MetalDetector: a web server for predicting metal-binding sites and disulfide bridges in proteins from sequence

The web server MetalDetector classifies histidine residues in proteins into one of two states (free or metal bound) and cysteines into one of three states (free, metal bound or disulfide bridged). A decision tree integrates predictions from two previously developed methods (DISULFIND and Metal Ligand Predictor). Cross-validated performance assessment indicates that our server predicts disulfide bonding state at 88.6% precision and 85.1% recall, while it identifies cysteines and histidines in transition metal-binding sites at 79.9% precision and 76.8% recall, and at 60.8% precision and 40.7% recall, respectively. AVAILABILITY: Freely available at http://metaldetector.dsi.unifi.it. SUPPLEMENTARY INFORMATION: Details and data can be found at http://metaldetector.dsi.unifi.it/help.php.     

Large-scale prediction of disulphide bridges using kernel methods, two-dimensional recursive neural networks, and weighted graph matching

The formation of disulphide bridges between cysteines plays an important role in protein folding, structure, function, and evolution. Here, we develop new methods for predicting disulphide bridges in proteins. We first build a large curated data set of proteins containing disulphide bridges to extract relevant statistics. We then use kernel methods to predict whether a given protein chain contains intrachain disulphide bridges or not, and recursive neural networks to predict the bonding probabilities of each pair of cysteines in the chain. These probabilities in turn lead to an accurate estimation of the total number of disulphide bridges and to a weighted graph matching problem that can be addressed efficiently to infer the global disulphide bridge connectivity pattern. This approach can be applied both in situations where the bonded state of each cysteine is known, or in ab initio mode where the state is unknown. Furthermore, it can easily cope with chains containing an arbitrary number of disulphide bridges, overcoming one of the major limitations of previous approaches. It can classify individual cysteine residues as bonded or nonbonded with 87% specificity and 89% sensitivity. The estimate for the total number of bridges in each chain is correct 71% of the times, and within one from the true value over 94% of the times. The prediction of the overall disulphide connectivity pattern is exact in about 51% of the chains. In addition to using profiles in the input to leverage evolutionary information, including true (but not predicted) secondary structure and solvent accessibility information yields small but noticeable improvements. Finally, once the system is trained, predictions can be computed rapidly on a proteomic or protein-engineering scale. The disulphide bridge prediction server (DIpro), software, and datasets are available through www.igb.uci.edu/servers/psss.html.     

Exploring synonymous codon usage preferences of disulfide-bonded and non-disulfide bonded cysteines in the E. coli genome

High-quality data about protein structures and their gene sequences are essential to the understanding of the relationship between protein folding and protein coding sequences. Firstly we constructed the EcoPDB database, which is a high-quality database of Escherichia coli genes and their corresponding PDB structures. Based on EcoPDB, we presented a novel approach based on information theory to investigate the correlation between cysteine synonymous codon usages and local amino acids flanking cysteines, the correlation between cysteine synonymous codon usages and synonymous codon usages of local amino acids flanking cysteines, as well as the correlation between cysteine synonymous codon usages and the disulfide bonding states of cysteines in the E. coli genome. The results indicate that the nearest neighboring residues and their synonymous codons of the C-terminus have the greatest influence on the usages of the synonymous codons of cysteines and the usage of the synonymous codons has a specific correlation with the disulfide bond formation of cysteines in proteins. The correlations may result from the regulation mechanism of protein structures at gene sequence level and reflect the biological function restriction that cysteines pair to form disulfide bonds. The results may also be helpful in identifying residues that are important for synonymous codon selection of cysteines to introduce disulfide bridges in protein engineering and molecular biology. The approach presented in this paper can also be utilized as a complementary computational method and be applicable to analyse the synonymous codon usages in other model organisms.     

Metallothioneins with unusual residues: histidines as modulators of zinc affinity and reactivity

For many years, paradigms regarding metallothioneins comprised the exclusive metal coordination by thiolates from cysteine residues and the absence of aromatic residues. As more sequence and in vitro data on metallothioneins, in particular from non-vertebrate organisms, has become available, both the occurrence of and metal coordination by histidine residues in metallothioneins is emerging as a more frequent feature than expected. We discuss the general implications of histidines versus cysteines in zinc binding sites, and review some recent results from literature and our own lab. We conclude that histidines can stabilise metallothionein clusters by reducing the overall charge, offering the ability to help with structural organisation by supplying H-bond donor and acceptor properties, reducing the likelihood for disulfide bond formation, whilst maintaining a high affinity towards metal ions, in particular the borderline zinc ion.     

Homology modeling and molecular dynamics simulations of the N-terminal domain of wheat high molecular weight glutenin subunit 10

High molecular weight glutenin subunits (HMW-GS) are of a particular interest because of their biomechanical properties, which are important in many food systems such as breadmaking. Using fold-recognition techniques, we identified a fold compatible with the N-terminal domain of HMW-GS Dy10. This fold corresponds to the one adopted by proteins belonging to the cereal inhibitor family. Starting from three known protein structures of this family as templates, we built three models for the N-terminal domain of HMW-GS Dy10. We analyzed these models, and we propose a number of hypotheses regarding the N-terminal domain properties that can be tested experimentally. In particular, we discuss two possible ways of interaction between the N-terminal domains of the y-type HMW glutenin subunits. The first way consists in the creation of interchain disulfide bridges. According to our models, we propose two plausible scenarios: (1) the existence of an intrachain disulfide bridge between cysteines 22 and 44, leaving the three other cysteines free of engaging in intermolecular bonds; and (2) the creation of two intrachain disulfide bridges (involving cysteines 22-44 and cysteines 10-55), leaving a single cysteine (45) for creating an intermolecular disulfide bridge. We discuss these scenarios in relation to contradictory experimental results. The second way, although less likely, is nevertheless worth considering. There might exist a possibility for the N-terminal domain of Dy10, Nt-Dy10, to create oligomers, because homologous cereal inhibitor proteins are known to exist as monomers, homodimers, and heterooligomers. We also discuss, in relation to the function of the cereal inhibitor proteins, the possibility that this N-terminal domain has retained similar inhibitory functions.     

Characterization of the zinc-binding site of the histidine-proline-rich glycoprotein associated with rabbit skeletal muscle AMP deaminase

The AMP deaminase-associated variant of histidine-proline-rich glycoprotein (HPRG) is isolated from rabbit skeletal muscle by a modification of the protocol previously used for the purification of AMP deaminase. This procedure yields highly pure HPRG suitable for investigation by x-ray absorption spectroscopy of the zinc-binding behavior of the protein. X-ray absorption spectroscopy analysis of a 2:1 zinc-HPRG complex shows that zinc is bound to the protein, most probably in a dinuclear cluster where each Zn(2+) ion is coordinated, on average, by three histidine ligands and one heavier ligand, likely a sulfur from a cysteine. 11 cysteines of HPRG from different species are totally conserved, suggesting that five disulfide bridges are essential for the proper folding of the protein. At least another cysteine is present at different positions in the histidine-proline-rich domain of HPRG in all species, suggesting that this cysteine is the candidate for zinc ligation in the muscle variant of HPRG. The same conclusion is likely to be true for the six histidines used by the protein as zinc ligands. The presence in muscle HPRG of a specific zinc-binding site permits us to envisage the addition of HPRG into the family of metallochaperones. In this view, HPRG may enhance the in vivo stability of metalloenzymes such as AMP deaminase.     

Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein.

To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease in the intrinsic fluorescence. Inter conversion between the two redox states could thus be followed in vitro as well as in vivo by non-invasive fluorimetric measurements. The 1.5 A crystal structure of the oxidized protein revealed a disulfide bond-induced distortion of the beta-barrel, as well as a structural reorganization of residues in the immediate chromophore environment. By combining this information with spectroscopic data, we propose a detailed mechanism accounting for the observed redox state-dependent fluorescence. The redox potential of the cysteine couple was found to be within the physiological range for redox-active cysteines. In the cytoplasm of Escherichia coli, the protein was a sensitive probe for the redox changes that occur upon disruption of the thioredoxin reductive pathway.     

Citrate lyase from Klebsiella pneumoniae. The complete primary structure of the acyl lyase subunit

The primary structure of the beta-subunit (acyl lyase subunit) of citrate lyase from Klebsiella pneumoniae (ATCC 13,882) was determined with protein chemical methods. The polypeptide chain consists of 289 amino acid residues and has a molecular mass of 31,352 Da. The two half-cystine residues of the subunit are present as cysteines and not involved in disulfide bridges. The sequence shows no homology to known sequences of proteins or nucleic acids and reads (sequence; see text)     

In HspA from Helicobacter pylori vicinal disulfide bridges are a key determinant of domain B structure

Helicobacter pylori produces a heat shock protein A (HspA) that is unique to this bacteria. While the first 91 residues (domain A) of the protein are similar to GroES, the last 26 (domain B) are unique to HspA. Domain B contains eight histidines and four cysteines and was suggested to bind nickel. We have produced HspA and two mutants: Cys94Ala and Cys94Ala/Cys111Ala and identified the disulfide bridge pattern of the protein. We found that the cysteines are engaged in three disulfide bonds: Cys51/Cys53, Cys94/Cys111 and Cys95/Cys112 that result in a unique closed loop structure for the domain B.     

Disulfide connectivity prediction using recursive neural networks and evolutionary information

MOTIVATION: We focus on the prediction of disulfide bridges in proteins starting from their amino acid sequence and from the knowledge of the disulfide bonding state of each cysteine. The location of disulfide bridges is a structural feature that conveys important information about the protein main chain conformation and can therefore help towards the solution of the folding problem. Existing approaches based on weighted graph matching algorithms do not take advantage of evolutionary information. Recursive neural networks (RNN), on the other hand, can handle in a natural way complex data structures such as graphs whose vertices are labeled by real vectors, allowing us to incorporate multiple alignment profiles in the graphical representation of disulfide connectivity patterns. RESULTS: The core of the method is the use of machine learning tools to rank alternative disulfide connectivity patterns. We develop an ad-hoc RNN architecture for scoring labeled undirected graphs that represent connectivity patterns. In order to compare our algorithm with previous methods, we report experimental results on the SWISS-PROT 39 dataset. We find that using multiple alignment profiles allows us to obtain significant prediction accuracy improvements, clearly demonstrating the important role played by evolutionary information. AVAILABILITY: The Web interface of the predictor is available at http://neural.dsi.unifi.it/cysteines     

半胱氨酸氧化还原状态的协同性

以2002年4月份的Culled Protein Data Bank数据库中的639条蛋白质多肽链为研究对象,统计分析了其含有的584条二硫键的形成特征,发现半胱氨酸氧化还原状态表现出明显的协同性现象：含有二硫键的蛋白质中几乎所有的半胱氨酸都以氧化态形式存在。这一协同性可以通过蛋白质全局水平上的20种氨基酸组分的百分含量很好地加以说明,由此来预测半胱氨酸的氧化还原状态准确率最高可达84．5％。结果表明半胱氨酸是否形成二硫键主要取决于蛋白质全局的而非局部的结构信息。     

Plant purple acid phosphatases - genes, structures and biological function

The properties of plant purple acid phosphatases (PAPs), metallophosphoesterases present in some bacteria, plants and animals are reviewed. All members of this group contain a characteristic set of seven amino-acid residues involved in metal ligation. Animal PAPs contain a binuclear metallic center composed of two irons, whereas in plant PAPs one iron ion is joined by zinc or manganese ion. Among plant PAPs two groups can be distinguished: small PAPs, monomeric proteins with molecular mass around 35 kDa, structurally close to mammalian PAPs, and large PAPs, homodimeric proteins with a single polypeptide of about 55 kDa. Large plant PAPs exhibit two types of structural organization. One type comprises enzymes with subunits bound by a disulfide bridge formed by cysteines located in the C-terminal region around position 350. In the second type no cysteines are located in this position and no disulfide bridges are formed between subunits. Differences in structural organisation are reflected in substrate preferences. Recent data reveal in plants the occurrence of metallophosphoesterases structurally different from small or large PAPs but with metal-ligating sequences characteristic for PAPs and expressing pronounced specificity towards phytate or diphosphate nucleosides and inorganic pyrophosphate.     

Reversible redox- and zinc-dependent dimerization of the Escherichia coli fur protein

Fur is a bacterial regulator using iron as a cofactor to bind to specific DNA sequences. This protein exists in solution as several oligomeric states, of which the dimer is generally assumed to be the biologically relevant one. We describe the equilibria that exist between dimeric Escherichia coli Fur and higher oligomers. The dissociation constant for the dimer-tetramer equilibrium is estimated to be in the millimolar range. Oligomerization is enhanced at low ionic strength and pH. The as-isolated monomeric form of Fur is not in equilibrium with the dimer and contains two disulfide bridges (C92-C95 and C132-C137). Binding of the monomer to DNA is metal-dependent and sequence specific with an apparent affinity 5.5 times lower than that of the dimer. Size exclusion chromatography, EDC cross-linking, and CD spectroscopy show that reconstitution of the dimer from the monomer requires reduction of the disulfide bridges and coordination of Zn2+. Reduction of the disulfide bridges or Zn2+ alone does not promote dimerization. EDC and DMA cross-links reveal that the N-terminal NH2 group of one subunit is in an ionic interaction with acidic residues of the C-terminal tail and close to Lys76 and Lys97 of the other. Furthermore, the yields of cross-link drastically decrease upon binding of metal in the activation site, suggesting that the N-terminus is involved in the conformational change. Conversely, oxidizing reagents, H2O2 or diamide, disrupt the dimeric structure leading to monomer formation. These results establish that coordination of the zinc ion and the redox state of the cysteines are essential for holding E. coli Fur in a dimeric state.     

Cooperativity of the oxidization of cysteines in globular proteins

Based on the 639 non-homologous proteins with 2910 cysteine-containing segments of well-resolved three-dimensional structures, a novel approach has been proposed to predict the disulfide-bonding state of cysteines in proteins by constructing a two-stage classifier combining a first global linear discriminator based on their amino acid composition and a second local support vector machine classifier. The overall prediction accuracy of this hybrid classifier for the disulfide-bonding state of cysteines in proteins has scored 84.1% and 80.1%, when measured on cysteine and protein basis using the rigorous jack-knife procedure, respectively. It shows that whether cysteines should form disulfide bonds depends not only on the global structural features of proteins but also on the local sequence environment of proteins. The result demonstrates the applicability of this novel method and provides comparable prediction performance compared with existing methods for the prediction of the oxidation states of cysteines in proteins.     

Cysteine cross-linking defines part of the dimer and B/C domain interface of the Escherichia coli mannitol permease

Part of the dimer and B/C domain interface of the Escherichia coli mannitol permease (EII(mtl)) has been identified by the generation of disulfide bridges in a single-cysteine EII(mtl), with only the activity linked Cys(384) in the B domain, and in a double-cysteine EII(mtl) with cysteines at positions 384 and 124 in the first cytoplasmic loop of the C domain. The disulfide bridges were formed in the enzyme in inside-out membrane vesicles and in the purified enzyme by oxidation with Cu(II)-(1,10-phenanthroline)(3), and they were visualized by SDS-polyacrylamide gel electrophoresis. Discrimination between possible disulfide bridges in the dimeric double-cysteine EII(mtl) was done by partial digestion of the protein and the formation of heterodimers, in which the cysteines were located either on different subunits or on one subunit. The disulfide bridges that were identified are an intersubunit Cys(384)-Cys(384), an intersubunit Cys(124)-Cys(124), an intersubunit Cys(384)-Cys(124), and an intrasubunit Cys(384)-Cys(124). The disulfide bridges between the B and C domain were observed with purified enzyme and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Mannitol did not influence the formation of the disulfide between Cys(384) and Cys(124). The close proximity of the two cysteines 124 was further confirmed with a separate C domain by oxidation with Cu(II)-(1,10-phenanthroline)(3) or by reactions with dimaleimides of different length. The data in combination with other work show that the first cytoplasmic loop around residue 124 is located at the dimer interface and involved in the interaction between the B and C domain.     

A Molecular Dynamics Study of Human Defensins HBD-1 and HNP-3 in Water

Abstract

Mammalian defensins are crucial components of the innate immune system. They are characterized by three disulfide bridges and exhibit broad spectrum antibacterial activity. The spacing between the cysteines and disulfide connectivities in the two classes of defensins, the α- and β-forms, are different. The structural motif of 3 β-strands appears to be conserved in α and β-defensins despite differences in disulfide connectivities and spacing between cysteines. In this study, Molecular Dynamics Simulations (MDS) have been carried out to study the conformational behavior of α- and β-defensins with and without disulfide bridges. Our results indicate that β-strands in the C-terminal region of HBD-1 and HNP-3 do not unfold during the course of MDS. The segment adopting α-helix in HBD-1 unfolds early during the simulations. The backbone hydrogen bonds in HBD-1 and HNP-3 are broken during MDS. When the disulfide bonds are absent, the N-terminal β-strand unfolds by 20 ns but β-strands are observed in the C-terminal region of HNP-3. HBD-1, without disulfide bridges, unfolds to a greater extent during the course of the MDS. Examination of distances between sulfur atoms of cysteines without disulfide bridges during the simulations indicate that there is no specific preference for native disulfide bridges, which could be the reason for the experimental observation of non-native disulfide bridge formation during chemical synthesis of human α- and β-defensins. Since defensins with non-native disulfide bridges are biologically active, the exact three dimensional structures observed for native HBD-1 and HNP-3 does not appear to be essential for exhibiting antibacterial activity.     

Structural analysis of ABC-family periplasmic zinc binding protein provides new insights into mechanism of ligand uptake and release

ATP-binding cassette superfamily of periplasmic metal transporters are known to be vital for maintaining ion homeostasis in several pathogenic and non-pathogenic bacteria. We have determined crystal structure of the high-affinity zinc transporter ZnuA from Escherichia coli at 1.8 A resolution. This structure represents the first native (non-recombinant) protein structure of a periplasmic metal binding protein. ZnuA reveals numerous conformational features, which occur either in Zn(2+) or in Mn(2+) transporters, and presents a unique conformational state. A comprehensive comparison of ZnuA with other periplasmic ligand binding protein structures suggests vital mechanistic differences between bound and release states of metal transporters. The key new attributes in ZnuA include a C-domain disulfide bond, an extra alpha-helix proximal to the highly charged metal chelating mobile loop region, alternate conformations of secondary shell stabilizing residues at the metal binding site, and domain movements potentially controlled by salt bridges. Based on in-depth structural analyses of five metal binding transporters, we present here a mechanistic model termed as "partial domain slippage" for binding and release of Zn(2+).