F. Akif Tezcan

Structural basis of biological nitrogen fixation

Biological nitrogen fixation is mediated by the nitrogenase enzyme system that catalyses the ATP dependent reduction of atmospheric dinitrogen to ammonia. Nitrogenase consists of two component metalloproteins, the MoFe-protein with the FeMo-cofactor that provides the active site for substrate reduction, and the Fe-protein that couples ATP hydrolysis to electron transfer. An overview of the nitrogenase system is presented that emphasizes the structural organization of the proteins and associated metalloclusters that have the remarkable ability to catalyse nitrogen fixation under ambient conditions. Although the mechanism of ammonia formation by nitrogenase remains enigmatic, mechanistic inferences motivated by recent developments in the areas of nitrogenase biochemistry, spectroscopy, model chemistry and computational studies are discussed within this structural framework.

Metal-directed, chemically-tunable assembly of one-, two- and three-dimensional crystalline protein arrays

Proteins represent the most sophisticated building blocks available to an organism or the laboratory chemist. Yet, in contrast to nearly all other types of molecular building blocks, the designed self-assembly of proteins has been largely inaccessible owing to the chemical and structural heterogeneity of protein surfaces. To circumvent the challenge of programming extensive non-covalent interactions for controlling protein self-assembly, we had previously exploited the directionality and strength of metal coordination interactions to guide the formation of closed, homoligomeric protein assemblies. Here, we extend this strategy to the generation of periodic protein arrays. We show that a monomeric protein with properly oriented coordination motifs on its surface can arrange upon metal binding into one-dimensional nanotubes, and two-or three-dimensional crystalline arrays whose dimensions collectively span nearly the entire nano- and micrometer length scale. The assembly of these arrays is predictably tuned by external stimuli, such as metal concentration and pH.

Metal-Directed Protein Self-Assembly

Proteins are natures premier building blocks for constructing sophisticated nanoscale architectures that carry out complex tasks and chemical transformations. Some 70%-80% of all proteins are thought to be permanently oligomeric; that is, they are composed of multiple proteins that are held together in precise spatial organization through noncovalent interactions. Although it is of great fundamental interest to understand the physicochemical basis of protein self-assembly, the mastery of protein-protein interactions (PPIs) would also allow access to novel biomaterials with natures favorite and most versatile building block. In this Account, we describe a new approach we have developed with this possibility in mind, metal-directed protein self-assembly (MDPSA), which utilizes the strength, directionality, and selectivity of metal-ligand interactions to control PPIs. At its core, MDPSA is inspired by supramolecular coordination chemistry, which exploits metal coordination for the self-assembly of small molecules into discrete, more-or-less predictable higher order structures. Proteins, however, are not exactly small molecules or simple metal ligands: they feature extensive, heterogeneous surfaces that can interact with each other and with metal ions in unpredictable ways. We begin by first describing the challenges of using entire proteins as molecular building blocks. We follow with an examination of our work on a model protein (cytochrome cb(562)), highlighting challenges toward establishing ground rules for MDPSA as well as progress in overcoming these challenges. Proteins are also natures metal ligands of choice. In MDPSA, once metal ions guide proteins into forming large assemblies, they are by definition embedded within extensive interfaces formed between protein surfaces. These complex surfaces make an inorganic chemists life somewhat difficult, yet they also provide a wide platform to modulate the metal coordination environment through distant, noncovalent interactions, exactly as natural metalloproteins and enzymes do. We describe our computational and experimental efforts toward restructuring the noncovalent interaction network formed between proteins surrounding the interfacial metal centers. This approach, of metal templating followed by the redesign of protein interfaces (metal-templated interface redesign, MeTIR), not only provides a route to engineer de novo PPIs and novel metal coordination environments but also suggests possible parallels with the evolution of metalloproteins.

A designed supramolecular protein assembly with in vivo enzymatic activity.

The generation of new enzymatic activities has mainly relied on repurposing the interiors of preexisting protein folds because of the challenge in designing functional, three-dimensional protein structures from first principles. Here we report an artificial metallo-β-lactamase, constructed via the self-assembly of a structurally and functionally unrelated, monomeric redox protein into a tetrameric assembly that possesses catalytic zinc sites in its interfaces. The designed metallo-β-lactamase is functional in the Escherichia coli periplasm and enables the bacteria to survive treatment with ampicillin. In vivo screening of libraries has yielded a variant that displays a catalytic proficiency [(kcat/Km)/kuncat] for ampicillin hydrolysis of 2.3 × 106 and features the emergence of a highly mobile loop near the active site, a key component of natural β-lactamases to enable substrate interactions. A monomeric redox protein can be engineered into a tetrameric β-lactamase that confers antibiotic resistance in vivo. Designing activity at an interface Enzymes are proteins that are the workhorses of the cell. Designing enzymes with new functions that are also manifested in living systems could be extremely valuable in bioengineering and synthetic biology applications. However, enzyme design is a challenging task and so far has mainly been restricted to repurposing natural enzymes and to in vitro systems. Song and Tezcan started with a monomeric redox protein and introduced mutations that cause it to assemble into a tetramer with catalytic zinc ions in its interfaces. This protein assembly displayed β-lactamase activity, the primary mechanism of antibiotic resistance, and enabled E. coli cells to survive ampicillin treatment. Science, this issue p. 1525

Re-engineering protein interfaces yields copper-inducible ferritin cage assembly

The ability to chemically control protein-protein interactions would allow the interrogation of dynamic cellular processes and lead to a better understanding and exploitation of self-assembling protein architectures. Here we introduce a new engineering strategy--reverse metal-templated interface redesign (rMeTIR)--that transforms a natural protein-protein interface into one that only engages in selective response to a metal ion. We have applied rMeTIR to render the self-assembly of the cage-like protein ferritin controllable by divalent copper binding, which has allowed the study of the structure and stability of the isolated ferritin monomer, the demonstration of the primary role of conserved hydrogen-bonding interactions in providing geometric specificity for cage assembly and the uniform chemical modification of the cage interior under physiological conditions. Notably, copper acts as a structural template for ferritin assembly in a manner that is highly reminiscent of RNA sequences that template virus capsid formation.

Metal templated design of protein interfaces

Metal coordination is a key structural and functional component of a large fraction of proteins. Given this dual role we considered the possibility that metal coordination may have played a templating role in the early evolution of protein folds and complexes. We describe here a rational design approach, Metal Templated Interface Redesign (MeTIR), that mimics the time course of a hypothetical evolutionary pathway for the formation of stable protein assemblies through an initial metal coordination event. Using a folded monomeric protein, cytochrome cb562, as a building block we show that its non-self-associating surface can be made self-associating through a minimal number of mutations that enable Zn coordination. The protein interfaces in the resulting Zn-directed, D2-symmetrical tetramer are subsequently redesigned, yielding unique protein architectures that self-assemble in the presence or absence of metals. Aside from its evolutionary implications, MeTIR provides a route to engineer de novo protein interfaces and metal coordination environments that can be tuned through the extensive noncovalent bonding interactions in these interfaces.

Electron tunneling in protein crystals.

The current understanding of electron tunneling through proteins has come from work on systems where donors and acceptors are held at fixed distances and orientations. The factors that control electron flow between proteins are less well understood, owing to uncertainties in the relative orientations and structures of the reactants during the very short time that tunneling occurs. As we report here, the way around such structural ambiguity is to examine oxidation–reduction reactions in protein crystals. Accordingly, we have measured and analyzed the kinetics of electron transfer between native and Zn-substituted tuna cytochrome c (cyt c) molecules in crystals of known structure. Electron transfer rates [(320 s−1 for *Zn-cyt c → Fe(III)-cyt c; 2000 s−1 for Fe(II)-cyt c → Zn-cyt c+)] over a Zn–Fe distance of 24.1 Å closely match those for intraprotein electron tunneling over similar donor–acceptor separations. Our results indicate that van der Waals interactions and water-mediated hydrogen bonds are effective coupling elements for tunneling across a protein–protein interface.

Self-assembly of coherently dynamic, auxetic, two-dimensional protein crystals

Two-dimensional (2D) crystalline materials possess unique structural, mechanical and electronic properties that make them highly attractive in many applications. Although there have been advances in preparing 2D materials that consist of one or a few atomic or molecular layers, bottom-up assembly of 2D crystalline materials remains a challenge and an active area of development. More challenging is the design of dynamic 2D lattices that can undergo large-scale motions without loss of crystallinity. Dynamic behaviour in porous three-dimensional (3D) crystalline solids has been exploited for stimuli-responsive functions and adaptive behaviour. As in such 3D materials, integrating flexibility and adaptiveness into crystalline 2D lattices would greatly broaden the functional scope of 2D materials. Here we report the self-assembly of unsupported, 2D protein lattices with precise spatial arrangements and patterns using a readily accessible design strategy. Three single- or double-point mutants of the C4-symmetric protein RhuA were designed to assemble via different modes of intermolecular interactions (single-disulfide, double-disulfide and metal-coordination) into crystalline 2D arrays. Owing to the flexibility of the single-disulfide interactions, the lattices of one of the variants (C98RhuA) are essentially defect-free and undergo substantial, but fully correlated, changes in molecular arrangement, yielding coherently dynamic 2D molecular lattices. C98RhuA lattices display a Poisson’s ratio of −1—the lowest thermodynamically possible value for an isotropic material—making them auxetic.

A Metal Organic Framework with Spherical Protein Nodes: Rational Chemical Design of 3D Protein Crystals.

We describe here the construction of a three-dimensional, porous, crystalline framework formed by spherical protein nodes that assemble into a prescribed lattice arrangement through metal-organic linker-directed interactions. The octahedral iron storage enzyme, ferritin, was engineered in its C3 symmetric pores with tripodal Zn coordination sites. Dynamic light scattering and crystallographic studies established that this Zn-ferritin construct could robustly self-assemble into the desired bcc-type crystals upon coordination of a ditopic linker bearing hydroxamic acid functional groups. This system represents the first example of a ternary protein-metal-organic crystalline framework whose formation is fully dependent on each of its three components.

Metal-mediated self-assembly of protein superstructures: influence of secondary interactions on protein oligomerization and aggregation.

We have previously demonstrated that non-self-associating protein building blocks can oligomerize to form discrete supramolecular assemblies under the control of metal coordination. We show here that secondary interactions (salt bridges and hydrogen bonds) can be critical in guiding the metal-induced self-assembly of proteins. Crystallographic and hydrodynamic measurements on appropriately engineered cytochrome cb562 variants pinpoint the importance of a single salt-bridging arginine side chain in determining whether the protein monomers form a discrete Zn-induced tetrameric complex or heterogeneous aggregates. The combined ability to direct PPIs through metal coordination and secondary interactions should provide the specificity required for the construction of complex protein superstructures and the selective control of cellular processes that involve protein-protein association reactions.