of a decrease in the latter, at least for weakly expressed TFs. Although decades of work on TFs have led to the impression that almost everything is known about the control of gene expression, we show that even the simplest models of transcription control have not delivered all their secrets yet.

Characterization Of β2‐containing Receptors in Rat Brainstem Using [125I]A‐85380

Nicotinic receptor diversity in brainstem has prompted us to determine the distribution and pharmacological characteristics of the β2‐containing. We used [125I]A‐85380 radioligand which is selective for receptors containing the β2 subunit. Our hypothesis was that β2‐containing nicotinic receptors would be present particularly in cardiorespiratory centers in the brainstem. First, we characterized the binding of [125I]A‐85380 in rat cerebral cortex (CTX). [125I]A‐85380 reached equilibrium in two hours and was found to have a Kd value of ~180pM and a Bmax of ~ 110 fmol/mg protein. In competition assays the rank order of potency for nicotinic receptor agonists was epibatidine > unlabeled A‐85380 > cytisine > nicotine > DMPP and for the antagonists the rank was DHbE > mecamylamine while upto 1mM k‐bungarotoxin did not displace any [125I]A‐85380 binding. In receptor binding and autoradiography studies where cytisine, DMPP, DHβE, epibatidine and nicotine were used to displace [125I]A‐85380, no additional binding sites were found, in all cases, binding was completely blocked by these ligands. Binding of [125I]A‐85380 was found at moderate to high levels in medial raphe areas some of which were insensitive to DHβE, and in most lateral regions of both caudal and rostral medulla.Support or Funding InformationAmerican Heart Association (MDG) 9930049N

Nucleic Acid Binding by Mason-Pfizer Monkey Virus CA Promotes Virus Assembly and Genome Packaging.

The Gag polyprotein of retroviruses drives immature virus assembly by forming hexameric protein lattices. The assembly is primarily mediated by protein-protein interactions between capsid (CA) domains and by interactions between nucleocapsid (NC) domains and RNA. Specific interactions between NC and the viral RNA are required for genome packaging. Previously reported cryoelectron microscopy analysis of immature Mason-Pfizer monkey virus (M-PMV) particles suggested that a basic region (residues RKK) in CA may serve as an additional binding site for nucleic acids. Here, we have introduced mutations into the RKK region in both bacterial and proviral M-PMV vectors and have assessed their impact on M-PMV assembly, structure, RNA binding, budding/release, nuclear trafficking, and infectivity using in vitro and in vivo systems. Our data indicate that the RKK region binds and structures nucleic acid that serves to promote virus particle assembly in the cytoplasm. Moreover, the RKK region appears to be important for recruitment of viral genomic RNA into Gag particles, and this function could be linked to changes in nuclear trafficking. Together these observations suggest that in M-PMV, direct interactions between CA and nucleic acid play important functions in the late stages of the viral life cycle. Assembly of retrovirus particles is driven by the Gag polyprotein, which can self-assemble to form virus particles and interact with RNA to recruit the viral genome into the particles. Generally, the capsid domains of Gag contribute to essential protein-protein interactions during assembly, while the nucleocapsid domain interacts with RNA. The interactions between the nucleocapsid domain and RNA are important both for identifying the genome and for self-assembly of Gag molecules. Here, we show that a region of basic residues in the capsid protein of the betaretrovirus Mason-Pfizer monkey virus (M-PMV) contributes to interaction of Gag with nucleic acid. This interaction appears to provide a critical scaffolding function that promotes assembly of virus particles in the cytoplasm. It is also crucial for packaging the viral genome and thus for infectivity. These data indicate that, surprisingly, interactions between the capsid domain and RNA play an important role in the assembly of M-PMV.

Muscle-Type Nicotinic Receptor Blockade by Diethylamine, the Hydrophilic Moiety of Lidocaine.

Lidocaine bears in its structure both an aromatic ring and a terminal amine, which can be protonated at physiological pH, linked by an amide group. Since lidocaine causes multiple inhibitory actions on nicotinic acetylcholine receptors (nAChRs), this work was aimed to determine the inhibitory effects of diethylamine (DEA), a small molecule resembling the hydrophilic moiety of lidocaine, on Torpedo marmorata nAChRs microtransplanted to Xenopus oocytes. Similarly to lidocaine, DEA reversibly blocked acetylcholine-elicited currents (IACh) in a dose-dependent manner (IC50 close to 70 μM), but unlike lidocaine, DEA did not affect IACh desensitization. IACh inhibition by DEA was more pronounced at negative potentials, suggesting an open-channel blockade of nAChRs, although roughly 30% inhibition persisted at positive potentials, indicating additional binding sites outside the pore. DEA block of nAChRs in the resting state (closed channel) was confirmed by the enhanced IACh inhibition when pre-applying DEA before its co-application with ACh, as compared with solely DEA and ACh co-application. Virtual docking assays provide a plausible explanation to the experimental observations in terms of the involvement of different sets of drug binding sites. So, at the nAChR transmembrane (TM) domain, DEA and lidocaine shared binding sites within the channel pore, giving support to their open-channel blockade; besides, lidocaine, but not DEA, interacted with residues at cavities among the M1, M2, M3, and M4 segments of each subunit and also at intersubunit crevices. At the extracellular (EC) domain, DEA and lidocaine binding sites were broadly distributed, which aids to explain the closed channel blockade observed. Interestingly, some DEA clusters were located at the α-γ interphase of the EC domain, in a cavity near the orthosteric binding site pocket; by contrast, lidocaine contacted with all α-subunit loops conforming the ACh binding site, both in α-γ and α-δ and interphases, likely because of its larger size. Together, these results indicate that DEA mimics some, but not all, inhibitory actions of lidocaine on nAChRs and that even this small polar molecule acts by different mechanisms on this receptor. The presented results contribute to a better understanding of the structural determinants of nAChR modulation.

The β2(+)/α4(-) Interfaces of (α4β2)2α4 Nicotinic Receptors Allosterically Contribute to Receptor Function

The α4β2 nicotinic acetylcholine receptor (nAChR) is the most abundant nAChR in the brain. Here it modulates the release of a wide range of neurotransmitters, affecting a wide range of brain functions such as cognition, mood and reward.The nAChR belongs to the Cys loop family of ligand gated ion channels. It assembles as a pentameric structure of one of two stoichiometries, (α4β2)2α4 and (α4β2)2β2. These receptors respond to ACh with low and high sensitivity, respectively. The ACh binding sites are located at the α4(+)/β2(-) interfaces in the N-terminal region of the protein, which is extracellularly positioned. The (α4β2)2α4 receptor has been recently shown to house an additional functional ACh binding site at its signature α4(+)/α4(+) interface. The remaining interfaces, β2(+)/α4(-) interfaces, are thought to bind allosteric modulators, but their contribution to receptor function is not known. However, it has been speculated, on the basis of the role of equivalent interfaces in allostery in other Cys loop ligand gated ion channels such as the GABAA receptor and α3β2 nAChR) that they may house binding sites for allosteric modulators.Using fully concatenated (α4β2)2α4 nACh receptors in conjunction with functional mutagenesis and substituted cystine accesibility methods (SCAM) we have examined whether the β2(+)/α4(-) interfaces impact receptor function. Our results suggest that β2(+)/α4(-) interface in the (α4β2)2α4 nAChR receptors may be a central part of an allosteric pathway that modulates the function of the α4(+)/α4(-) interface.

Analysis of regulatory mechanism after ErbB4 gene mutation based on local modeling methodology.

ErbB4 is an oncogene belonging to the epidermal growth factor receptor family and contributes to the occurrence and development of multiple cancers, such as gastric, breast, and colorectal cancers. Therefore, studies of the regulation of ErbB4 in cancerigenic pathway will advance molecular targeted therapy. Advanced bioinformatic analysis softwares, such as ExPASy, Predictprotei, QUARK, and I-TASSER, were used to analyze the regulatory mechanism after ErbB4 gene mutation in terms of amino acid sequence, primary, secondary, and tertiary structure of the protein and upstream-downstream receptor/ligands. Mutation of the 19th and 113th amino acids at the carboxyl terminus of ErbB4 protein did not affect its biological nature, but its secondary structure changed and protein binding sites were near 2 mutational sites; moreover, after mutation introduction, additional binding sites were observed. Tertiary structure modeling indicated that local structure of ErbB4 was changed from an α helical conformation into a β chain folding structure; the α helical conformation is the functional site of protein, while active sites are typically near junctions between helical regions, thus the helical structures are easily destroyed and change into folding structures or other structures after stretching. Mutable sites of ErbB4 is exact binding sites where dimer formed with other epidermal growth factor family proteins; mutation enabled the ErbB4 receptor to bind to neuregulin 1 ligand without dimer formation, disrupting the signal transduction pathway and affecting ErbB4 function.

Detection of secondary binding sites in proteins using fragment screening

Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets.

Cytoplasmic poly(A) binding protein-1 binds to genomically encoded sequences within mammalian mRNAs

The functions of the major mammalian cytoplasmic poly(A) binding protein, PABPC1, have been characterized predominantly in the context of its binding to the 3′ poly(A) tails of mRNAs. These interactions play important roles in post-transcriptional gene regulation by enhancing translation and mRNA stability. Here, we performed transcriptome-wide CLIP-seq analysis to identify additional PABPC1 binding sites within genomically encoded mRNA sequences that may impact on gene regulation. From this analysis, we found that PABPC1 binds directly to the canonical polyadenylation signal in thousands of mRNAs in the mouse transcriptome. PABPC1 binding also maps to translation initiation and termination sites bracketing open reading frames, exemplified most dramatically in replication-dependent histone mRNAs. Additionally, a more restricted subset of PABPC1 interaction sites comprised A-rich sequences within the 5′ UTRs of mRNAs, including Pabpc1 mRNA itself. Functional analyses revealed that these PABPC1 interactions in the 5′ UTR mediate both auto- and trans-regulatory translational control. In total, these findings reveal a repertoire of PABPC1 binding that is substantially broader than previously recognized with a corresponding potential to impact and coordinate post-transcriptional controls critical to a broad array of cellular functions.

Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function.

Messenger RNA acts as an informational molecule between DNA and translating ribosomes. Emerging evidence places mRNA in central cellular processes beyond its major function as informational entity. Although individual examples show that specific structural features of mRNA regulate translation and transcript stability, their role and function throughout the bacterial transcriptome remains unknown. Combining three sequencing approaches to provide a high resolution view of global mRNA secondary structure, translation efficiency and mRNA abundance, we unraveled structural features in E. coli mRNA with implications in translation and mRNA degradation. A poorly structured site upstream of the coding sequence serves as an additional unspecific binding site of the ribosomes and the degree of its secondary structure propensity negatively correlates with gene expression. Secondary structures within coding sequences are highly dynamic and influence translation only within a very small subset of positions. A secondary structure upstream of the stop codon is enriched in genes terminated by UAA codon with likely implications in translation termination. The global analysis further substantiates a common recognition signature of RNase E to initiate endonucleolytic cleavage. This work determines for the first time the E. coli RNA structurome, highlighting the contribution of mRNA secondary structure as a direct effector of a variety of processes, including translation and mRNA degradation.

Structure of HCMV glycoprotein B in the postfusion conformation bound to a neutralizing human antibody.

Human cytomegalovirus (HCMV) poses a significant threat to immunocompromised individuals and neonates infected in utero. Glycoprotein B (gB), the herpesvirus fusion protein, is a target for neutralizing antibodies and a vaccine candidate due to its indispensable role in infection. Here we show the crystal structure of the HCMV gB ectodomain bound to the Fab fragment of 1G2, a neutralizing human monoclonal antibody isolated from a seropositive subject. The gB/1G2 interaction is dominated by aromatic residues in the 1G2 heavy chain CDR3 protruding into a hydrophobic cleft in the gB antigenic domain 5 (AD-5). Structural analysis and comparison with HSV gB suggest the location of additional neutralizing antibody binding sites on HCMV gB. Finally, immunoprecipitation experiments reveal that 1G2 can bind to HCMV virion gB suggesting that its epitope is exposed and accessible on the virus surface. Our data will support the development of vaccines and therapeutic antibodies against HCMV infection.

Discovery and information-theoretic characterization of transcription factor binding sites that act cooperatively

Transcription factor binding to the surface of DNA regulatory regions is one of the primary causes of regulating gene expression levels. A probabilistic approach to model protein–DNA interactions at the sequence level is through position weight matrices (PWMs) that estimate the joint probability of a DNA binding site sequence by assuming positional independence within the DNA sequence. Here we construct conditional PWMs that depend on the motif signatures in the flanking DNA sequence, by conditioning known binding site loci on the presence or absence of additional binding sites in the flanking sequence of each siteʼs locus. Pooling known sites with similar flanking sequence patterns allows for the estimation of the conditional distribution function over the binding site sequences. We apply our model to the Dorsal transcription factor binding sites active in patterning the Dorsal–Ventral axis of Drosophila development. We find that those binding sites that cooperate with nearby Twist sites on average contain about 0.5 bits of information about the presence of Twist transcription factor binding sites in the flanking sequence. We also find that Dorsal binding site detectors conditioned on flanking sequence information make better predictions about what is a Dorsal site relative to background DNA than detection without information about flanking sequence features.

INHIBITORY POTENTIAL OF POLYHYDROXYLATED FULLERENES AGAINST PROTEIN TYROSINE PHOSPHATASE 1B.

Inhibition of PTP1B by polyhydroxylated fullerenes was studied in silico and in vitro. The enzyme kinetics in the presence of polyhydroxy small gap fullerenes showed that reciprocal value of maximum velocity non-linearly increases with increasing the inhibitor concentration. Analysis of the dose-dependent curve of PTP1B inhibition suggests an apparent positive cooperativity with involvement of at least two binding sites for the hydroxylated fullerene cages. Molecular docking calculations indicated that highly hydroxylated fullerene C60 may occupy the active site and additional allosteric binding site with similar affinity. In silico analysis of a number of fullerenols with 6, 12, 18, 24, 30, and 36 hydroxyl groups showed that the inhibitory activity may depend on the degree of hydroxylation of the nanoparticles surface. These data provide some understanding of the mechanisms of inhibitory action of fullerenols on activity of protein tyrosine phosphatases.

Copper Environment in Artificial Metalloproteins Probed by Electron Paramagnetic Resonance Spectroscopy.

The design of binding sites for divalent metals in artificial proteins is a productive platform for examining the characteristics of metal-ligand interactions. In this report, we investigate the spectroscopic properties of small peptides and four-helix bundles that bind Cu(II). Three small peptides, consisting of 15 amino acid residues, were designed to have two arms, each containing a metal-binding site comprised of different combinations of imidazole and carboxylate side chains. Two four-helix bundles each had a binding site for a central dinuclear metal cofactor, with one design incorporating additional potential metal ligands at two identical sites. The small peptides displayed pH-dependent, metal-induced changes in the circular dichroism spectra, consistent with large changes in the secondary structure upon metal binding, while the spectra of the four-helix bundles showed a predominant α-helix content but only small structural changes upon metal binding. Electron paramagnetic resonance spectra were measured at X-band revealing classic Cu(II) axial patterns with hyperfine coupling peaks for the small peptides and four-helix bundles exhibiting a range of values that were related to the specific chemical natures of the ligands. The variety of electronic structures allow us to define the distinctive environment of each metal-binding site in these artificial systems, including the designed additional binding sites in one of the four-helix bundles.

Positive modulation of synaptic and extrasynaptic GABAA receptors by an antagonist of the high affinity benzodiazepine binding site

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs such as the benzodiazepines. Benzodiazepines act at the high-affinity binding site at the α+/γ− subunit interface. Previously, an additional low affinity binding site for diazepam located in the transmembrane (TM) domain has been described. The compound SJM-3 was recently identified in a prospective screening of ligands for the benzodiazepine binding site and investigated for its site of action. We determined the binding properties of SJM-3 at GABAA receptors recombinantly expressed in HEK-cells using radioactive ligand binding assays. Impact on function was assessed in Xenopus laevis oocytes with electrophysiological experiments using the two-electrode voltage clamp method. SJM-3 was shown to act as an antagonist at the α+/γ− site. At the same time it strongly potentiated GABA currents via the binding site for diazepam in the transmembrane domain. Mutation of a residue in M2 of the α subunit strongly reduced receptor modulation by SJM-3 and a homologous mutation in the β subunit abolished potentiation. SJM-3 acts as a more efficient modulator than diazepam at the site in the trans-membrane domain. In contrast to low concentrations of benzodiazepines, SJM-3 modulates both synaptic and extrasynaptic receptors. A detailed exploration of the membrane site may provide the basis for the design and identification of subtype-selective modulatory drugs.

Fluorescence Resonance Energy Transfer (FRET) and Proximity Ligation Assays Reveal Functionally Relevant Homo- and Heteromeric Complexes among Hyaluronan Synthases HAS1, HAS2, and HAS3

In vertebrates, hyaluronan is produced in the plasma membrane from cytosolic UDP-sugar substrates by hyaluronan synthase 1-3 (HAS1-3) isoenzymes that transfer N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) in alternative positions in the growing polysaccharide chain during its simultaneous extrusion into the extracellular space. It has been shown that HAS2 immunoprecipitates contain functional HAS2 homomers and also heteromers with HAS3 (Karousou, E., Kamiryo, M., Skandalis, S. S., Ruusala, A., Asteriou, T., Passi, A., Yamashita, H., Hellman, U., Heldin, C. H., and Heldin, P. (2010) The activity of hyaluronan synthase 2 is regulated by dimerization and ubiquitination. J. Biol. Chem. 285, 23647-23654). Here we have systematically screened in live cells, potential interactions among the HAS isoenzymes using fluorescence resonance energy transfer (FRET) and flow cytometric quantification. We show that all HAS isoenzymes form homomeric and also heteromeric complexes with each other. The same complexes were detected both in Golgi apparatus and plasma membrane by using FRET microscopy and the acceptor photobleaching method. Proximity ligation assays with HAS antibodies confirmed the presence of HAS1-HAS2, HAS2-HAS2, and HAS2-HAS3 complexes between endogenously expressed HASs. C-terminal deletions revealed that the enzymes interact mainly via uncharacterized N-terminal 86-amino acid domain(s), but additional binding site(s) probably exist in their C-terminal parts. Of all the homomeric complexes HAS1 had the lowest and HAS3 the highest synthetic activity. Interestingly, HAS1 transfection reduced the synthesis of hyaluronan obtained by HAS2 and HAS3, suggesting functional cooperation between the isoenzymes. These data indicate a general tendency of HAS isoenzymes to form both homomeric and heteromeric complexes with potentially important functional consequences on hyaluronan synthesis.

Computational modeling and biological validation of novel non-steroidal ligands for the cholesterol recognition/interaction amino acid consensus (CRAC) motif of the mitochondrial translocator protein (TSPO)

Mitochondria play a critical role in the physiological homeostasis of the cell, contributing to numerous cellular processes, including bioenergetics, metabolism and cell life and death. Owing to their keystone role, mitochondria have gained much attention as pharmacological targets. The outer mitochondrial integral membrane translocator protein (TSPO) has attracted a significant degree of pharmacological interest owing to its ability to bind a number of classes of drugs with high affinity and specificity. In addition to its well-characterized drug binding site, TSPO possess an additional high-affinity ligand binding site, originally identified for its ability to bind the lipid cholesterol, which was named the cholesterol recognition/interaction amino acid consensus (CRAC) motif. Previous investigations from our laboratory identified additional ligands targeted to TSPO's CRAC motif which are able to potently inhibit mitochondrial cholesterol transport and steroid biosynthesis, processes for which TSPO has been well-characterized. However, all of these compounds possessed the steroidal backbone common to cholesterol and steroid hormones. In our efforts to expand our understanding of TSPO's CRAC motif, we performed studies aimed at identifying non-steroidal ligands for this motif. Molecular modeling and in silico screening of large chemical libraries identified a panel of compounds which were subsequently screened for bioactivity in a number of steroidogenic model systems. These efforts identified a family of non-steroidal CRAC ligands able to potently inhibit steroidogenesis, and at higher concentrations, promote apoptosis. In addition, the best candidate in this family was able to suppress testosterone synthesis when administered to rats, indicating that this novel family of non-steroidal CRAC ligands may serve as prototypes for the development of drugs useful for treatment of diseases of steroid overproduction, such as Cushing's syndrome and steroidogenic cell tumors in humans and animals.

New complexation behaviour of the potentially bis-terdentate triazole based ligands PMAT and PMPT: FeIII4 oxo-bridged metallomacrocycles

The reaction of the potentially bis-terdentate ligand PMPT with [FeII(py)4(NCS)2] in methanol under an inert atmosphere gives [FeIIn(PMPT)n(NCS)2n] (1), where PMPT=4-pyrrolyl-3,5-bis{[(2-pyridylmethyl)-amino]methyl}-4H-1,2,4-triazole. In contrast, conducting this reaction in dimethylformamide (DMF), or recrystallizing 1 from DMF, leads to the formation of the tetranuclear FeIII metallomacrocycle [FeIII4(PMPT)2(O)2(NCS)8(DMF)2] (2). This is the first structurally characterised complex of a ligand of this type that does not bind in a bis-terdentate manner with a 1:1 metal:ligand stoichiometry and N1N2 bridging of the bound metal ions. Instead, each ligand strand binds in an asymmetric fashion, providing two well separated (no triazole bridge) bidentate and terdentate binding pockets, with the additional binding sites on the iron(III) centres occupied by N-bound thiocyanate anions (NB. previous complexations have involved non-coordinating anions), oxide bridges, and/or DMF. Analogous reactions of [FeII(py)4(NCS)2] and the closely-related ligand PMAT appear to result in ligand decomposition, although a few crystals of the complex [FeIII4(PMAT)2(O)2(NCS)8(DMF)2]·solvents, (3·solvents) were obtained (PMAT=4-amino-3,5-bis{[(2-pyridylmethyl)-amino]methyl}-4H-1,2,4-triazole).

Structures of the hydrolase domain of zebrafish 10-formyltetrahydrofolate dehydrogenase and its complexes reveal a complete set of key residues for hydrolysis and product inhibition.

10-Formyltetrahydrofolate dehydrogenase (FDH), which is composed of a small N-terminal domain (Nt-FDH) and a large C-terminal domain, is an abundant folate enzyme in the liver and converts 10-formyltetrahydrofolate (10-FTHF) to tetrahydrofolate (THF) and CO2. Nt-FDH alone possesses a hydrolase activity, which converts 10-FTHF to THF and formate in the presence of β-mercaptoethanol. To elucidate the catalytic mechanism of Nt-FDH, crystal structures of apo-form zNt-FDH from zebrafish and its complexes with the substrate analogue 10-formyl-5,8-dideazafolate (10-FDDF) and with the products THF and formate have been determined. The structures reveal that the conformations of three loops (residues 86-90, 135-143 and 200-203) are altered upon ligand (10-FDDF or THF) binding in the active site. The orientations and geometries of key residues, including Phe89, His106, Arg114, Asp142 and Tyr200, are adjusted for substrate binding and product release during catalysis. Among them, Tyr200 is especially crucial for product release. An additional potential THF binding site is identified in the cavity between two zNt-FDH molecules, which might contribute to the properties of product inhibition and THF storage reported for FDH. Together with mutagenesis studies and activity assays, the structures of zNt-FDH and its complexes provide a coherent picture of the active site and a potential THF binding site of zNt-FDH along with the substrate and product specificity, lending new insights into the molecular mechanism underlying the enzymatic properties of Nt-FDH.

Competitive adsorption of cadmium and aluminum onto fresh and oxidized biochars during aging processes

Fresh biochar was shown to effectively reduce concentrations of heavy metal in soil, but the influences of the aging processes of biochar and competitions from other elements are warranted to precisely evaluate the long-term effect of biochar. This work investigated the effects of water washing, oxidation, and coexistence of aluminum (Al) on cadmium (Cd) adsorption by biochars and oxidized biochars. The Cd adsorption and the competitive adsorption of Cd and Al to rice straw-derived biochars, before and after oxidation by HNO3/H2SO4, were investigated. Meanwhile, the structural characteristics and surface charges of primary and oxidized biochars, with and without Cd loading, were analyzed by scanning electron microscopy, fourier-transform infrared spectroscopy, and zeta potential. The adsorption of Cd onto fresh biochars was dominated by surface complexation of oxygen-containing functional groups via esterification reactions, which was regulated by solution pH. Oxidization (aging) introduced carboxylic functional groups to biochar surfaces, which served as additional binding sites for Cd. The Cd binding to biochars was significantly affected by the coexistence of Al via acidification and competition for adsorption sites. The biochars exhibited high sorption capacities of Cd in soil, but soil acidification led to a counteractive of biochar’s liming effect and a reduction of Cd-binding sites; thus, the long-term effect of biochar for heavy metal immobilization should be paid more attention in acidic soil.