Metal Binding Sites In Proteins Research Articles

Development of METAL-ACTIVE SITE and ZINCCLUSTER tool to predict active site pockets.

The advent of whole genome sequencing leads to increasing number of proteins with known amino acid sequences. Despite many efforts, the number of proteins with resolved three dimensional structures is still low. One of the challenging tasks the structural biologists face is the prediction of the interaction of metal ion with any protein for which the structure is unknown. Based on the information available in Protein Data Bank, a site (METALACTIVE INTERACTION) has been generated which displays information for significant high preferential and low-preferential combination of endogenous ligands for 49 metal ions. User can also gain information about the residues present in the first and second coordination sphere as it plays a major role in maintaining the structure and function of metalloproteins in biological system. In this paper, a novel computational tool (ZINCCLUSTER) is developed, which can predict the zinc metal binding sites of proteins even if only the primary sequence is known. The purpose of this tool is to predict the active site cluster of an uncharacterized protein based on its primary sequence or a 3D structure. The tool can predict amino acids interacting with a metal or vice versa. This tool is based on the occurrence of significant triplets and it is tested to have higher prediction accuracy when compared to that of other available techniques.

Prediction of zinc binding sites in proteins using sequence derived information

Zinc is one the most abundant catalytic cofactor and also an important structural component of a large number of metallo-proteins. Hence prediction of zinc metal binding sites in proteins can be a significant step in annotation of molecular function of a large number of proteins. Majority of existing methods for zinc-binding site predictions are based on a data-set of proteins, which has been compiled nearly a decade ago. Hence there is a need to develop zinc-binding site prediction system using the current updated data to include recently added proteins. Herein, we propose a support vector machine-based method, named as ZincBinder, for prediction of zinc metal-binding site in a protein using sequence profile information. The predictor was trained using fivefold cross validation approach and achieved 85.37% sensitivity with 86.20% specificity during training. Benchmarking on an independent non-redundant data-set, which was not used during training, showed better performance of ZincBinder vis-à-vis existing methods. Executable versions, source code, sample datasets, and usage instructions are available at http://proteininformatics.org/mkumar/znbinder/

Site-Selective Conjugation of Native Proteins with DNA.

Conjugation of DNA to proteins is increasingly used in academia and industry to provide proteins with tags for identification or handles for hybridization to other DNA strands. Assay technologies such as immuno-PCR and proximity ligation and the imaging technology DNA-PAINT require DNA-protein conjugates. In DNA nanotechnology, the DNA handle is exploited to precisely position proteins by self-assembly. For these applications, site-selective conjugation is almost always desired because fully functional proteins are required to maintain the specificity of antibodies and the activity of enzymes. The introduction of a bioorthogonal handle at a specific position of a protein by recombinant techniques provides an excellent approach to site-specific conjugation, but for many laboratories and for applications where several proteins are to be labeled, the expression of recombinant proteins may be cumbersome. In recent years, a number of chemical methods that target conjugation to specific sites at native proteins have become available, and an overview of these methods is provided in this Account. Our laboratory has investigated DNA-templated protein conjugation (DTPC), which offers an alternative approach to site-selective conjugation of DNA to proteins. The method is inspired by the concept of DNA-templated synthesis where functional groups conjugated to DNA strands are preorganized by DNA hybridization to dramatically increase the reaction rate. In DPTC, we target metal binding sites in proteins to template selective covalent conjugation reactions. By chelation of a DNA-metal complex with a metal binding site of the protein, an electrophile on a second DNA strand is aligned for reaction with a lysine side chain on the protein in the proximity of the metal binding site. The method is quite general because approximately one-third of all wild-type proteins contain metal-binding sites, including many IgG antibodies, and it is also applicable to His-tagged proteins. This emerging field provides direct access to site-selective conjugates of DNA to commercially available proteins. In this Account, we introduce these methods to the reader and describe current developments and future aspects.

The involvement of coordinative interactions in the binding of dihydrolipoamide dehydrogenase to titanium dioxide-Localization of a putative binding site.

Titanium (Ti) and its alloys are widely used in orthodontic and orthopedic implants by virtue to their high biocompatibility, mechanical strength, and high resistance to corrosion. Biointegration of the implants with the tissue requires strong interactions, which involve biological molecules, proteins in particular, with metal oxide surfaces. An exocellular high-affinity titanium dioxide (TiO2 )-binding protein (TiBP), purified from Rhodococcus ruber, has been previously studied in our lab. This protein was shown to be homologous with the orthologous cytoplasmic rhodococcal dihydrolipoamide dehydrogenase (rhDLDH). We have found that rhDLDH and its human homolog (hDLDH) share the TiO2 -binding capabilities with TiBP. Intrigued by the unique TiO2 -binding properties of hDLDH, we anticipated that it may serve as a molecular bridge between Ti-based medical structures and human tissues. The objective of the current study was to locate the region and the amino acids of the protein that mediate the protein-TiO2 surface interaction. We demonstrated the role of acidic amino acids in the nonelectrostatic enzyme/dioxide interactions at neutral pH. The observation that the interaction of DLDH with various metal oxides is independent of their isoelectric values strengthens this notion. DLDH does not lose its enzymatic activity upon binding to TiO2 , indicating that neither the enzyme undergoes major conformational changes nor the TiO2 binding site is blocked. Docking predictions suggest that both rhDLDH and hDLDH bind TiO2 through similar regions located far from the active site and the dimerization sites. The putative TiO2 -binding regions of both the bacterial and human enzymes were found to contain a CHED (Cys, His, Glu, Asp) motif, which has been shown to participate in metal-binding sites in proteins.

Chelation behavior of various flavonols and transfer of flavonol-chelated zinc(II) to alanylaspartic dipeptide: A PCM/DFT investigation

Alanylaspartic dipeptide (AlaAsp) and zinc(II)-flavonol complex could represent a metal-binding site in proteins and a metal-ion releasing agent, respectively. Chelation of zinc(II) by either AlaAsp or flavonol ligands in aqueous solution has been examined using DFT methods with polarizable continuum model (PCM/DFT). Coordination geometry, complexation stoichiometry, coordination bond strength, preferable metal-binding site on ligands and effect of water coordination on the stability of complexes have been addressed. In several cases, the long-range corrected density functional CAM-B3LYP allows the most accurate prediction of both structural and spectroscopic data. The preferential transfer of flavonol-chelated zinc(II) to AlaAsp under solvation is attainable through the ligand-exchange reaction. The energy barrier of such reaction is significantly dependent on the degree of hydrogen bonding within the transition state. In summary, either hydroxylation or methoxylation at particular positions on the 3-hydroxyflavone backbone significantly affects the reactivity of flavonol chelates in the metal-ion transfer.

Structural basis of an engineered dual-specific antibody: conformational diversity leads to a hypervariable loop metal-binding site.

To explore dual-specificity in a small protein interface, we previously generated a 'metal switch' anti-RNase A VHH antibody using a combinatorial histidine library approach. While most metal-binding sites in proteins are found within rigid secondary structure, the engineered VHH antibody (VHH(metal)), which contained three new histidine residues, possessed metal-binding residues within the flexible hypervariable loops. Here, crystal structure analysis of the free and bound states of VHH(metal) reveals the structural determinants leading to dual-function. Most notably, CDR1 is observed in two distinct conformations when adopting the metal or RNase A bound states. Furthermore, mutagenesis studies revealed that one of the engineered residues, not located in the metal-binding pocket, contributed indirectly to metal recognition, likely through influencing CDR1 conformation. Despite these changes, VHH(metal) possesses a relatively minor energetic penalty toward binding the original antigen, RNase A (~1 kcal/mol), where the engineered gain-of-function metal-binding residues are observed to possess a mix of favorable and unfavorable contributions towards RNase A recognition. Ultimately, the conformationally distinct metal-switch interface architecture reflects the robust, library-based strategy used to produce VHH(metal). These results also suggest that even small protein interfaces, such as VHH, may be structurally and energetically forgiving in adopting novel function, while maintaining original function.

Modeling Metal Binding Sites in Proteins by Quantum Chemical Calculations

Modeling Metal Binding Sites in Proteins by Quantum Chemical Calculations

Role of a remote leucine residue in the catalytic function of polyol dehydrogenase.

Studies on the protein-metal binding sites have mainly focused on the residues immediately surrounding the reacting substrate, cofactors, and metal ions. The contribution of residues in remote layers to the highly optimized microenvironments of catalytic active sites is not well understood. To improve our understanding, the present study examined the role of remote residues on the structure and function of zinc-dependent polyol dehydrogenases. We used an integrated computational and biochemical approach to determine the role of L136 in the third shell of the l-arabinitol 4-dehydrogenase (LAD) from Neurospora crassa. Substitution of L136 with charged (Asp, Lys, or His) and bulky (Trp) side chain amino acids abolished enzyme activity. Whereas the L136A mutant exhibited a 95% decrease in catalytic efficiency (kcat/Km), the L136C mutant exhibited a 39% decrease in kcat/Km. Additionally, molecular docking and dynamic simulations on the mutant (L136A, L136C, L136H, and L136P) complexes showed the loss of crucial H-bonds between G77 and the corresponding mutated residue. It is evident from theoretical and biochemical studies that the L136 is part of the extensive hydrogen bonding network coupled to the reaction catalyzed at the active site. We propose that L136, critically positioned behind the active site residues H78 and E79 in the third shell of LAD, plays a crucial role in modulating catalysis or substrate binding by stabilizing the GHE motif in the LAD active site.

A minimalist chemical model of matrix metalloproteinases — Can small peptides mimic the more rigid metal binding sites of proteins?

In order to mimic the active center of matrix metalloproteinases (MMPs), we synthesized a pentadecapeptide (Ac-KAHEFGHSLGLDHSK-NH2) corresponding to the catalytic zinc(II) binding site of human MMP-13. The multi-domain structural organization of MMPs fundamentally determines their metal binding affinity, catalytic activity and selectivity. Our potentiometric, UV-visible, CD, EPR, NMR, mass spectrometric and kinetic studies are aimed to explore the usefulness of such flexible peptides to mimic the more rigid metal binding sites of proteins, to examine the intrinsic metal binding properties of this naked sequence, as well as to contribute to the development of a minimalist, peptide-based chemical model of MMPs, including the catalytic properties. Since the multiimidazole environment is also characteristic for copper(II), and recently copper(II) containing variants of MMPs have been identified, we also studied the copper(II) complexes of the above peptide. Around pH6–7 the peptide, similarly to MMPs, offers a {3Nim} coordination binding site for both zinc(II) and copper(II). In the case of copper(II), the formation of amide coordinated species at higher pH abolished the analogy with the copper(II) containing MMP variant. On the other hand, the zinc(II)-peptide system mimics some basic features of the MMP active sites: the main species around pH7 (ZnH2L) possesses a {3Nim,H2O} coordination environment, the deprotonation of the zinc-bound water takes place near the physiological pH, it forms relatively stable ternary complexes with hydroxamic acids, and the species ZnH2L(OH) and ZnH2L(OH)2 have notable hydrolytic activity between pH7 and 9.

Web Tools for Predicting Metal Binding Sites in Proteins

AbstractApproximately one third of proteins bind metal ions for stability and/or enzymatic function. However, on a structural level, only a small fraction of binding sites have been resolved. Metal binding site predictions can serve as a first step in putative function assignment for many unannotated proteins. Sequence based and structure based methods for metal binding site predictions are reviewed here. The CHED and SeqCHED methods of prediction from apo protein structures and translated gene sequences, respectively, are described in detail, including their web server applications. The relevance of SeqCHED to the analysis of single nucleotide polymorphisms (SNPs) associated with disease related metal binding sites is illustrated.

MetalPDB: a database of metal sites in biological macromolecular structures

We present here MetalPDB (freely accessible at http://metalweb.cerm.unifi.it), a novel resource aimed at conveying the information available on the three-dimensional (3D) structures of metal-binding biological macromolecules in a consistent and effective manner. This is achieved through the systematic and automated representation of metal-binding sites in proteins and nucleic acids by way of Minimal Functional Sites (MFSs). MFSs are 3D templates that describe the local environment around the metal(s) independently of the larger context of the macromolecular structure embedding the site(s), and are the central objects of MetalPDB design. MFSs are grouped into equistructural (broadly defined as sites found in corresponding positions in similar structures) and equivalent sites (equistructural sites that contain the same metals), allowing users to easily analyse similarities and variations in metal–macromolecule interactions, and to link them to functional information. The web interface of MetalPDB allows access to a comprehensive overview of metal-containing biological structures, providing a basis to investigate the basic principles governing the properties of these systems. MetalPDB is updated monthly in an automated manner.

Predicting Protein Metal Binding Sites with RBF Networks based on PSSM Profiles and Additional Properties

Predicting Protein Metal Binding Sites with RBF Networks based on PSSM Profiles and Additional Properties

Tools for Predicting Metal Binding Sites in Protein: A Review

The rapid growth of the Protein Data Bank (PDB) highlights a great challenge for researchers to predict the binding sites of protein for specific metal ion(s). Experimental determination of functional features of a protein is expensive, time consuming and difficult to automate. Therefore, there is a great demand of computational methods for predicting functional features of protein. This review sheds light on currently available in-silico methods including different tools and databases which are based on various information of metal ion and their binding sites (protein residue length, amino acid composition, geometrical and molecular information etc.) and determines the efficiency, speed and accuracy by using diverse algorithms which make the tools beneficial.

First- and second-shell metal binding residues in human proteins are disproportionately associated with disease-related SNPs

Protein structure serves as a key determinant for revealing the molecular basis of human disease. Metal ions are among the most frequently bound heterogroups in proteins affecting structure and function. We analyzed the relationship between single nucleotide polymorphisms (SNPs) associated with human disease and metal binding sites in proteins on a database scale, using structural models and predictive tools. A match was identified for 586 disease-associated SNPs (dSNPs) located at 135 predicted metal binding sites and associated with 126 diverse diseases. For 104 diseases, a metal is known to bind at the predicted site in the homologue; for 22, the analysis gives a first indication for metal involvement in the disease. As second-shell residues play an important part in metal ion binding, our analysis included protein space up to 4.5 Å from metal binding sites. The ratio of disease-associated versus nondisease-associated SNPs (dSNP/ndSNP) for first-shell residues is 7.4 and for second-shell residues, 3.1. In addition, over 13% of all dSNPs were found to be associated with first- and second-shell residues, although these residues occupy only about 3% of protein space. These results show a disproportionate association of dSNPs and metal binding sites over a wide variety of diseases.

Preparation, characterization and biological relevance of calcium(II)/diethylmalonate(-1,-2) complexes

Reactions of Ca(NO 3) 2·4H 2O and diethylmalonic acid (Et 2malH 2, its anions represent functional side-chain analogs of γ-carboxyglutamic acid residues which are implicated as essential Ca 2+-binding ligands in a variety of proteins) in aqueous media have afforded compounds [Ca(Et 2malH) 2(H 2O) 3] n ( 1) and [Ca(Et 2mal)(H 2O)] n ( 2) at pH 4 and 8, respectively. The structure of 1 was determined by single-crystal, X-ray crystallography, which revealed an 1D coordination polymer. The diethylmalonate ligands exist in their monoanionic form and present two different coordination modes. The Ca II ion is 7-coordinate with a pentagonal bipyramidal geometry. IR data are discussed in terms of the known ( 1) and proposed ( 2) structures of the complexes. The role of the carboxylate binding modes in determining the affinity of Ca 2+ for the various metal binding sites in proteins containing the γ-carboxyglutamate residue is discussed in the light of our and previous results.

Development of Tools and Database for Analysis of Metal Binding Sites in Protein

In this study, we have developed a standalone tool called as ANAMBS (Analysis of Metal Binding Site) to derive metal neighbourhood information using PERL as the programming language. The tool accepts the structures in the pdb format. The cut off distance to define the metal binding region can be specified. The metal binding site composition, orientation of various amino acids and atoms along with the Hydropathy index within the metal binding site region can be measured. Its speed and efficiency makes it a beneficial tool for various structural biology projects, especially when the characterization of the metal binding domain is needed. Additionally, the database MEDB (Metal Environment Database) was developed which presents quantitative information on metal-binding sites in protein structures. It can be used for identification of trends or patterns in the metal-binding sites. The information obtained can be used to generate structural templates from metal binding sites of known enzymes and to develop constraints for computational modeling of metalloproteins. The tool and database are available at http://www.uohyd.ernet.in/anambs/

Rational design of a structural and functional nitric oxide reductase

SummaryProtein design provides an ultimate test of our knowledge about proteins and allows the creation of novel enzymes for biotechnological applications. While progress has been made in designing proteins that mimic native proteins structurally1–3, it is more difficult to design functional proteins4–8. In comparison to recent successes in designing non-metalloproteins4,6,7,9,10, it is even more challenging to rationally design metalloproteins that reproduce both the structure and function of native metalloenzymes5,8,11–20, since protein metal binding sites are much more varied than non-metal containing sites, in terms of different metal ion oxidation states, preferred geometry and metal ion ligand donor sets. Because of their variability, it has been difficult to predict metal binding site properties in silico, as many of the parameters for metal binding sites, such as force fields are ill-defined. Therefore, the successful design of a structural and functional metalloprotein will greatly advance the field of protein design and our understanding of enzymes. Here, we report a successful, rational design of a structural and functional model of a metalloprotein, nitric oxide reductase (NOR), by introducing three histidines and one glutamate, predicted as ligands in the active site of NOR, into the distal pocket of myoglobin. A crystal structure of the designed protein confirms that the minimized computer model contains a heme/non-heme FeB center that is remarkably similar to that in the crystal structure. This designed protein also exhibits NOR activity. This is the first designed protein that models both the structure and function of NOR, offering insight that the active site glutamate is required for both iron binding and activity. These results show that structural and functional metalloproteins can be rationally designed in silico.

Prediction of 3D metal binding sites from translated gene sequences based on remote‐homology templates

Database-scale analysis was performed to determine whether structural models, based on remote homologues, are effective in predicting 3D transition metal binding sites in proteins directly from translated gene sequences. The extent by which side chain modeling alone reduces sensitivity and selectivity is shown to be <10%. Surprisingly, selectivity was not dependent on the level of sequence homology between template and target, or on the presence of a metal ion in the structural template. Applying a modification of the CHED algorithm (Babor et al., Proteins 2008;70:208-217) and machine learning filters, a selectivity of approximately 90% was achieved for protein sequences using unrelated structural templates over a sequence identity range of 18-100%. Below approximately 18% identity, the number of analyzable target-template pairs and predictability of metal binding sites falls off sharply. A full third of structural templates were found to have target partners only in the remote homology range of 18-30%. In this range, nonmetal-binding templates are calculated to be the majority and serve to predict with 50% sensitivity at the geometric level. Overall, sensitivity at the geometric level for targets having templates in the 18-30% sequence identity range is 73%, with an average of one false positive site per true site. Protein sequences described as "unknown" in the UniProt database and composed largely of unidentified genome project sequences were studied and metal binding sites predicted. A web server for prediction of metal binding sites from protein sequence is provided.

MetalMine: a database of functional metal-binding sites in proteins

We present a new database called MetalMine that contains the classification of metal-binding sites derived from the structures of protein-metal-ion complexes. Metal-binding sites were automatically extracted from Protein Data Bank (PDB) structures, classified based on the protein domains in which the metal-binding sites are incorporated, and then manually curated. Tentative or artificial metal ion coordinations were excluded during this curation process. On the web pages of the database, the following information about metal-binding sites is presented in a hierarchical manner: the kind of metal ion, metal-binding site typically specified by the name of the protein, and each instance of metal-binding coordinates in the PDB structures. The database search engine currently supports the following two types of queries. First, given the PDB code of a protein–metal–ion complex, it provides a list of metal-binding sites incorporated in the structure file. Second, given an amino acid sequence as a query, it looks for matches with metal-binding residues in the Basic Local Alignment Search Tool (BLAST) sequence alignment. As of October 2009, MetalMine contained 412 classified entries of functional metal-binding sites, which we believe is the largest number of entries in databases available to the public.

The role of the histidine residue in the coordination abilities of peptides with a multi-histidine sequence towards copper(II) ions

Cu 2+ complexes with peptides containing three histidine residues have very specific metal binding abilities and can mimic the structures of various multi-histidine metal binding sites in proteins. The main goal of the work concerns the investigations of coordination abilities of the group of N-terminally protected Ac-His-Arg-His-Gly-His-Gly, Ac-His-Gly-His-Arg-His-Gly, Ac-Gly-His-His-Arg-His-Gly and Ac-His-His-Gly-His-Arg-Gly, and their unprotected analogs His-Arg-His-Gly-His-Gly, His-Gly-His-Arg-His-Gly, Gly-His-His-Arg-His-Gly and His-His-Gly-His-Arg-Gly towards Cu 2+ ions. Detailed spectroscopic (UV/Vis, CD and EPR) and potentiometric studies have been made. The stoichiometry and binding mode for each ligand–Cu 2+ system were determined. In the case of N-terminally protected peptides the coordination begins at the imidazole nitrogen(s) which act as the anchoring groups. At physiological pH the {3N im, 2N −} binding pattern is suggested and at high pH three amide nitrogens are involved in Cu 2+ binding. According to the coordination abilities of the unprotected peptides, the position of the histidine residue determines the coordination mode. For His 1 peptides the histamine-like {NH 2, N im} mode for the first species is suggested. The final albumin-like coordination mode is proposed not only for HGHRHG and HRHGHG but also for GHHRHG. For ligands with the His-Xaa-His motif the {2N −, 2N im} binding mode is formed easier. For ligands with His 2 the 3N complex with the {NH 2, N −, N im} binding pattern is a dominant species within the physiological pH range. The protection of the amine group caused a significant decrease of the coordination abilities of the peptides.