Toward the de Novo Design of a Catalytically Active Helix Bundle:  A Substrate-Accessible Carboxylate-Bridged Dinuclear Metal Center

Costanzo, Luigi Di; Wade, Herschel; Geremia, Silvano; Randaccio, Lucio; Pavone, Vincenzo; DeGrado, William F.; Lombardi, Angela

doi:10.1021/ja010506x

Cited by 101 publications

(135 citation statements)

References 64 publications

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“…Previously, we examined the structural and thermodynamic effects of opening the substrate-access cavity of DF1, by changing a pair of residues from Leu to Ala (15), as well as the helix-breaking residue, Gly (16). The mutations had relatively small (Ͻ1 Å) effects on the crystal structure of the protein, although they significantly changed its thermodynamic stability (14).…”

Section: Resultsmentioning

confidence: 99%

“…By combining two Fe(II͞III) ions within a single site, it is possible to bias the reaction toward two-electron chemistry, thereby avoiding the formation of oxygen radicals. The starting point for the present design is the dueferri (DF) family of de novo-designed diiron proteins (11-13), whose structures have been extensively characterized by NMR and x-ray crystallography (11,(14)(15)(16). Here, we focus on DF tet , a four-chain heterotetrameric helical bundle whose structure was originally designed beginning with a mathematical equation describing the positions of the backbone atoms (12).…”

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confidence: 99%

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De novo design of catalytic proteins

Kaplan

DeGrado

2004

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

308

290

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The de novo design of catalytic proteins provides a stringent test of our understanding of enzyme function, while simultaneously laying the groundwork for the design of novel catalysts. Here we describe the design of an O2-dependent phenol oxidase whose structure, sequence, and activity are designed from first principles. The protein catalyzes the two-electron oxidation of 4-aminophenol (kcat͞KM ‫؍‬ 1,500 M ؊1 ⅐min ؊1 ) to the corresponding quinone monoimine by using a diiron cofactor. The catalytic efficiency is sensitive to changes of the size of a methyl group in the protein, illustrating the specificity of the design.T he de novo design of proteins provides a stringent test of our understanding of their molecular mechanisms of action (1-3). Recently, it has become possible to design proteins with novel three-dimensional structures (4), which has laid the groundwork for the elaboration of function. Catalysis provides a particularly challenging function to achieve, because a successfully designed protein catalyst must bind and precisely orient substrates, transition states, and intermediates adjacent to catalytic groups such as metal ions, general acids, and͞or general bases. Two approaches to the design of catalytic proteins include automated sequence design, in which a novel catalytic site is engineered into a natural protein by mutating a subset of its side chains (5-7), and de novo protein design, which requires the simultaneous design of the entire backbone structure and sequence (2). The first method has the advantage of separating the problem of protein design and folding from the more restricted problem of designing an active site. The second approach has the potential advantage of greater applicability in terms of the sizes and shapes of substrates that can be accommodated. Furthermore, de novo protein design critically tests our understanding of how an amino acid sequence dictates both the folding as well as the activity of a protein.To date, most work on the design of catalytic proteins has focused on hydrolysis of activated 4-nitrophenyl esters, using an active site histidine side chain as a nucleophilic catalyst. Automated sequence design methods have been used to design a variant of thioredoxin that hydrolyses 4-ntirophenyl acetate with a rate enhancement of Ϸ25-fold (7), when the value of k cat ͞K M was compared to the second order rate constant for hydrolysis of 4-methyl imidazole. Proteins with similar or greater catalytic efficiencies were observed frequently in a library of four-helix bundle proteins, whose polar exterior residues were randomly selected from Lys, His, Glu, Gln, Asp, and Asn (8). Baltzer and Nilsson (1) have designed a series of helical bundles that employ His residues to promote hydrolysis and intrapeptide acyl transfer reactions. It has also been possible to design helical bundles that catalyze decarboxylation of oxaloacetate (9), in one case by recognizing a key aldamine intermediate in the reaction (10).Automated sequence design has also been used to design catalytic meta...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

De novo design of catalytic proteins

Kaplan

DeGrado

2004

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

308

290

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“…The metal-binding site was partially preorganized in the absence of metal ions, as assessed from a comparison of the geometries of the liganding residues in the di-Zn(II) versus the apo structures. Glu-10 adopts the g ϩ , g ϩ conformation (40) in almost all crystal structures of DF1 and its mutants ( 1 ϭ Ϫ65 Ϯ 5°, 2 ϭ Ϫ78 Ϯ 12°) (8)(9)(10). A g ϩ , g Ϫ conformation was found only in one monomer of the asymmetric unit in di-Zn-DF1 (8).…”

Section: Discussionmentioning

confidence: 97%

“…Here we trace the energetic consequences of introducing an active-site access channel into a designed diiron protein, DF1 (Dueferri 1) (7)(8)(9)(10). This protein is a small, 48-residue homodimeric model for the family of O 2 -using diiron proteins that includes methane monooxygenase and the radical-forming R2 subunit of the ribonucleotide reductase from Escherichia coli (11)(12)(13)(14).…”

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Preorganization of molecular binding sites in designed diiron proteins

Maglio

Nastri²,

Pavone³

et al. 2003

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

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De novo protein design provides an attractive approach to critically test the features that are required for metalloprotein structure and function. Previously we designed and crystallographically characterized an idealized dimeric model for the four-helix bundle class of diiron and dimanganese proteins [Dueferri 1 (DF1)]. Although the protein bound metal ions in the expected manner, access to its active site was blocked by large bulky hydrophobic residues. Subsequently, a substrate-access channel was introduced proximal to the metal-binding center, resulting in a protein with properties more closely resembling those of natural enzymes. Here we delineate the energetic and structural consequences associated with the introduction of these binding sites. To determine the extent to which the binding site was preorganized in the absence of metal ions, the apo structure of DF1 in solution was solved by NMR and compared with the crystal structure of the di-Zn(II) derivative. The overall fold of the apo protein was highly similar to that of the di-Zn(II) derivative, although there was a rotation of one of the helices. We also examined the thermodynamic consequences associated with building a small molecule-binding site within the protein. The protein exists in an equilibrium between folded dimers and unfolded monomers. DF1 is a highly stable protein (Kdiss ‫؍‬ 0.001 fM), but the dissociation constant increases to 0.6 nM (⌬⌬G ‫؍‬ 5.4 kcal͞ mol monomer) as the active-site cavity is increased to accommodate small molecules.T he requirements for protein stability versus function are often diametrically opposed. The folded conformations of proteins are stabilized by maximizing the burial of hydrophobic groups, minimizing voids, and forming intramolecular hydrogen bonds (1, 2). In contrast, binding and enzymatic functions generally require active-site clefts replete with solventexposed hydrophobic groups and hydrogen-bonding groups, which are essential for proper binding of substrates. This tradeoff between conformational stability and function presents a particularly large challenge to protein design (3), forcing the designer to skirt the waters between Scylla and Charybdis.The stability͞function tradeoff is particularly apparent in metalloproteins. Structural metal-binding sites in proteins frequently achieve stability by binding metal ions in coordinately saturated ligand environments with idealized ligand-metal bond geometries (4, 5). However, functional sites in metalloenzymes frequently contain coordinately unsaturated metal ions that are positioned appropriately for binding substrates; they also sometimes display geometries not frequently observed in simple, small-molecule metal-ligand complexes (4, 5). Further, the active sites of metalloproteins are frequently preorganized in the absence of metal ions, which requires the burial of polar ligands at the expense of folding free energy (6). The preorganization imparts tight and geometrically specific metal binding by assuring that the protein imparts its own structural pr...

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Metalloprotein Design & Engineering

2005

Encyclopedia of Inorganic Chemistry

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This review covers recent advances in metalloprotein design, with focus on different approaches to the design. Impressive progress has been made in designing metal‐binding sites in peptides, de novo designed proteins, and native protein scaffolds. The approach can be rational or combinatorial. Under rational design, redesigning an existing metal‐binding site to a new site with dramatically different structure and function complements well the design of new metal‐binding sites by revealing the role of specific residues responsible for a particular structural or functional feature of the metal‐binding site of interest. To create a new metal‐binding site, several approaches have been used, including design based on structural homology, by inspection, using automated computer search algorithms, or combination of the above approaches. In addition, modular approach by transplanting a conserved structural unit from one protein into another has also been shown to be effective. Design through combinatorial and evolution methods has also been successful as it requires little prior knowledge of the protein structure. Finally, introducing unnatural amino acids or nonnative metal ions/prosthetic groups to expand the repertoires of metalloproteins have been demonstrated. Successful examples of each of the approaches are given; advantages and disadvantages of the approaches are discussed; the outlook for future research is also presented.

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Toward the de Novo Design of a Catalytically Active Helix Bundle: A Substrate-Accessible Carboxylate-Bridged Dinuclear Metal Center

Cited by 101 publications

References 64 publications

De novo design of catalytic proteins

De novo design of catalytic proteins

Preorganization of molecular binding sites in designed diiron proteins

Metalloprotein Design & Engineering

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