Pecoraro, V.L.

Angew. Chem. Int. Ed. 2014, 53, 7900-7903, 10.1002/anie.201404925

Protein design will ultimately allow for the creation of artificial enzymes with novel functions and unprecedented stability. To test our current mastery of nature’s approach to catalysis, a ZnII metalloenzyme was prepared using de novo design. α3DH3 folds into a stable single‐stranded three‐helix bundle and binds ZnII with high affinity using His3O coordination. The resulting metalloenzyme catalyzes the hydration of CO2 better than any small molecule model of carbonic anhydrase and with an efficiency within 1400‐fold of the fastest carbonic anhydrase isoform, CAII, and 11‐fold of CAIII.

Pecoraro, V.L.

Curr. Opin. Chem. Biol. 2015, 25, 65-70, 10.1016/j.cbpa.2014.12.034

Three-helix bundles and coiled-coil motifs are well-established de novo designed scaffolds that have been investigated for their metal-binding and catalytic properties. Satisfaction of the primary coordination sphere for a given metal is sufficient to introduce catalytic activity and a given structure may catalyze different reactions dependent on the identity of the incorporated metal. Here we describe recent contributions in the de novo design of metalloenzymes based on three-helix bundles and coiled-coil motifs, focusing on non-heme systems for hydrolytic and redox chemistry.

Pecoraro, V.L.

Angew. Chem. Int. Ed. 2020, 59, 7678-7699, 10.1002/anie.201907502

The relationship between protein structure and function is one of the greatest puzzles within biochemistry. De novo metalloprotein design is a way to wipe the board clean and determine what is required to build in function from the ground up in an unrelated structure. This Review focuses on protein design efforts to create de novo metalloproteins within alpha-helical scaffolds. Examples of successful designs include those with carbonic anhydrase or nitrite reductase activity by incorporating a ZnHis3 or CuHis3 site, or that recapitulate the spectroscopic properties of unique electron-transfer sites in cupredoxins (CuHis2Cys) or rubredoxins (FeCys4). This work showcases the versatility of alpha helices as scaffolds for metalloprotein design and the progress that is possible through careful rational design. Our studies cover the invariance of carbonic anhydrase activity with different site positions and scaffolds, refinement of our cupredoxin models, and enhancement of nitrite reductase activity up to 1000-fold.

Pecoraro, V.L.

Acc. Chem. Res. 2015, 48, 2388-2396, 10.1021/acs.accounts.5b00175

The major advances in molecular and structural biology and automated peptide and DNA synthesis of the 1970s and 1980s generated fertile conditions in the 1990s for the exploration of designed proteins as a new approach for inorganic chemists to generate biomolecular mimics of metalloproteins. This Account follows the development of the TRI peptide family of three-stranded coiled coils (3SCC) and α3D family of three-helix bundles (3HB) as scaffolds for the preparation of metal binding sites within de novo designed constructs. The 3SCC were developed using the concept of a heptad repeat (abcdefg) putting hydrophobes in the a and d positions. The TRI peptides contain four heptads with capping glycines. Via substitution of leucine hydrophobes, metal ligands can be introduced into the a or d sites in order to bind metals. First, the ability to use cysteine-substituted 3SCC aggregates to impose higher or lower coordination numbers on Hg(II) and Cd(II) or matching the coordination preferences of As(III) and Pb(II) is discussed. Then, methods to develop dual site peptides capable of discriminating metals based on their type (e.g., Cd(II) vs Pb(II)), their preference for a vs d sites, and then their coordination number is described. Once these principles of metal site differentiation are described, we shift to building dual site peptides using both cysteine and histidine metal binding sites. This approach provides a construct with both a Hg(II) structural and a Zn(II) hydrolytic center, the latter of which is capable of hydrating CO2. With these Zn(II) proteins, we consider the relative importance of the location of the catalytic center along the primary sequence of the peptide and show that only minor perturbations in catalytic efficiencies are observed based on metal location. We then assess the feasibility of preparing enzymes competent to reduce nitrite with copper centers in a histidine-rich environment. As part of this discussion, we examine the influence of surface residues on catalyst reduction potentials and catalytic efficiencies. We end describing approaches to prepare asymmetric proteins that can incorporate acid–base catalysts or water channels. In this respect, we highlight modifications of a helix–turn–helix–turn–helix motif called α3D and show how this 3HB can be modified to bind heavy metals or to make Zn(II) centers, which are active hydrolytic catalysts. A comparison is made to the comparable parallel 3SCC.

Pecoraro, V.L.

Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 21234-21239, 10.1073/pnas.1212893110

One of the ultimate objectives of de novo protein design is to realize systems capable of catalyzing redox reactions on substrates. This goal is challenging as redox-active proteins require design considerations for both the reduced and oxidized states of the protein. In this paper, we describe the spectroscopic characterization and catalytic activity of a de novo designed metallopeptide Cu(I/II)(TRIL23H)3+/2+, where Cu(I/II) is embeded in α-helical coiled coils, as a model for the CuT2 center of copper nitrite reductase. In Cu(I/II)(TRIL23H)3+/2+, Cu(I) is coordinated to three histidines, as indicated by X-ray absorption data, and Cu(II) to three histidines and one or two water molecules. Both ions are bound in the interior of the three-stranded coiled coils with affinities that range from nano- to micromolar [Cu(II)], and picomolar [Cu(I)]. The Cu(His)3 active site is characterized in both oxidation states, revealing similarities to the CuT2 site in the natural enzyme. The species Cu(II)(TRIL23H)32+ in aqueous solution can be reduced to Cu(I)(TRIL23H)3+ using ascorbate, and reoxidized by nitrite with production of nitric oxide. At pH 5.8, with an excess of both the reductant (ascorbate) and the substrate (nitrite), the copper peptide Cu(II)(TRIL23H)32+ acts as a catalyst for the reduction of nitrite with at least five turnovers and no loss of catalytic efficiency after 3.7 h. The catalytic activity, which is first order in the concentration of the peptide, also shows a pH dependence that is described and discussed.

Pecoraro, V.L.

Coord. Chem. Rev. 2013, 257, 2565-2588, 10.1016/j.ccr.2013.02.007

Metalloenzymes efficiently catalyze some of the most important and difficult reactions in nature. For many years, coordination chemists have effectively used small molecule models to understand these systems. More recently, protein design has been shown to be an effective approach for mimicking metal coordination environments. Since the first designed proteins were reported, much success has been seen for incorporating metal sites into proteins and attaining the desired coordination environment but until recently, this has been with a lack of significant catalytic activity. Now there are examples of designed metalloproteins that, although not yet reaching the activity of native enzymes, are considerably closer. In this review, we highlight work leading up to the design of a small metalloprotein containing two metal sites, one for structural stability (HgS3) and the other a separate catalytic zinc site to mimic carbonic anhydrase activity (ZnN3O). The first section will describe previous studies that allowed for a high affinity thiolate site that binds heavy metals in a way that stabilizes three-stranded coiled coils. The second section will examine ways of preparing histidine-rich environments that lead to metal-based hydrolytic catalysts. We will also discuss other recent examples of the design of structural metal sites and functional metalloenzymes. Our work demonstrates that attaining the proper first coordination geometry of a metal site can lead to a significant fraction of catalytic activity, apparently independent of the type of secondary structure of the surrounding protein environment. We are now in a position to begin to meet the challenge of building a metalloenzyme systematically from the bottom-up by engineering and analyzing interactions directly around the metal site and beyond.

Pecoraro, V.L.

Biochemistry 2014, 53, 957-978, 10.1021/bi4016617

Zinc is an essential element required for the function of more than 300 enzymes spanning all classes. Despite years of dedicated study, questions regarding the connections between primary and secondary metal ligands and protein structure and function remain unanswered, despite numerous mechanistic, structural, biochemical, and synthetic model studies. Protein design is a powerful strategy for reproducing native metal sites that may be applied to answering some of these questions and subsequently generating novel zinc enzymes. From examination of the earliest design studies introducing simple Zn(II)-binding sites into de novo and natural protein scaffolds to current studies involving the preparation of efficient hydrolytic zinc sites, it is increasingly likely that protein design will achieve reaction rates previously thought possible only for native enzymes. This Current Topic will review the design and redesign of Zn(II)-binding sites in de novo-designed proteins and native protein scaffolds toward the preparation of catalytic hydrolytic sites. After discussing the preparation of Zn(II)-binding sites in various scaffolds, we will describe relevant examples for reengineering existing zinc sites to generate new or altered catalytic activities. Then, we will describe our work on the preparation of a de novo-designed hydrolytic zinc site in detail and present comparisons to related designed zinc sites. Collectively, these studies demonstrate the significant progress being made toward building zinc metalloenzymes from the bottom up.

Pecoraro, V.L.

ACS Catal. 2018, 8, 8046-8057, 10.1021/acscatal.8b02153

The development of redox-active metalloprotein catalysts is a challenging objective of de novo protein design. Within this Perspective we detail our efforts to create a redox-active Cu nitrite reductase (NiR) by incorporating Cu into the hydrophobic interior of well-defined three-stranded coiled coils (3SCCs). The scaffold contains three histidine residues that provide a layer of three nitrogen donors that mimic the type 2 catalytic site of NiR. We have found that this strategy successfully produces an active and stable CuNiR model that functions for over 1000 turnovers. Spectroscopic evidence indicates that the Cu(I) site has a lower coordination number in comparison to the enzyme, whereas the Cu(II) geometry may more faithfully reproduce the NiR type 2 center. Mutations at the helical interface successfully produce a hydrogen bond between an interfacial Glu residue and the Cu-ligating His residue, which allows for the tuning of the redox potential over a 100 mV range. We successfully created constructs with as much as a 120-fold improvement from the original design by modifying the steric bulk above or below the Cu binding site. These systems are now the most active water-soluble and stable artificial NiR catalysts yet produced. Several avenues for improving the catalytic efficiency of later designs are detailed within this Perspective, including adjustment of their resting oxidation state, the use of asymmetric scaffolds to allow for single amino acid mutation within the second coordination sphere, and the design of hydrogen-bonding networks to tune residue orientation and electronics. Through these studies the TRI-H system has given insight into the difficulties that arise in creating a de novo redox active enzyme. Work to improve upon this model will provide strategies by which redox-active de novo enzymes may be tuned and detail how native enzymes accomplish catalytic efficiencies through proton gated redox catalysis.

Pecoraro, V.L.

Nat. Chem. 2020, 12, 405-411, 10.1038/s41557-020-0423-6

Three-stranded coiled coils are peptide structures constructed from amphipathic heptad repeats. Here we show that it is possible to form pure heterotrimeric three-stranded coiled coils by combining three distinct characteristics: (1) a cysteine sulfur layer for metal coordination, (2) a thiophilic, trigonal pyramidal metalloid (Pb(ii)) that binds to these sulfurs and (3) an adjacent layer of reduced steric bulk generating a cavity where water can hydrogen bond to the cysteine sulfur atoms. Cysteine substitution in an a site yields Pb(ii)A2B heterotrimers, while d sites provide pure Pb(ii)C2D or Pb(ii)CD2 scaffolds. Altering the metal from Pb(ii) to Hg(ii) or shifting the relative position of the sterically less demanding layer removes heterotrimer specificity. Because only two of the eight or ten hydrophobic layers are perturbed, catalytic sites can be introduced at other regions of the scaffold. A Zn(ii)(histidine)3(H2O) centre can be incorporated at a remote location without perturbing the heterotrimer selectivity, suggesting a unique strategy to prepare dissymmetric catalytic sites within self-assembling de novo-designed proteins.

Pecoraro, V.L.

Nat. Chem. 2012, 4, 118-123, 10.1038/NCHEM.1201

Metal ions are an important part of many natural proteins, providing structural, catalytic and electron transfer functions. Reproducing these functions in a designed protein is the ultimate challenge to our understanding of them. Here, we present an artificial metallohydrolase, which has been shown by X-ray crystallography to contain two different metal ions—a Zn(II) ion, which is important for catalytic activity, and a Hg(II) ion, which provides structural stability. This metallohydrolase displays catalytic activity that compares well with several characteristic reactions of natural enzymes. It catalyses p-nitrophenyl acetate (pNPA) hydrolysis with an efficiency only ~100-fold less than that of human carbonic anhydrase (CA)II and at least 550-fold better than comparable synthetic complexes. Similarly, CO2 hydration occurs with an efficiency within ~500-fold of CAII. Although histidine residues in the absence of Zn(II) exhibit pNPA hydrolysis, miniscule apopeptide activity is observed for CO2 hydration. The kinetic and structural analysis of this first de novo designed hydrolytic metalloenzyme reveals necessary design features for future metalloenzymes containing one or more metals.

Pecoraro, V.L.

J. Am. Chem. Soc. 2013, 135, 5895-5903, 10.1021/ja401537t

While metalloprotein design has now yielded a number of successful metal-bound and even catalytically active constructs, the question of where to put a metal site along a linear, repetitive sequence has not been thoroughly addressed. Often several possibilities in a given sequence may exist that would appear equivalent but may in fact differ for metal affinity, substrate access, or protein dynamics. We present a systematic variation of active site location for a hydrolytically active ZnHis3O site contained within a de novo-designed three-stranded coiled coil. We find that the maximal rate, substrate access, and metal-binding affinity are dependent on the selected position, while catalytic efficiency for p-nitrophenyl acetate hydrolysis can be retained regardless of the location of the active site. This achievement demonstrates how efficient, tailor-made enzymes which control rate, pKa, substrate and solvent access (and selectivity), and metal-binding affinity may be realized. These findings may be applied to the more advanced de novo design of constructs containing secondary interactions, such as hydrogen-bonding channels. We are now confident that changes to location for accommodating such channels can be achieved without location-dependent loss of catalytic efficiency. These findings bring us closer to our ultimate goal of incorporating the secondary interactions we believe will be necessary in order to improve both active site properties and the catalytic efficiency to be competitive with the native enzyme, carbonic anhydrase.

Pecoraro, V.L.

Chem. Rev. 2014, 114, 3495-3578, 10.1021/cr400458x

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Pecoraro, V.L.

J. Am. Chem. Soc. 2020, 142, 15282-15294, 10.1021/jacs.0c04757

Blue copper proteins have a constrained Cu(II) geometry that has proven difficult to recapitulate outside native cupredoxin folds. Previous work has successfully designed green copper proteins which could be tuned blue using exogenous ligands, but the question of how one can create a self-contained blue copper site within a de novo scaffold, especially one removed from a cupredoxin fold, remained. We have recently reported a red copper protein site within a three helical bundle scaffold which we later revisited and determined to be a nitrosocyanin mimic, with a CuHis2CysGlu binding site. We now report efforts to rationally design this construct toward either green or blue copper chromophores using mutation strategies that have proven successful in native cupredoxins. By rotating the metal binding site, we created a de novo green copper protein. This in turn was converted to a blue copper protein by removing an axial methionine. Following this rational sequence, we have successfully created red, green, and blue copper proteins within an alpha helical fold, enabling comparisons for the first time of their structure and function disconnected from the overall cupredoxin fold.