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

Abstract

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...

Identifying important structural characteristics of arsenic resistance proteins by using designed three-stranded coiled coils

“…Additionally, a 1.9-Å structure of a di-Mn(II)-DF2 derivative contains four crystallographically independent bundles that unveil subtle differences in ligand coordination to the dinuclear centers (29). The analysis of these structures and those of other due-ferri-family derivatives provides insight into how changes in the hydrophobic core affect ligand coordination to these dinuclear sites and has allowed for the design of catalytic functionality into this type of simple designed framework (27,31).…”

“…The class of designed metallopeptides that has been structurally characterized most thor-oughly is four-helix bundles comprised of dimers of helix-turnhelix peptides. A 2.8-Å resolution structure of a heme-binding four-helix bundle maquette has been reported, but by far the most detailed structural information has been obtained for the due-ferri (DF) family of peptides designed by DeGrado et al, which contain carboxylate-bridged dinuclear metal centers in the center of a four-helix bundle (26,27). NMR structures of both the apo and the di-Zn(II) forms of DF1 reveal slight changes in the protein fold upon metal binding (28).…”

De novo design of catalytic proteins

“…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).…”

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

“…More complex coordination sites were engineered with the aid of computational searching, for example, the Cys 2 -His 2 zinc binding sites and Fe 4 -S 4 sites (Lu et al, 2001). Retrostructural analysis for designing diiron binding protein is the most complex approach by far (Lombardi et al, 2000). The natural diiron binding site was analyzed first to obtain the desired chelation geometry, the symmetry of the structure, and other rules for forming the binding site.…”