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

Clarke, Neil D.; Yuan, Shao-Min

doi:10.1002/prot.340230214

Cited by 49 publications

(35 citation statements)

References 14 publications

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“…The method described in this manuscript-in which the geometry of the active site helps dictate the overall fold of the protein-differs from the computational approaches of Hellinga (15,17) and Clark (16,18). Their methods instead begin with a fixed backbone structure (generally of a natural protein) and then search for convenient locations for introducing a metal ion-binding site.…”

Section: Discussionmentioning

confidence: 99%

“…This problem may be circumvented by grafting inorganic cofactor-binding sites into the structures of natural proteins that normally do not bind metal ions. Automated methods have been developed recently for engineering such ion-binding sites (15,16), and it has been possible to build a number of structural as well as redox-active metal ion-binding sites within several different proteins (17)(18)(19)(20). Another successful approach has been to design small flexible peptides that are able to fold around metal sites such as Cu(II)-binding motifs (21)(22)(23)(24) and small peptides that assemble into Fe 4 S 4 clusters (25,26).…”

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confidence: 99%

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Retrostructural analysis of metalloproteins: Application to the design of a minimal model for diiron proteins

Lombardi

Summa

Geremia

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

232

279

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De novo protein design provides an attractive approach for the construction of models to probe the features required for function of complex metalloproteins. The metal-binding sites of many metalloproteins lie between multiple elements of secondary structure, inviting a retrostructural approach to constructing minimal models of their active sites. The backbone geometries comprising the metal-binding sites of zinc fingers, diiron proteins, and rubredoxins may be described to within approximately 1 Å rms deviation by using a simple geometric model with only six adjustable parameters. These geometric models provide excellent starting points for the design of metalloproteins, as illustrated in the construction of Due Ferro 1 (DF1), a minimal model for the GluXxx-Xxx-His class of dinuclear metalloproteins. This protein was synthesized and structurally characterized as the di-Zn(II) complex by x-ray crystallography, by using data that extend to 2.5 Å. This four-helix bundle protein is comprised of two noncovalently associated helix-loop-helix motifs. The dinuclear center is formed by two bridging Glu and two chelating Glu side chains, as well as two monodentate His ligands. The primary ligands are mostly buried in the protein interior, and their geometries are stabilized by a network of hydrogen bonds to second-shell ligands. In particular, a Tyr residue forms a hydrogen bond to a chelating Glu ligand, similar to a motif found in the diiron-containing R2 subunit of Escherichia coli ribonucleotide reductase and the ferritins. DF1 also binds cobalt and iron ions and should provide an attractive model for a variety of diiron proteins that use oxygen for processes including iron storage, radical formation, and hydrocarbon oxidation. P roteins use a limited repertoire of metal ion cofactors to help catalyze a multitude of reactions. For example, diiron sites (1-4) mediate reversible oxygen binding in hemerythrins, whereas they function as hydrolytic centers in phosphatases. Structurally similar diiron sites also mediate a number of oxygendependent oxidative processes. Ferritins serve as ferroxidases, while other diiron proteins catalyze hydroxylation, epoxidation, and desaturation reactions. Further, a diiron site in Escherichia coli ribonucleotide reductase is responsible for the formation of a Tyr radical. How do the structures of these proteins tune the chemical properties of a common diiron center to obtain such a diversity of highly specific catalysts? This question is being addressed through the study of the natural proteins as well as the study of small-molecule diiron complexes (1-4). Although impressive progress has been made on both fronts, these approaches have inherent limitations. The study of large proteins is hampered by their extreme complexity, and it is difficult to synthesize small-molecule models capable of simultaneously binding diiron, oxygen, and various substrates. Recently, we and others have sought a molecular middle ground between these two extremes through the design of small proteins and peptid...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Retrostructural analysis of metalloproteins: Application to the design of a minimal model for diiron proteins

Lombardi

Summa

Geremia

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

232

279

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“…The first step in identifying potential tetrahedrally coordinated metal-binding sites in proteins of known structure involves a computer search. The program Metal Search (Clarke & Yuan, 1995) uses backbone coordinates of the crystal or average NMR structure as input, and it replaces the natural amino acids with His and Cys residues. The program then searches all possible side-chain rotamers and stores them in an array.…”

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confidence: 99%

The de novo design of a rubredoxin‐like fe site

Farinas

Regan

1998

Protein Science

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A redox center similar to that of rubredoxin was designed into the 56 amino acid immunoglobulin binding B 1 domain of Streptococcals protein G. The redox center in rubredoxin contains an iron ion tetrahedrally coordinated by four cysteine residues, [ F e ( S -c~s )~] ",-'. The design criteria for the target site included taking backbone movements into account, tetrahedral metal-binding, and maintaining the structure and stability of the wild-type protein. The optical absorption spectrum of the Co(I1) complex of the metal-binding variant is characteristic of tetrahedral chelation by four cysteine residues. Circular dichroism and nuclear magnetic resonance measurements reveal that the metal-free and Cd(I1)-bound forms of the variant are folded correctly and are stable. The Fe(II1) complex of the metal-binding mutant reproduces the optical and the electron paramagnetic resonance spectra of oxidized rubredoxin. This demonstrates that the engineered protein chelates Fe(II1) in a tetrahedral array, and the resulting center is similar to that of oxidized rubredoxin.Keywords: de novo design; metalloprotein; protein engineering; rubredoxin Protein engineering and de novo design approaches have begun to afford a more detailed understanding of the balance of the forces that determine protein structure and function (Regan & DeGrado, 1988;Regan, 1993;Bryson et al., 1995;Regan, 1995;Dalal et al., 1997;Smith & Regan, 1997;Hellinga, 1998). To date, there are just a few examples of designed proteins that adopt a specified fold, and number which display novel activities is still smaller (Dill, 1990; Schafmeister et al., 1993;Jackson et al., 1994;Quinn et al., 1994;Yan & Erickson, 1994;Baltzer et al., 1996;Dahiyat & Mayo, 1997;Hill & DeGrado, 1998;QuCnCneur et al., 1998). A particular focus of functional design efforts has been the engineering of novel metal-binding sites. Metal-binding sites represent an attractive target because they play a variety of functions from stabilization of protein structure to catalytic roles such as the activation of water for nucleophilic attack, electron transfer, and redox activity. More recently, several groups have focused strongly on the design of metal-binding sites with particular activities in mind. For example, two metal-binding histidine residues have been engineered into rat trypsin to "switch" the catalytic activity on or off by disrupting the catalytic triad. The protein can be turned off by Cu(I1) chelation in the designed site and reactivated upon removal of Cu(I1) with EDTA (Halfon & Craik, 1996). In another design, a metal-binding site, based on the metal center of carbonic

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“…This approach is different from previous metalloprotein design programs which only search in one rigid conformation (Hellinga and Richards, 1991;Clarke and Yuan, 1995). Three types of zinc coordination sites were considered: Cys 4 , Cys 3 His and Cys 2 His 2 , and their ideal coordination parameters were obtained by statistical analysis of natural zinc binding proteins in the Protein Data Bank (Berman et al, 2000).…”

Section: Resultsmentioning

confidence: 99%

Engineering a zinc binding site into the de novo designed protein DS119 with a βαβ structure

Zhu¹,

Liang²,

Lai³

2011

Protein Cell

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Functional proteins designed de novo have potential application in chemical engineering, agriculture and healthcare. Metal binding sites are commonly used to incorporate functions. Based on a de novo designed protein DS119 with a βαβ structure, we have computationally engineered zinc binding sites into it using a home-made searching program. Seven out of the eight designed sequences tested were shown to bind Zn 2+ with micromolar affinity, and one of them bound Zn 2+ with 1:1 stoichiometry. This is the first time that metalloproteins with an α, β mixed structure have been designed from scratch.

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

Cited by 49 publications

References 14 publications

Retrostructural analysis of metalloproteins: Application to the design of a minimal model for diiron proteins

Retrostructural analysis of metalloproteins: Application to the design of a minimal model for diiron proteins

The de novo design of a rubredoxin‐like fe site

Engineering a zinc binding site into the de novo designed protein DS119 with a βαβ structure

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