Prediction of transition metal-binding sites from apo protein structures

Metal ions are crucial for protein function. They participate in enzyme catalysis, play regulatory roles, and help maintain protein structure. Current tools for predicting metal-protein interactions are based on proteins crystallized with their metal ions present (holo forms). However, a majority of resolved structures are free of metal ions (apo forms). Moreover, metal binding is a dynamic process, often involving conformational rearrangement of the binding pocket. Thus, effective predictions need to be based on the structure of the apo state. Here, we report an approach that identifies transition metal-binding sites in apo forms with a resulting selectivity >95%. Applying the approach to apo forms in the Protein Data Bank and structural genomics initiative identifies a large number of previously unknown, putative metal-binding sites, and their amino acid residues, in some cases providing a first clue to the function of the protein.     

Mapping metal-binding sites in the catalytic domain of bacterial RNase P RNA

Ribonuclease P (RNase P) is a ribonucleoprotein enzyme that contains a universally conserved, catalytically active RNA component. RNase P RNA requires divalent metal ions for folding, substrate binding, and catalysis. Despite recent advances in understanding the structure of RNase P RNA, no comprehensive analysis of metal-binding sites has been reported, in part due to the poor crystallization properties of this large RNA. We have developed an abbreviated yet still catalytic construct, Bst P7Δ RNA, which contains the catalytic domain of the bacterial RNase P RNA and has improved crystallization properties. We use this mutant RNA as well as the native RNA to map metal-binding sites in the catalytic core of the bacterial RNase P RNA, by anomalous scattering in diffraction analysis. The results provide insight into the interplay between RNA structure and focalization of metal ions, and a structural basis for some previous biochemical observations with RNase P. We use electrostatic calculations to extract the potential functional significance of these metal-binding sites with respect to binding Mg²⁺. The results suggest that with at least one important exception of specific binding, these sites mainly map areas of diffuse association of magnesium ions.     

Exploiting 3D structural templates for detection of metal-binding sites in protein structures

High throughput structural genomics efforts have been making the structures of proteins available even before their function has been fully characterized. Therefore, methods that exploit the structural knowledge to provide evidence about the functions of proteins would be useful. Such methods would be needed to complement the sequence-based function annotation approaches. The current study describes generation of 3D-structural motifs for metal-binding sites from the known metalloproteins. It then scans all the available protein structures in the PDB database for putative metal-binding sites. Our analysis predicted more than 1000 novel metal-binding sites in proteins using three-residue templates, and more than 150 novel metal-binding sites using four-residue templates. Prediction of metal-binding site in a yeast protein YDR533c led to the hypothesis that it might function as metal-dependent amidopeptidase. The structural motifs identified by our method present novel metal-binding sites that reveal newer mechanisms for a few well-known proteins.     

Protein metal-binding sites.

Metal ions have a role in a variety of important functions in proteins including protein folding, assembly, stability, conformational change, and catalysis. The presence or absence of a given metal ion is crucial to the conformation or activity of over one third of all proteins. Recent developments have been made in the understanding and design of metal-binding sites in proteins, an important and rapidly advancing area of protein engineering.     

醇脱氢酶金属离子绑定位点的可替换性

金属酶通过其极性氨基酸残基侧链所形成的共价键去锚定金属离子,目前鲜有报道替换金属绑定位点本身是否影响原有酶催化性能.以来源于Thermoanaerobacter brockii的锌离子依赖型醇脱氢酶TbSADH为研究对象,对其绑定锌离子的3个氨基酸残基位点Cys37、His59及Asp150进行序列保守性分析并构建突变...     

High-resolution X-ray structures of human apoferritin H-chain mutants correlated with their activity and metal-binding sites

Ferritins are a family of proteins distributed widely in nature. In bacterial, plant, and animal cells, ferritin appears to serve as a soluble, bioavailable, and non-toxic form of iron provider. Ferritins from animal sources are heteropolymers composed of two types of subunit, H and L, which differ mainly by the presence (H) or absence (L) of active ferroxidase centres. We report the crystallographic structures of four human H apoferritin variants at a resolution of up to 1.5 Angstrom. Crystal derivatives using Zn(II) as redox-stable alternative for Fe(II), allows us to characterize the different metal-binding sites. The ferroxidase centre, which is composed of sites A and B, binds metal with a preference for the A site. In addition, distinct Zn(II)-binding sites were found in the 3-fold axes, 4-fold axes and on the cavity surface near the ferroxidase centre. To study the importance of the distance of the two metal atoms in the ferroxidase centre, single and double replacement of glutamate 27 (site A) and glutamate 107 (site B), the two axial ligands, by aspartate residues have been carried out. The consequences for metal binding and the correlation with Fe(II) oxidation rates are discussed.     

Designed metal-binding sites in biomolecular and bioinorganic interactions

The design of metal-binding functionality in proteins is expanding into many different areas with a wide range of practical and research applications. Here we review several developing areas of metal-related protein design, including the use of metals to induce protein-protein interactions or facilitate the assembly of extended nanostructures; the design of metallopeptides that bind metal and other inorganic surfaces, an area with potential in diverse applications ranging from nanoelectronics and photonics to biotechnology and biomedicine; and, the creation of sensitive and selective metal sensors for use both in vivo and in vitro.     

Cationic probes: Specificity of distribution of metal-binding sites in bovine sperm

Salts of transition elements that alter the rate of sperm cell movement act at or near calcium-binding sites. After living bull sperm cells had been preincubated in VO₄^3?, Ni²⁺, Zn²⁺, Mn²⁺, and also La³⁺, they were then fixed. Crisply defined organelles and the absence of particulate deposits in the morphological controls contrasted sharply with the treated specimens; the latter contained regions of increased electron density, the nature and distribution of which depended on the test substance, reflecting the differential affinities of the specific ions. La³⁺ formed fine dense areas, mainly at the exocytic surface of the plasma membrane. VO₄^3? marks the cell surface but also left particulate densities within the cell. Ni²⁺ caused a nearly uniformly dense deposit at the surface and on the satellite fibers and axonemal microtubules. Zn²⁺ formed less uniform but coarser deposits, while in Mn²⁺ the distribution was similar to that in Zn²⁺ but much denser in the axonemal matrix and on the satellite fibers. Verapamil restricted the size and number of the opacities, while procaine permitted a similar distribution of slightly larger size reaction product. The differences in size and distribution of the enhanced densities were consistent and replicable for the individual assay substances. Vanadate, which specifically inhibits Na, K-ATPase, bound to ouabain-sensitive enzyme loci, however, completely disrupting the axonemal complex. This suggests that an important role of dynein in flagellar motion may relate to intracellular transport of Ca²⁺.     

Beyond structural genomics: computational approaches for the identification of ligand binding sites in protein structures

Structural genomics projects have revealed structures for a large number of proteins of unknown function. Understanding the interactions between these proteins and their ligands would provide an initial step in their functional characterization. Binding site identification methods are a fast and cost-effective way to facilitate the characterization of functionally important protein regions. In this review we describe our recently developed methods for binding site identification in the context of existing methods. The advantage of energy-based approaches is emphasized, since they provide flexibility in the identification and characterization of different types of binding sites.     

Localization and functional characterization of metal-binding sites in phytochelatin synthases

Metal-binding domains consisting of short, contiguous stretches of amino acids are found in many proteins mediating the transport, buffering, trafficking or detoxification of metal ions. Phytochelatin synthases are metal-activated enzymes that function in the detoxification of Cd²⁺ and other toxic metal and metalloid ions. In order to localize Cd²⁺-binding sites, peptide libraries of two diverse phytochelatin synthases were synthesized and incubated with ¹⁰⁹Cd²⁺. Distinct binding sites and binding motifs could be localized based on the patterns of Cd²⁺-binding. The number of binding sites was consistent with previous findings for recombinant protein. Positions of binding sites appeared to be conserved even among diverse phytochelatin synthases. Mutant peptide analysis was used to assess the contribution of exemplary amino acids to binding. Several binding motifs contain cysteines or glutamates. For cysteines a strong correlation was found between binding activity and degree of conservation among known phytochelatin synthases. These findings indicate the suitability of peptide scanning for the identification of metal-binding sites. The functional role of several cysteines was investigated by expression of hemagglutinin-tagged phytochelatin synthases in phytochelatin synthase-deficient, Cd²⁺-hypersensitive Schizosaccharomyces pombe cells. The data are consistent with a model suggesting functionally essential metal-binding activation sites in the N-terminal catalytic part of phytochelatin synthases and additional binding sites at the C-terminus not essential for activity.Abbreviations EMM Edinburgh's minimal medium - GSH glutathione - HA hemagglutinin - PC phytochelatin - PCS phytochelatin synthase     

Architecture of the two metal-binding sites in prolactin

Metal binding by members of the growth hormone (GH) family of hematopoietic cytokines has been a subject of considerable interest. However, beyond appreciation of its role in reversible packing of GH proteins in secretory granules, the molecular mechanisms of metal binding and granule formation remain poorly understood. Here, we investigate metal binding by a GH family member prolactin (PRL) using paramagnetic metal titration and chelation experiments. Cu²⁺-mediated paramagnetic relaxation enhancement measurements identified two partial metal-binding sites on the opposite faces of PRL composed of residues H30/H180 and E93/H97, respectively. Coordination of metal ions by these two sites causes formation of inter-molecular bridges between the PRL protomers and enables formation of reversible higher aggregates. These findings in vitro suggest a model for reversible packaging of PRL in secretory granules. The proposed mechanism of metal-promoted PRL aggregation lends insight and support to the previously suggested role of metal coordination in secretory granule formation by GH proteins.     

From knotted to nested RNA structures: a variety of computational methods for pseudoknot removal

Pseudoknots are abundant in RNA structures. Many computational analyses require pseudoknot-free structures, which means that some of the base pairs in the knotted structure must be disregarded to obtain a nested structure. There is a surprising diversity of methods to perform this pseudoknot removal task, but these methods are often poorly described and studies can therefore be difficult to reproduce (in part, because different procedures may be intuitively obvious to different investigators). Here we provide a variety of algorithms for pseudoknot removal, some of which can incorporate sequence or alignment information in the removal process. We demonstrate that different methods lead to different results, which might affect structure-based analyses. This work thus provides a starting point for discussion of the extent to which these different methods recapture the underlying biological reality. We provide access to reference implementations through a web interface (at http://www.ibi.vu.nl/programs/k2nwww), and the source code is available in the PyCogent project.     

Characterization of transferrin metal-binding sites by diffusion-enhanced energy transfer

The distance from the protein surface to ferric or manganic ions in the two specific metal-binding sites of human serum transferrin has been estimated by measuring energy transfer from freely diffusing terbium chelaters in aqueous solution to transferrin-bound metal ions. In addition, both monoferric forms of the protein were studied, as well as the diferric complex formed by using oxalate instead of (bi)carbonate as the auxiliary anion in binding of iron(III) to transferrin. Second-order rate constants for energy transfer between electrically neutral terbium(III)--N-(2-hydroxy-ethyl)ethylenediaminetriacetate and the FeA, FeB, and Fe2 forms of transferrin were 0.9 X 10(5) M-1 S-1, 1.4 X 10(5) M-1 S-1, and 2.6 X 10(5) M-1 S-1, respectively (based on iron concentraton). For the Fe2 species, substitution of oxalate for (bi)carbonate has the effect of decreasing the accessibility of both electrically neutral and negatively charged terbium chelates to the protein-bound iron chromophores. Theoretical considerations of the effect of acceptor location in the protein on energy transfer suggest that the iron chromophores are not on the surface of the protein but are less than 1.7 nm below the surface. The use of diterbium transferrin as energy donor to a small cobalt chelate in solution or to diferric transferrin corroborates these results.     

Protein-coding structured RNAs: A computational survey of conserved RNA secondary structures overlapping coding regions in drosophilids

Functional RNA elements can be embedded also within exonic sequences coding for functional proteins. While not uncommon in viruses, only a few examples of this type have been described in some detail for eukaryotic genomes. Here we use RNAz and RNAcode, two comparative genomics methods that measure signatures of stabilizing selection acting on RNA secondary structure and peptide sequence, resp., to survey the fruit fly genomes. We estimate that there might be on the order of 1000 loci that are subject to dual selection pressure. The used genome-wide screens also expose the limitations of the currently available methods.     

Molego-based definition of the architecture and specificity of metal-binding sites

Decomposing proteins into "molegos," building blocks that are conserved in sequence and 3D-structure, can identify functional elements. To demonstrate the specificity of the decomposition method, the PCPMer program suite was used to numerically define physical chemical property motifs corresponding to the molegos that make up the metal-containing active sites of three distinct enzyme families, from the dimetallic phosphatases, DNase 1 related nucleases/phosphatases, and dioxygenases. All three superfamilies bind metal ions in a beta-strand core region but differ in the number and type of ions needed for activity. The motifs were then used to automatically identify proteins in the ASTRAL40 database that contained similar motifs. The proteins with the highest PCPMer score in the database were primarily metal-binding enzymes that were related in function to those in the alignment used to generate the PCPMer motif lists. The proteins that contained motifs similar to the dioxygenases differed from those found with PCP-motifs for phosphatases and nucleases. Relatively few metal-binding enzymes were detected when the search was done with PCP-motifs defined for interleukin-1 related proteins, which have a beta-strand core but do not bind metal ions. While the box architecture was constant in each superfamily, the specificity for the metal ion preferred for enzymatic activity is determined by the pattern of carbonyl, hydroxyl or imadazole groups in key positions in the molegos. These results have implications for the design of metal-binding enzymes, and illustrate the ability of the PCPMer approach to distinguish, at the sequence level, structural and functional elements.     

Metal search: A computer program that helps design tetrahedral metal-binding sites

We describe a computer program (Metal Search) that helps design tetrahedrally coordinated metal binding sites in proteins of known structure. The program takes as input the backbone coordinates of a protein and outputs lists of four residues that might form tetrahedral sites if wild-type amino acids were replaced by cysteine or histidine. The program also outputs the side chain dihedral angles of the amino acids and the coordinates of the predicted metal ion. The only function evaluated by Metal Search is the ability of side chains to meet simple geometric criteria for formation of a tetrahedral site, but these criteria are sufficient to produce a manageably small list that can then be evaluated by other means. The program has been used in the introduction of zinc binding sites in the designed four-helix bundle protein α 4 and in the B1 domain of streptococcal protein G, and in both cases the tetrahedral coordination of a bound metal ion has been confirmed¹ (Klemba, M., Gardner, K. H., Marino, S., Clarke, N. D., and Regan, L., Nature: Structural Biology 2:368–373, 1995). © 1995 Wiley-Liss, Inc.     

Compensatory evolution in RNA secondary structures increases substitution rate variation among sites

There is growing evidence that interactions between biological molecules (e.g., RNA-RNA, protein-protein, RNA-protein) place limits on the rate and trajectory of molecular evolution. Here, by extending Kimura's model of compensatory evolution at interacting sites, we show that the ratio of transition to transversion substitutions (kappa) at interacting sites should be equal to the square of the ratio at independent sites. Because transition mutations generally occur at a higher rate than transversions, the model predicts that kappa should be higher at interacting sites than at independent sites. We tested this prediction in 10 RNA secondary structures by comparing phylogenetically derived estimates of kappa in paired sites within stems (kappa(p)) and unpaired sites within loops (kappa(u)). Eight of the 10 structures showed an excellent match to the quantitative predictions of the model, and 9 of the 10 structures matched the qualitative prediction kappa(p) > kappa(u). Only the Rev response element from the human immunovirus (HIV) genome showed the reverse pattern, with kappa(p) < kappa(u). Although a variety of evolutionary forces could produce quantitative deviations from the model predictions, the reversal in magnitude of kappa(p) and kappa(u) could be achieved only by violating the model assumption that the underlying transition (or transversion) mutation rates were identical in paired and unpaired regions of the molecule. We explore the ability of the APOBEC3 enzymes, host defense mechanisms against retroviruses, which induce transition mutations preferentially in single-stranded regions of the HIV genome, to explain this exception to the rule. Taken as a whole, our findings suggest that kappa may have utility as a simple diagnostic to evaluate proposed secondary structures.     

Terbium as a luminescent probe of metal-binding sites in protein kinase C

In the present report, we demonstrate that Tb3+ binds to protein kinase C and serves as a luminescent reporter of certain cationic metal-binding sites. Tb3+ titration of 50 nM protein kinase C results in a 20-fold enhancement of Tb3+ luminescence which is half-maximal at 12 microM Tb3+. A Kd of approximately 145 nM was determined for Tb3+ binding to the enzyme. The excitation spectrum of bound Tb3+ exhibits a peak at 280 nm characteristic of energy transfer from protein tryptophan or tyrosine residues. The luminescence of this complex can be markedly decreased by other metals, including Pb2+ (IC50 = 25 microM), La3+ (IC50 = 50 microM), Hg2+ (IC50 = 300 microM), Ca2+ (IC50 = 6 mM), and Zn2+ (IC50 greater than 10 mM), and chelation of Tb3+ by 2 mM EGTA. Tb3+ binding to protein kinase C is correlated with its inhibition of protein kinase activity (IC50 = 8 microM), r = 0.99) and phorbol ester binding (IC50 = 15 microM, r = 0.98). Tb3+ inhibition of protein kinase C activity cannot be overcome by excess Ca2+, but can be partially overcome with excess phosphatidylserine or by chelation of Tb3+ with EGTA. Tb3+ noncompetitively inhibits phorbol ester binding by decreasing the maximal extent of binding without significantly altering binding affinity. The results suggest that the Tb3(+)-binding site is at or allosterically related to the enzyme's phosphatidylserine-binding site, but is distinct from the phorbol ester-binding domain and the Ca2(+)-binding site that regulates enzyme activity.