Metal Ion Binding Sites Research Articles

Mutations Found in the pncA Gene of Mycobacterium tuberculosis in Clinical Pyrazinamide-resistant Isolates from a Local Region of China

This study was designed to investigate the presence of mutations in the pncA gene, minimum inhibitory concentrations and pyrazinamidase activity of pyrazinamide-resistant Mycobacterium tuberculosis. In total, 47 M. tuberculosis clinical isolates from a local region of China were assayed. Pyrazinamidase activity was measured by pyrazinamide deamination to pyrazinoic acid and ammonia, and a 721 bp region, including the entire pncA open-reading frame, 104 bp of the upstream sequence and 59 bp of the downstream sequence, was determined by DNA sequencing of purified polymerase chain reaction products. Of the 47 isolates resistant to pyrazinamide, 44 lost pyrazinamidase activity and had pncA mutations that occurred mainly near pyrazinamidase's active or metal ion binding sites; nine of them have not been reported previously. Three pyrazinamide-resistant isolates carried the wild-type pncA sequence and retained pyrazinamidase activity. These results show the molecular mechanism of pyrazinamide resistance in China and may also contribute towards the prevention of tuberculosis in China.

Short-distance probes for protein backbone structure based on energy transfer between bimane and transition metal ions

The structure and dynamics of proteins underlies the workings of virtually every biological process. Existing biophysical methods are inadequate to measure protein structure at atomic resolution, on a rapid time scale, with limited amounts of protein, and in the context of a cell or membrane. FRET can measure distances between two probes, but depends on the orientation of the probes and typically works only over long distances comparable with the size of many proteins. Also, common probes used for FRET can be large and have long, flexible attachment linkers that position dyes far from the protein backbone. Here, we improve and extend a fluorescence method called transition metal ion FRET that uses energy transfer to transition metal ions as a reporter of short-range distances in proteins with little orientation dependence. This method uses a very small cysteine-reactive dye monobromobimane, with virtually no linker, and various transition metal ions bound close to the peptide backbone as the acceptor. We show that, unlike larger fluorophores and longer linkers, this donor-acceptor pair accurately reports short-range distances and changes in backbone distances. We further extend the method by using cysteine-reactive metal chelators, which allow the technique to be used in protein regions of unknown secondary structure or when native metal ion binding sites are present. This improved method overcomes several of the key limitations of classical FRET for intramolecular distance measurements.

Probing the Cu2+ and Zn2+ binding affinity of histidine-rich glycoprotein

The zinc(II) and copper(II) binding ability of two oligopeptide fragments, Ac-HHPHG-NH(2) and Ac-HHPHGHHPHG-NH(2), derived from the repeat-region of the His-Pro-rich domain of histidine-rich glycoprotein (HRG) and the structure of the formed complexes have been investigated by potentiometry, NMR-, UV-visible-, CD-, SRCD- and EPR spectroscopy. Exclusive coordination of the side-chain imidazoles of the peptides has been observed with both metal ions in the acidic and neutral pH range. While the three His units of the pentapeptide resulted in a modest stability of the ML complexes, the decapeptide with its increased number of His residues offered a high-affinity metal binding site for both metal ions with the participation of at least four nitrogen donors. Due to the high number of potential donor groups, the formation of binding isomers of the protonated and parent complexes is very likely. Both peptides show a synchrotron radiation (SR) CD-pattern resembling to that of the polyproline II structure, similarly to that of the His-Pro-rich domain of the HRG protein. The longer sequence was shown to bind a second metal ion in the slightly acidic pH-range. The determined stability data suggest a remarkable extra stabilization emerging in the decapeptide for the coordination of the second metal ions, as compared to the ML complexes of the pentapeptide. Whether the observed cooperativity has similarities to the cooperative metal binding feature of HRG or the two phenomena have different sources is a question yet to be clarified.

RNA biophysics has come of age

RNA biophysics has come of age

ONIOM Calculation on Azurin: Effect of Metal Ion Substitutions

In this study an attempt has been made to investigate the effect of metal ion substitution on the structure and spectra of azurin using two-layer ONIOM (our own N-layered integrated molecular orbital + molecular mechanics) approach. It is evident from the results that the overall structure of the protein is not altered by metal ion substitution; nevertheless, the metal ion binding site undergoes noticeable changes. The present study highlights the importance of protein milieu in the prediction of structure, electronic, and spectral properties of native and substituted azurins and illustrates the usefulness of ONIOM approach in the designing and engineering of metalloproteins.

Chitosan(chitin)/cellulose composite biosorbents prepared using ionic liquid for heavy metal ions adsorption

AbstractChitosan(chitin)/cellulose composites as biodegradable biosorbents were prepared under an environment‐friendly preparation processes using ionic liquids. Infrared and X‐ray photoelectron spectra indicated the stronger intermolecular hydrogen bond between chitosan and cellulose, and the hydroxyl and amine groups were believed to be the metal ion binding sites. Among the prepared biosorbents, freeze‐dried composite had higher adsorption capacity and better stability. The capacity of adsorption was found to be Cu(II) (0.417 mmol/g) > Zn(II) (0.303 mmol/g) > Cr(VI) (0.251 mmol/g) > Ni(II) (0.225 mmol/g) > Pb(II) (0.127 mmol/g) at the same initial concentration 5 mmol L−1. In contrast to some other chitosan‐type biosorbenrts, preparation and component of the biosorbent were obviously more environment friendly. Moreover, adsorption capacity of chitosan in the blending biosorbent could be fully shown. © 2009 American Institute of Chemical Engineers AIChE J, 2009

Structural Basis for DNase Activity of a Conserved Protein Implicated in CRISPR-Mediated Genome Defense

Acquired immunity in prokaryotes is achieved by integrating short fragments of foreign nucleic acids into clustered regularly interspaced short palindromic repeats (CRISPRs). This nucleic acid-based immune system is mediated by a variable cassette of up to 45 protein families that represent distinct immune system subtypes. CRISPR-associated gene 1 (cas1) encodes the only universally conserved protein component of CRISPR immune systems, yet its function is unknown. Here we show that the Cas1 protein is a metal-dependent DNA-specific endonuclease that produces double-stranded DNA fragments of approximately 80 base pairs in length. The 2.2 A crystal structure of the Cas1 protein reveals a distinct fold and a conserved divalent metal ion-binding site. Mutation of metal ion-binding residues, chelation of metal ions, or metal-ion substitution inhibits Cas1-catalyzed DNA degradation. These results provide a foundation for understanding how Cas1 contributes to CRISPR function, perhaps as part of the machinery for processing foreign nucleic acids.

Spiropyran modified micro-fluidic chip channels as photonically controlled self-indicating system for metal ion accumulation and release

In this paper, we show how through integrating the beneficial characteristics of micro-fluidic devices and spiropyrans dyes, a simple and very innovative chip configured as an on-line photonically controlled self-indicating system for metal ion accumulation and release can be realised. The micro-fluidic device consists of five independent 94 μm depth, 150 μm width channels fabricated in polydimethylsiloxane. The spiropyran 1′-(3-carboxypropyl)-3,3′-dimethyl-6-nitrospiro-1-benzopyran-2,2′-indoline is immobilised by physical adsorption into a polydimethylsiloxane matrix and covalently on the ozone plasma activated polydimethylsiloxane micro-channel walls. When the colourless, inactive, spiropyran coating absorbs UV light it switches to the highly coloured merocyanine form, which also has an active binding site for certain metal ions. Therefore metal ion uptake can be triggered using UV light and subsequently reversed on demand by shining white light on the coloured complex, which regenerates the inactive spiropyran form, and releases the metal ion. When stock solutions of several metal ions (Ca 2+, Zn 2+, Hg 2+, Cu 2+, Co 2+) are pumped independently through the five channels, different optical responses were observed for each metal, and the platform can therefore be regarded as a micro-structured device for photo-controlled self-indicating metal ion complexation, accumulation and release.

Molecular mechanics and molecular dynamics study on azurin using extensible and systematic force field (ESFF)

A systematic study on the assessment of predictive power of extensible and systematic force field (ESFF) has been carried out by taking azurin as a model metalloprotein. Both molecular mechanics (MM) and molecular dynamics (MD) calculations have been performed on native azurin. In addition, the effects of metal ion substitution and mutation of axial methionine have been investigated. MM calculations with axial ligand restraints reasonably predict the structures of native and engineered azurins. It is also evident from the MD simulations that the average structures are similar to that of corresponding X-ray structures. The overall conformation of the engineered and mutated azurins is also akin to that of native protein. However, significant changes are observed in metal ion binding site upon substitution and axial mutation. It is found that the calculated change in strain energy linearly varies with the corresponding experimental redox potentials of engineered azurins.

Facilitating chromophore formation of engineered Ca 2+ binding green fluorescent proteins

Green fluorescent protein (GFP) containing a self-coded chromophore has been applied in protein trafficking and folding, gene expression, and as sensors in living cells. While the “cycle3” mutation denoted as C3 mutation (F99S/M153T/V163A) offers the ability to increase GFP fluorescence at 37 °C, it is not clear whether such mutations will also be able to assist the folding and formation of the chromophore upon the addition of metal ion binding sites. Here, we investigate in both bacterial and mammalian systems, the effect of C2 (M153T/V163A) and C3 (F99S/M153T/V163A) mutations on the folding of enhanced GFP (EGFP, includes F64L/S65T) and its variants engineered with two types of Ca 2+ binding sites: (1) a designed discontinuous Ca 2+ binding site and (2) a grafted continuous Ca 2+ binding motif. We show that, for the constructed EGFP variants, the C2 mutation is sufficient to facilitate the production of fluorescence in both bacterial and mammalian cells. Further addition of the mutation F99S decreases the folding efficiency of these variants although a similar effect is not detectable for EGFP, likely due to the already greatly enhanced mutation F64L/S65T from the original GFP, which hastens the chromophore formation. The extinction coefficient and quantum yield of purified proteins of each construct were also examined to compare the effects of both C2 and C3 mutations on protein spectroscopic properties. Our quantitative analyses of the effect of C2 and C3 mutations on the folding and formation of GFP chromophore that undergoes different folding trajectories in bacterial versus mammalian cells provide insights into the development of fluorescent protein-based analytical sensors.

Characterization of recombinant thiamine diphosphate-dependent phosphonopyruvate decarboxylase from Streptomyces viridochromogenes Tü494

Phosphonopyruvate decarboxylase (PPDC) catalyzes the thiamine diphosphate-dependent non-oxidative decarboxylation of phosphonopyruvate to phosphonoacetaldehyde and carbon dioxide. The enzyme of S. viridochromogenes Tü494 was expressed as a recombinant fusion protein with an N-terminal 10× histidine-tag in Escherichia coli cells, and was purified to homogeneity by nickel affinity chromatography. The biochemical properties of the recombinant enzyme were characterized, measuring phosphonoacetaldehyde formation by two newly developed coupled enzyme assays. PPDC has a high affinity to its cofactors ThDP, and Mg 2+ (both K m ∼ 40 μM). The metal ions Ca 2+ and Mn 2+ ( K m ∼ 3 μM) could substitute for Mg 2+. In coupled enzyme assays at pH 8.0 (HEPES buffer) and at 30 °C, PPDC followed Michaelis–Menten kinetics with phosphonopyruvate, with a K m value of 3.2 ± 0.35 μM and a v max value of 0.81 ± 0.01 U/mg. Neither pyruvate, β-hydroxypyruvate, nor fluoropyruvate served as alternative substrates. Gel filtration chromatography indicated a molecular mass of 72,100 ± 220 Da. Taking into account a subunit size of about 43,600 Da, the quaternary structure of the Ppd appears to be homodimeric. A comparison with the pyruvate decarboxylase (PDC) from Zymomonas mobilis allowed the identification of several amino acid residues, whose potential functions were examined by site-directed mutagenesis. Based on kinetic data of various site-directed PPDC variants and by comparison to 3D structures of PDC and benzoylformate decarboxylase, a model of the active site was generated. As in most other ThDP-dependent enzymes a glutamate residue (Glu-48 of PPDC) appears to be responsible for ThDP activation. Residues Ser-25, His-110, and Asp-297 affect the catalytic activity and are probably located in the direct vicinity of the substrate binding site. Residues Asp-265, Asn-293, and Gly-294 were found within the conserved ThDP-binding motif and mark the binding site of the Me 2+ ion that is responsible for ThDP anchoring. Asp-263 is conserved in PPDC sequences and in sulfopyruvate decarboxylase, and appears to contribute to the metal ion binding site, too. Glu-224, another conserved residue is essential for catalytic activity. The results for Glu-224 and Asp-263 are contrary to the description of the corresponding residues in the PPDC of the gram-negative bacterium Bacteroides fragilis [G. Zhang, J. Dai, Z. Lu, D. Dunaway-Mariano, J. Biol. Chem. 278 (2003) 41302–41308].

A Lysine-Tyrosine Pair Carries Out Acid−Base Chemistry in the Metal Ion-Dependent Pyridine Dinucleotide-Linked β-Hydroxyacid Oxidative Decarboxylases

This work reviews published structural and kinetic data on the pyridine nucleotide-linked beta-hydroxyacid oxidative decarboxylases. The family of metal ion-dependent pyridine nucleotide-linked beta-hydroxyacid oxidative decarboxylases can be divided into two structural families with the malic enzyme, which has an (S)-hydroxyacid substrate, comprising one subfamily and isocitrate dehydrogenase, isopropylmalate dehydrogenase, homoisocitrate dehydrogenase, and tartrate dehydrogenase, which have an (R)-hydroxyacid substrate, comprising the second subclass. Multiple-sequence alignment of the members of the (R)-hydroxyacid family indicates a high degree of sequence identity with most of the active site residues conserved. The three-dimensional structures of the members of the (R)-hydroxyacid family with structures available superimpose on one another, and the active site structures of the enzymes have a similar overall geometry of residues in the substrate and metal ion binding sites. In addition, a number of residues in the malic enzyme active site are also conserved, and the arrangement of these residues has a similar geometry, although the (R)-hydroxyacid and (S)-hydroxyacid family sites are geometrically mirror images of one another. The active sites of the (R)-hydroxyacid family have a higher positive charge density when compared to those of the (S)-hydroxyacid family, largely due to the number of arginine residues in the vicinity of the substrate alpha-carboxylate and one fewer carboxylate ligand to the divalent metal ion. Data available for all of the enzymes in the family have been considered, and a general mechanism that makes use of a lysine (general base)-tyrosine (general acid) pair is proposed. Differences exist in the mechanism for generating the neutral form of lysine so that it can act as a base.

Exploring Metal Ion Coordination to Nucleic Acids by NMR

Metal ions play a crucial role in charge compensation, folding and stabilization of tertiary structures of large nucleic acids. In addition, they may be directly involved in the catalytic mechanism of ribozymes. Most metal ions applied in the context of nucleic acids in vivo and in vitro bind in a kinetically labile fashion. Hence, the detection of metal ion binding sites, not to mention the elucidation of the specific coordination sphere, still poses largely unresolved problems. Here we describe the different strategies applied and the progress made over the last years to characterize metal ion coordination to large nucleic acids by NMR.

Investigating the Origins of Fractional ψ-values in Protein Folding Transition States

ψ-analysis has been used to characterize the inter-residue contacts that define the structure of the transition state ensemble (TSE) for three protein systems. ψ-values identify the degree to which an engineered bi-Histidine metal ion binding site is formed in the TSE. Values of zero or one indicate that the site is fully unfolded-like or native-like, respectively, while fractional values reflect a partial recovery of the binding-induced stabilization in the TSE. This method has been applied to three proteins, protein A (Baxa et al., 2008), Ubiquitin (Ub) (Krantz et al., 2004), and acyl-phosphotase (Pandit et al., 2006). In all three cases, the TSE captures ∼70% of the respective native-state topology, as quantified by the relative contact order (RCO) metric. In light of the proposed “70% rule”, a re-evaluation of the TSE of many small proteins, especially those characterized as polarized by mutational f-analysis, must be considered. While this “70% rule” is believed to be a general feature of most small proteins, the potential origin of fractional ψ-values remains to be investigated. All-atom Langevin dynamics (LD) simulations of TS models of Ub are performed with distance constraints on residue pairs having experimentally-determined ψ-values of unity. An analysis of the trajectories indicates that the fractional ψ-values of sites adjacent to unity sites tend to reflect distorted site geometries, while the residues for more distal, fractional values indicate that the sites are able to sample configurations where they are unfolded-like. Nevertheless, the simulations indicate that the unity ψ-values alone are sufficient to generate a TSE with a highly native-like topology. Furthermore, the calculation of f-values based on sidechain-sidechain contacts made in the TSE indicate that experimental f-values can dramatically under-report the amount of structure present even for highly buried residues.

A Catalytic Metal Ion Interacts with the Cleavage Site G·U Wobble in the HDV Ribozyme

The HDV ribozyme self-cleaves by a chemical mechanism involving general acid-base catalysis to generate 2',3'-cyclic phosphate and 5'-hydroxyl termini. Biochemical studies from several laboratories have implicated C75 as the general acid and hydrated magnesium as the general base. We have previously shown that C75 has a pK(a) shifted >2 pH units toward neutrality [Gong, B., Chen, J. H., Chase, E., Chadalavada, D. M., Yajima, R., Golden, B. L., Bevilacqua, P. C., and Carey, P. R. (2007) J. Am. Chem. Soc. 129, 13335-13342], while in crystal structures, it is well-positioned for proton transfer. However, no evidence for a hydrated magnesium poised to serve as a general base in the reaction has been observed in high-resolution crystal structures of various reaction states and mutants. Herein, we use solution kinetic experiments and parallel Raman crystallographic studies to examine the effects of pH on the rate and Mg(2+) binding properties of wild-type and 7-deazaguanosine mutants of the HDV ribozyme. These data suggest that a previously unobserved hydrated magnesium ion interacts with N7 of the cleavage site G.U wobble base pair. Integrating this metal ion binding site with the available crystal structures provides a new three-dimensional model for the active site of the ribozyme that accommodates all available biochemical data and appears competent for catalysis. The position of this metal is consistent with a role of a magnesium-bound hydroxide as a general base as dictated by biochemical data.

Mononuclear Ferrocenophane Structural Motifs with Two Thiourea Arms Acting as a Dual Binding Site for Anions and Cations

The synthesis of a new type of mononuclear ferrocenophane-based thiourea, in which the ferrocene moiety is simultaneously attached to two thiourea groups directly from 1,1'-bis(isothiocyanato)ferrocene, is reported. These nitrogen-rich structural motifs show remarkable ion-sensing properties because of the presence of the redox active ferrocene unit and the thiourea bridges, which unexpectedly act as a dual binding site for anions and metal ions. They display a selective downfield shift of the thiourea protons and a remarkable cathodic shift of the ferrocene/ferrocenium redox couple with F(-), AcO(-), H(2)PO(4)(-), and HP(2)O(7)(3-) anions, whereas the selective recognition of Hg(2+) metal cations is achieved either by electrochemical or by spectral measurements. The preferred binding modes are proposed for the most representative complexes by means of density functional theory based theoretical calculations showing the Janus-like faces of the receptor.

ψ-Constrained Simulations of Protein Folding Transition States: Implications for Calculating ϕ

ψ-analysis has been used to identify interresidue contacts in the transition state ensemble (TSE) of ubiquitin and other proteins. The magnitude of ψ depends on the degree to which an inserted bihistidine (biHis) metal ion binding site is formed in the TSE. A ψ equal to zero or one indicates that the biHis site is absent or fully native-like, respectively, while a fractional ψ implies that in the TSE, the biHis site recovers only part of the binding-induced stabilization of the native state. All-atom Langevin dynamics simulations of the TSE are performed with restrictions imposed only on the distances between the pairs of residues with experimentally determined ψ of unity. When a site with a fractional ψ lies adjacent to a site with ψ = 1, the fractional ψ generally signifies that the “fractional site” has a distorted geometry in the TSE. When a fractional site is distal to the sites with ψ = 1, however, the histidines sample configurations in which the site is absent. The simulations indicate that the ψ = 1 sites by themselves can be used to generate a well-defined TSE having near-native topology. ϕ values calculated from the TS simulations exhibit mixed agreement with the experimental values. The origin and implication of the disparities are discussed.

Mutagenesis identifies the critical amino acid residues of human endonuclease G involved in catalysis, magnesium coordination, and substrate specificity

BackgroundEndonuclease G (EndoG), a member of DNA/RNA nonspecific ββα-Me-finger nucleases, is involved in apoptosis and normal cellular proliferation. In this study, we analyzed the critical amino acid residues of EndoG and proposed the catalytic mechanism of EndoG.MethodsTo identify the critical amino acid residues of human EndoG, we replaced the conserved histidine, asparagine, and arginine residues with alanine. The catalytic efficacies of Escherichia coli-expressed EndoG variants were further analyzed by kinetic studies.ResultsDiethyl pyrocarbonate modification assay revealed that histidine residues were involved in EndoG activity. His-141, Asn-163, and Asn-172 in the H-N-H motif of EndoG were critical for catalysis and substrate specificity. H141A mutant required a higher magnesium concentration to achieve its activity, suggesting the unique role of His-141 in both catalysis and magnesium coordination. Furthermore, an additional catalytic residue (Asn-251) and an additional metal ion binding site (Glu-271) of human EndoG were identified.ConclusionBased on the mutational analysis and homology modeling, we proposed that human EndoG shared a similar catalytic mechanism with nuclease A from Anabaena.

Structure of a Complete Integrin Ectodomain in a Physiologic Resting State and Activation and Deactivation by Applied Forces

The complete ectodomain of integrin alpha(IIb)beta(3) reveals a bent, closed, low-affinity conformation, the beta knee, and a mechanism for linking cytoskeleton attachment to high affinity for ligand. Ca and Mg ions in the recognition site, including the synergistic metal ion binding site (SyMBS), are loaded prior to ligand binding. Electrophilicity of the ligand-binding Mg ion is increased in the open conformation. The beta(3) knee passes between the beta(3)-PSI and alpha(IIb)-knob to bury the lower beta leg in a cleft, from which it is released for extension. Different integrin molecules in crystals and EM reveal breathing that appears on pathway to extension. Tensile force applied to the extended ligand-receptor complex stabilizes the closed, low-affinity conformation. By contrast, an additional lateral force applied to the beta subunit to mimic attachment to moving actin filaments stabilizes the open, high-affinity conformation. This mechanism propagates allostery over long distances and couples cytoskeleton attachment of integrins to their high-affinity state.

Dual-mode recognition of transition metal ions by bis-triazoles chained pyrenes

Fluorescent chemosensors 7−10, with variable methylene chain length as spacers between the two triazole methyl ether units, have been synthesized under ‘Click’ condition, where the bistriazoles are used as the metal ion binding sites and the pyrenes as the fluorophores. Compound 10, having the longest methylene chain among 7−10, shows monomer and excimer fluorescence quenching in acetonitrile toward Ni 2+, Pb 2+, Cu 2+, Hg 2+, and Cr 3+ ions, however, it shows an enhanced monomer but a decreased excimer emission when complexed with Cd 2+ and Zn 2+ ions.