11 publications

11 publications

Allosteric Cooperation in a De Novo-Designed Two-Domain Protein

DeGrado, W.F.; Lombardi, A.

Proc. Natl. Acad. Sci. U.S.A. 2020, 117, 33246-33253, 10.1073/pnas.2017062117

We describe the de novo design of an allosterically regulated protein, which comprises two tightly coupled domains. One domain is based on the DF (Due Ferri in Italian or two-iron in English) family of de novo proteins, which have a diiron cofactor that catalyzes a phenol oxidase reaction, while the second domain is based on PS1 (Porphyrin-binding Sequence), which binds a synthetic Zn-porphyrin (ZnP). The binding of ZnP to the original PS1 protein induces changes in structure and dynamics, which we expected to influence the catalytic rate of a fused DF domain when appropriately coupled. Both DF and PS1 are four-helix bundles, but they have distinct bundle architectures. To achieve tight coupling between the domains, they were connected by four helical linkers using a computational method to discover the most designable connections capable of spanning the two architectures. The resulting protein, DFP1 (Due Ferri Porphyrin), bound the two cofactors in the expected manner. The crystal structure of fully reconstituted DFP1 was also in excellent agreement with the design, and it showed the ZnP cofactor bound over 12 Å from the dimetal center. Next, a substrate-binding cleft leading to the diiron center was introduced into DFP1. The resulting protein acts as an allosterically modulated phenol oxidase. Its Michaelis–Menten parameters were strongly affected by the binding of ZnP, resulting in a fourfold tighter Km and a 7-fold decrease in kcat. These studies establish the feasibility of designing allosterically regulated catalytic proteins, entirely from scratch.


Metal: Fe; Zn
Ligand type: Amino acid
Host protein: Due Ferri
Anchoring strategy: Amino acid
Optimization: ---
Max TON: 10
ee: ---
PDB: 7JH6
Notes: diFe-DFP3: Km 2.9 mM, kcat 0.7 min-1, 10 turnovers for 1 mM substrate, 20 uM protein. On binding ZnP, Km decreased 4x, and kcat decreased 7x, resulting in a lower kcat/Km overall.

Alteration of the Oxygen-Dependent Reactivity of De Novo Due Ferri Proteins

DeGrado, W.F.

Nat. Chem. 2012, 4, 900-906, 10.1038/NCHEM.1454

De novo proteins provide a unique opportunity to investigate the structure–function relationships of metalloproteins in a minimal, well-defined and controlled scaffold. Here, we describe the rational programming of function in a de novo designed di-iron carboxylate protein from the Due Ferri family. Originally created to catalyse the O2-dependent, two-electron oxidation of hydroquinones, the protein was reprogrammed to catalyse the selective N-hydroxylation of arylamines by remodelling the substrate access cavity and introducing a critical third His ligand to the metal-binding cavity. Additional second- and third-shell modifications were required to stabilize the His ligand in the core of the protein. These structural changes resulted in at least a 106-fold increase in the relative rate between the arylamine N-hydroxylation and hydroquinone oxidation reactions. This result highlights the potential for using de novo proteins as scaffolds for future investigations of the geometric and electronic factors that influence the catalytic tuning of di-iron active sites.


Metal: Fe
Ligand type: Amino acid
Host protein: Due Ferri
Anchoring strategy: Dative
Optimization: Genetic
Reaction: N-Hydroxylation
Max TON: ---
ee: ---
PDB: 2LFD
Notes: ---

An Artificial Di-Iron Oxo-Orotein with Phenol Oxidase Activity

DeGrado, W.F.; Lombardi, A.

Nat. Chem. Biol. 2009, 5, 882-884, 10.1038/nchembio.257

Here we report the de novo design and NMR structure of a four-helical bundle di-iron protein with phenol oxidase activity. The introduction of the cofactor-binding and phenol-binding sites required the incorporation of residues that were detrimental to the free energy of folding of the protein. Sufficient stability was, however, obtained by optimizing the sequence of a loop distant from the active site.


Metal: Fe
Ligand type: Amino acid
Host protein: Due Ferri
Anchoring strategy: Dative
Optimization: Genetic
Reaction: Alcohol oxidation
Max TON: >50
ee: ---
PDB: 2KIK
Notes: kcat/KM ≈ 1380 M-1*min-1

Metal: Fe
Ligand type: Amino acid
Host protein: Due Ferri
Anchoring strategy: Dative
Optimization: Genetic
Reaction: Amine oxidation
Max TON: ---
ee: ---
PDB: 2KIK
Notes: kcat/KM ≈ 83 M-1*min-1

Artificial Diiron Enzymes with a De Novo Designed Four-Helix Bundle Structure

Review

DeGrado, W.F.; Lombardi, A.

Eur. J. Inorg. Chem. 2015, 2015, 3371-3390, 10.1002/ejic.201500470

A single polypeptide chain may provide an astronomical number of conformers. Nature selected only a trivial number of them through evolution, composing an alphabet of scaffolds, that can afford the complete set of chemical reactions needed to support life. These structural templates are so stable that they allow several mutations without disruption of the global folding, even having the ability to bind several exogenous cofactors. With this perspective, metal cofactors play a crucial role in the regulation and catalysis of several processes. Nature is able to modulate the chemistry of metals, adopting only a few ligands and slightly different geometries. Several scaffolds and metal‐binding motifs are representing the focus of intense interest in the literature. This review discusses the widespread four‐helix bundle fold, adopted as a scaffold for metal binding sites in the context of de novo protein design to obtain basic biochemical components for biosensing or catalysis. In particular, we describe the rational refinement of structure/function in diiron–oxo protein models from the due ferri (DF) family. The DF proteins were developed by us through an iterative process of design and rigorous characterization, which has allowed a shift from structural to functional models. The examples reported herein demonstrate the importance of the synergic application of de novo design methods as well as spectroscopic and structural characterization to optimize the catalytic performance of artificial enzymes.


Notes: ---

Catalytic Efficiency of Designed Catalytic Proteins

Review

DeGrado, W.F.; Korendovych, I.V.

Curr. Opin. Struct. Biol. 2014, 27, 113-121, 10.1016/j.sbi.2014.06.006

The de novo design of catalysts that mimic the affinity and specificity of natural enzymes remains one of the Holy Grails of chemistry. Despite decades of concerted effort we are still unable to design catalysts as efficient as enzymes. Here we critically evaluate approaches to (re)design of novel catalytic function in proteins using two test cases: Kemp elimination and ester hydrolysis. We show that the degree of success thus far has been modest when the rate enhancements seen for the designed proteins are compared with the rate enhancements by small molecule catalysts in solvents with properties similar to the active site. Nevertheless, there are reasons for optimism: the design methods are ever improving and the resulting catalyst can be efficiently improved using directed evolution.


Notes: ---

De Novo Design of Catalytic Proteins

DeGrado, W.F.

Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 11566-11570, 10.1073/pnas.0404387101

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 (k cat/K M = 1,500 M·1·min·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.


Metal: Fe
Ligand type: Amino acid
Host protein: Due Ferri
Anchoring strategy: Dative
Optimization: Genetic
Reaction: Alcohol oxidation
Max TON: >100
ee: ---
PDB: ---
Notes: kcat/KM ≈ 1540 M-1*min-1

De Novo Design of Four-Helix Bundle Metalloproteins: One Scaffold, Diverse Reactivities

DeGrado, W.F.

Acc. Chem. Res. 2019, 10.1021/acs.accounts.8b00674

De novo protein design represents anattractive approach for testing and extending our under-standing of metalloprotein structure and function. Here, we describe our work on the design of DF (Due Ferri or two-ironin Italian), a minimalist model for the active sites of muchlarger and more complex natural diiron and dimanganeseproteins. In nature, diiron and dimanganese proteins protypi-cally bind their ions in 4-Glu, 2-His environments, and theycatalyze diverse reactions, ranging from hydrolysis, to O2-dependent chemistry, to decarbonylation of aldehydes. In the design of DF, the position of each atom including the backbone, the first-shell ligands, the second-shell hydrogen-bonded groups, and the well-packed hydrophobic core was bespoke using precise mathematical equations and chemical principles. The first member of the DF family was designed to be of minimal size and complexity and yet to display the quintessential elements required for binding the dimetal cofactor. After thoroughly characterizing its structural, dynamic, spectroscopic, and functional properties, we added additional complexity in a rational stepwise manner to achieve increasingly sophisticated catalytic functions, ultimately demonstrating substrate-gated four-electron reduction of O2to water. We also briefly describe the extension of these studies to the design of proteins that bind non biological metal cofactors (a synthetic porphyrin and a tetranuclear cluster), and a Zn2+/proton antiporting membrane protein. Together these studies demonstrate a successful and generally applicable strategy for de novo metalloprotein design, which might indeed mimic the process by which primordial metalloproteins evolved. We began the design process with a highly symmetrical backbone and binding site, by using point-group symmetry to assemble the secondary structures that position the amino acid side chains required for binding. The resulting models provided a rough starting point and initial parameters for the subsequent precise design of thefinal protein using modern methods of computational protein design. Unless the desired site is itself symmetrical, this process requires reduction of the symmetry or lifting it altogether. Nevertheless, the initial symmetrical structure can be helpful to restrain the search space during assembly of the backbone. Finally, the methods described here should be generally applicable to the design of highly stable and robust catalysts and sensors. There is considerable potential in combining the efficiency and knowledge base associated with homogeneous metal catalysis with the programmability, biocompatibility, and versatility of proteins. While the work reported here focuses on testing and learning the principles of natural metalloproteins by designing and studying proteins one at a time, there is also considerable potential for using designed proteins that incorporate both biological and non biological metal ion cofactors for the evolution of novel catalysts.


Metal: Fe
Ligand type: Amino acid
Host protein: Due Ferri
Anchoring strategy: Dative
Optimization: Computational design
Reaction: Oxidation
Max TON: ---
ee: ---
PDB: 1EC5
Notes: Additional PDB: 1LT1

De Novo Design, Solution Characterization, and Crystallographic Structure of an Abiological Mn–Porphyrin-Binding Protein Capable of Stabilizing a Mn(V) Species

DeGrado, W.F.

J. Am. Chem. Soc. 2021, 143, 252-259, 10.1021/jacs.0c10136

De novo protein design offers the opportunity to test our understanding of how metalloproteins perform difficult transformations. Attaining high-resolution structural information is critical to understanding how such designs function. There have been many successes in the design of porphyrin-binding proteins; however, crystallographic characterization has been elusive, limiting what can be learned from such studies as well as the extension to new functions. Moreover, formation of highly oxidizing high-valent intermediates poses design challenges that have not been previously implemented: (1) purposeful design of substrate/oxidant access to the binding site and (2) limiting deleterious oxidation of the protein scaffold. Here we report the first crystallographically characterized porphyrin-binding protein that was programmed to not only bind a synthetic Mn–porphyrin but also maintain binding site access to form high-valent oxidation states. We explicitly designed a binding site with accessibility to dioxygen units in the open coordination site of the Mn center. In solution, the protein is capable of accessing a high-valent Mn(V)–oxo species which can transfer an O atom to a thioether substrate. The crystallographic structure is within 0.6 Å of the design and indeed contained an aquo ligand with a second water molecule stabilized by hydrogen bonding to a Gln side chain in the active site, offering a structural explanation for the observed reactivity.


Metal: Mn
Ligand type: Porphyrin
Anchoring strategy: Covalent
Optimization: ---
Reaction: ---
Max TON: ---
ee: ---
PDB: 7JRQ
Notes: ---

De Novo Metalloprotein Design

Review

DeGrado, W.F.

Nat. Rev. Chem. 2022, 6, 31-50, 10.1038/s41570-021-00339-5

Natural metalloproteins perform many functions — ranging from sensing to electron transfer and catalysis — in which the position and property of each ligand and metal are dictated by protein structure. De novo protein design aims to define an amino acid sequence that encodes a specific structure and function, providing a critical test of the hypothetical inner workings of (metallo)proteins. To date, de novo metalloproteins have used simple, symmetric tertiary structures — uncomplicated by the large size and evolutionary marks of natural proteins — to interrogate structure–function hypotheses. In this Review, we discuss de novo design applications, such as proteins that induce complex, increasingly asymmetric ligand geometries to achieve function, as well as the use of more canonical ligand geometries to achieve stability. De novo design has been used to explore how proteins fine-tune redox potentials and catalyse both oxidative and hydrolytic reactions. With an increased understanding of structure–function relationships, functional proteins including O2-dependent oxidases, fast hydrolases and multi-proton/multielectron reductases have been created. In addition, proteins can now be designed using xenobiological metals or cofactors and principles from inorganic chemistry to derive new-to-nature functions. These results and the advances in computational protein design suggest a bright future for the de novo design of diverse, functional metalloproteins.


Notes: ---

Design of a Switchable Eliminase

DeGrado, W.F.

Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 6823-6827, 10.1073/pnas.1018191108

The active sites of enzymes are lined with side chains whose dynamic, geometric, and chemical properties have been finely tuned relative to the corresponding residues in water. For example, the carboxylates of glutamate and aspartate are weakly basic in water but become strongly basic when dehydrated in enzymatic sites. The dehydration of the carboxylate, although intrinsically thermodynamically unfavorable, is achieved by harnessing the free energy of folding and substrate binding to reach the required basicity. Allosterically regulated enzymes additionally rely on the free energy of ligand binding to stabilize the protein in a catalytically competent state. We demonstrate the interplay of protein folding energetics and functional group tuning to convert calmodulin (CaM), a regulatory binding protein, into AlleyCat, an allosterically controlled eliminase. Upon binding Ca(II), native CaM opens a hydrophobic pocket on each of its domains. We computationally identified a mutant that (i) accommodates carboxylate as a general base within these pockets, (ii) interacts productively in the Michaelis complex with the substrate, and (iii) stabilizes the transition state for the reaction. Remarkably, a single mutation of an apolar residue at the bottom of an otherwise hydrophobic cavity confers catalytic activity on calmodulin. AlleyCat showed the expected pH-rate profile, and it was inactivated by mutation of its active site Glu to Gln. A variety of control mutants demonstrated the specificity of the design. The activity of this minimal 75-residue allosterically regulated catalyst is similar to that obtained using more elaborate computational approaches to redesign complex enzymes to catalyze the Kemp elimination reaction.


Metal: Ca
Ligand type: Amino acid
Anchoring strategy: Dative
Optimization: Genetic
Reaction: Kemp elimination
Max TON: >40
ee: ---
PDB: 2KZ2
Notes: Ca acts as allosteric regulator, catalytically active site contains no metal

Diiron-Containing Metalloproteins: Developing Functional Models

Review

DeGrado, W.F.; Lombardi, A.

C. R. Chim. 2007, 10, 703-720, 10.1016/j.crci.2007.03.010

A major objective in protein science is the design of enzymes with novel catalytic activities that are tailored to specific applications. Such enzymes may have great potential in biocatalysis and biosensor technology, such as in degradation of pollutants and biomass, and in drug and food processing. To reach this objective, investigations into the basic biochemical functioning of metalloproteins are still required. In this perspective, metalloprotein design provides a powerful approach first to contribute to a more comprehensive understanding of the way metalloproteins function in biology, with the ultimate goal of developing novel biocatalysts and sensing devices. Metalloprotein mimetics have been developed through the introduction of novel metal-binding sites into naturally occurring proteins as well as through de novo protein design. We have approached the challenge of reproducing metalloprotein active sites by using a miniaturization process. We centered our attention on iron-containing proteins, and we developed models for heme proteins and diiron–oxo proteins. In this paper we summarize the results we obtained on the design, structural, and functional properties of DFs, a family of artificial diiron proteins.


Notes: ---