Overcoming universal restrictions on metal selectivity by protein design.

Selective metal coordination is central to the functions of metalloproteins:1,2 each metalloprotein must pair with its cognate metallocofactor to fulfil its biological role3. However, achieving metal selectivity solely through a three-dimensional protein structure is a great challenge, because there is a limited set of metal-coordinating amino acid functionalities and proteins are inherently flexible, which impedes steric selection of metals3,4. Metal-binding affinities of natural proteins are primarily dictated by the electronic properties of metal ions and follow the Irving-Williams series5 (Mn2+ < Fe2+ < Co2+ < Ni2+ < Cu2+ > Zn2+) with few exceptions6,7. Accordingly, metalloproteins overwhelmingly bind Cu2+ and Zn2+ in isolation, regardless of the nature of their active sites and their cognate metal ions1,3,8. This led organisms to evolve complex homeostatic machinery and non-equilibrium strategies to achieve correct metal speciation1,3,8-10. Here we report an artificial dimeric protein, (AB)2, that thermodynamically overcomes the Irving-Williams restrictions in vitro and in cells, favouring the binding of lower-Irving-Williams transition metals over Cu2+, the most dominant ion in the Irving-Williams series. Counter to the convention in molecular design of achieving specificity through structural preorganization, (AB)2 was deliberately designed to be flexible. This flexibility enabled (AB)2 to adopt mutually exclusive, metal-dependent conformational states, which led to the discovery of structurally coupled coordination sites that disfavour Cu2+ ions by enforcing an unfavourable coordination geometry. Aside from highlighting flexibility as a valuable element in protein design, our results illustrate design principles for constructing selective metal sequestration agents.

Constructing protein polyhedra via orthogonal chemical interactions.

Many proteins exist naturally as symmetrical homooligomers or homopolymers1. The emergent structural and functional properties of such protein assemblies have inspired extensive efforts in biomolecular design2-5. As synthesized by ribosomes, proteins are inherently asymmetric. Thus, they must acquire multiple surface patches that selectively associate to generate the different symmetry elements needed to form higher-order architectures1,6-a daunting task for protein design. Here we address this problem using an inorganic chemical approach, whereby multiple modes of protein-protein interactions and symmetry are simultaneously achieved by selective, 'one-pot' coordination of soft and hard metal ions. We show that a monomeric protein (protomer) appropriately modified with biologically inspired hydroxamate groups and zinc-binding motifs assembles through concurrent Fe3+ and Zn2+ coordination into discrete dodecameric and hexameric cages. Our cages closely resemble natural polyhedral protein architectures7,8 and are, to our knowledge, unique among designed systems9-13 in that they possess tightly packed shells devoid of large apertures. At the same time, they can assemble and disassemble in response to diverse stimuli, owing to their heterobimetallic construction on minimal interprotein-bonding footprints. With stoichiometries ranging from [2 Fe:9 Zn:6 protomers] to [8 Fe:21 Zn:12 protomers], these protein cages represent some of the compositionally most complex protein assemblies-or inorganic coordination complexes-obtained by design.

Strengthening of enterococcal biofilms by Esp.

Multidrug-resistant (MDR) Enterococcus faecalis are major causes of hospital-acquired infections. Numerous clinical strains of E. faecalis harbor a large pathogenicity island that encodes enterococcal surface protein (Esp), which is suggested to promote biofilm production and virulence, but this remains controversial. To resolve this issue, we characterized the Esp N-terminal region, the portion implicated in biofilm production. Small angle X-ray scattering indicated that the N-terminal region had a globular head, which consisted of two DEv-Ig domains as visualized by X-ray crystallography, followed by an extended tail. The N-terminal region was not required for biofilm production but instead significantly strengthened biofilms against mechanical or degradative disruption, greatly increasing retention of Enterococcus within biofilms. Biofilm strengthening required low pH, which resulted in Esp unfolding, aggregating, and forming amyloid-like structures. The pH threshold for biofilm strengthening depended on protein stability. A truncated fragment of the first DEv-Ig domain, plausibly generated by a host protease, was the least stable and sufficient to strengthen biofilms at pH ≤ 5.0, while the entire N-terminal region and intact Esp on the enterococcal surface was more stable and required a pH ≤ 4.3. These results suggested a virulence role of Esp in strengthening enterococcal biofilms in acidic abiotic or host environments.

Hyperexpandable, self-healing macromolecular crystals with integrated polymer networks.

The formation of condensed matter typically involves a trade-off between structural order and flexibility. As the extent and directionality of interactions between atomic or molecular components increase, materials generally become more ordered but less compliant, and vice versa. Nevertheless, high levels of structural order and flexibility are not necessarily mutually exclusive; there are many biological (such as microtubules1,2, flagella 3 , viruses4,5) and synthetic assemblies (for example, dynamic molecular crystals6-9 and frameworks10-13) that can undergo considerable structural transformations without losing their crystalline order and that have remarkable mechanical properties8,14,15 that are useful in diverse applications, such as selective sorption 16 , separation 17 , sensing 18 and mechanoactuation 19 . However, the extent of structural changes and the elasticity of such flexible crystals are constrained by the necessity to maintain a continuous network of bonding interactions between the constituents of the lattice. Consequently, even the most dynamic porous materials tend to be brittle and isolated as microcrystalline powders 14 , whereas flexible organic or inorganic molecular crystals cannot expand without fracturing. Owing to their rigidity, crystalline materials rarely display self-healing behaviour 20 . Here we report that macromolecular ferritin crystals with integrated hydrogel polymers can isotropically expand to 180 per cent of their original dimensions and more than 500 per cent of their original volume while retaining periodic order and faceted Wulff morphologies. Even after the separation of neighbouring ferritin molecules by 50 ångströms upon lattice expansion, specific molecular contacts between them can be reformed upon lattice contraction, resulting in the recovery of atomic-level periodicity and the highest-resolution ferritin structure reported so far. Dynamic bonding interactions between the hydrogel network and the ferritin molecules endow the crystals with the ability to resist fragmentation and self-heal efficiently, whereas the chemical tailorability of the ferritin molecules enables the creation of chemically and mechanically differentiated domains within single crystals.

Author Correction: Hyperexpandable, self-healing macromolecular crystals with integrated polymer networks.

Change history: In this Letter, Alexander Groisman should have been listed as an author. This error has been corrected online.

Ion-dependent protein-surface interactions from intrinsic solvent response.

The phyllosilicate mineral muscovite mica is widely used as a surface template for the patterning of macromolecules, yet a molecular understanding of its surface chemistry under varying solution conditions, required to predict and control the self-assembly of adsorbed species, is lacking. We utilize all-atom molecular dynamics simulations in conjunction with an electrostatic analysis based in local molecular field theory that affords a clean separation of long-range and short-range electrostatics. Using water polarization response as a measure of the electric fields that arise from patterned, surface-bound ions that direct the adsorption of charged macromolecules, we apply a Landau theory of forces induced by asymmetrically polarized surfaces to compute protein-surface interactions for two muscovite-binding proteins (DHR10-mica6 and C98RhuA). Comparison of the pressure between surface and protein in high-concentration KCl and NaCl aqueous solutions reveals ion-specific differences in far-field protein-surface interactions, neatly capturing the ability of ions to modulate the surface charge of muscovite that in turn selectively attracts one binding face of each protein over all others.

Atomically Accurate Design of Metalloproteins with Predefined Coordination Geometries.

We report a new computational protein design method for the construction of oligomeric protein assemblies around metal centers with predefined coordination geometries. We apply this method to design two homotrimeric assemblies, Tet4 and TP1, with tetrahedral and trigonal-pyramidal tris(histidine) metal coordination geometries, respectively, and demonstrate that both assemblies form the targeted metal centers with ≤0.2 Å accuracy. Although Tet4 and TP1 are constructed from the same parent protein building block, they are distinct in terms of their overall architectures, the environment surrounding the metal centers, and their metal-based reactivities, illustrating the versatility of our approach.

Spatially Patterned, Porous Protein Crystals as Multifunctional Materials.

While the primary use of protein crystals has historically been in crystallographic structure determination, they have recently emerged as promising materials with many advantageous properties such as high porosity, biocompatibility, stability, structural and functional versatility, and genetic/chemical tailorability. Here, we report that the utility of protein crystals as functional materials can be further augmented through their spatial patterning and control of their morphologies. To this end, we took advantage of the chemically and kinetically controllable nature of ferritin self-assembly and constructed core-shell crystals with chemically distinct domains, tunable structural patterns, and morphologies. The spatial organization within ferritin crystals enabled the generation of patterned, multi-enzyme frameworks with cooperative catalytic behavior. We further exploited the differential growth kinetics of ferritin crystal facets to assemble Janus-type architectures with an anisotropic arrangement of chemically distinct domains. These examples represent a step toward using protein crystals as reaction vessels for complex multi-step reactions and broadening their utility as functional, solid-state materials. Our results demonstrate that morphology control and spatial patterning, which are key concepts in materials science and nanotechnology, can also be applied for engineering protein crystals.

Protein Assembly by Design.

Proteins are nature's primary building blocks for the construction of sophisticated molecular machines and dynamic materials, ranging from protein complexes such as photosystem II and nitrogenase that drive biogeochemical cycles to cytoskeletal assemblies and muscle fibers for motion. Such natural systems have inspired extensive efforts in the rational design of artificial protein assemblies in the last two decades. As molecular building blocks, proteins are highly complex, in terms of both their three-dimensional structures and chemical compositions. To enable control over the self-assembly of such complex molecules, scientists have devised many creative strategies by combining tools and principles of experimental and computational biophysics, supramolecular chemistry, inorganic chemistry, materials science, and polymer chemistry, among others. Owing to these innovative strategies, what started as a purely structure-building exercise two decades ago has, in short order, led to artificial protein assemblies with unprecedented structures and functions and protein-based materials with unusual properties. Our goal in this review is to give an overview of this exciting and highly interdisciplinary area of research, first outlining the design strategies and tools that have been devised for controlling protein self-assembly, then describing the diverse structures of artificial protein assemblies, and finally highlighting the emergent properties and functions of these assemblies.

Computationally Guided Redesign of a Heme-free Cytochrome with Native-like Structure and Stability.

Metals can play key roles in stabilizing protein structures, but ensuring their proper incorporation is a challenge when a metalloprotein is overexpressed in a non-native cellular environment. Here, we have used computational protein design tools to redesign cytochrome b562 (cyt b562), which relies on the binding of its heme cofactor to achieve its proper fold, into a stable, heme-free protein. The resulting protein, ApoCyt, features only four mutations and no metal-ligand or covalent bonds, yet displays improved stability over cyt b562. Mutagenesis studies and X-ray crystal structures reveal that the increase in stability is due to the computationally prescribed mutations, which stabilize the protein fold through a combination of hydrophobic packing interactions, hydrogen bonds, and cation-π interactions. Upon installation of the relevant mutations, ApoCyt is capable of assembling into previously reported, cytochrome-based trimeric and tetrameric assemblies, demonstrating that ApoCyt retains the structure and assembly properties of cyt b562. The successful design of ApoCyt therefore enables further functional diversification of cytochrome-based assemblies and demonstrates that structural metal cofactors can be replaced by a small number of well-designed, non-covalent interactions.

Design of a Flexible, Zn-Selective Protein Scaffold that Displays Anti-Irving-Williams Behavior.

Selective metal binding is a key requirement not only for the functions of natural metalloproteins but also for the potential applications of artificial metalloproteins in heterogeneous environments such as cells and environmental samples. The selection of transition-metal ions through protein design can, in principle, be achieved through the appropriate choice and the precise positioning of amino acids that comprise the primary metal coordination sphere. However, this task is made difficult by the intrinsic flexibility of proteins and the fact that protein design approaches generally lack the sub-Å precision required for the steric selection of metal ions. We recently introduced a flexible/probabilistic protein design strategy (MASCoT) that allows metal ions to search for optimal coordination geometry within a flexible, yet covalently constrained dimer interface. In an earlier proof-of-principle study, we used MASCoT to generate an artificial metalloprotein dimer, (AB)2, which selectively bound CoII and NiII over CuII (as well as other first-row transition-metal ions) through the imposition of a rigid octahedral coordination geometry, thus countering the Irving-Williams trend. In this study, we set out to redesign (AB)2 to examine the applicability of MASCoT to the selective binding of other metal ions. We report here the design and characterization of a new flexible protein dimer, B2, which displays ZnII selectivity over all other tested metal ions including CuII both in vitro and in cellulo. Selective, anti-Irving-Williams ZnII binding by B2 is achieved through the formation of a unique trinuclear Zn coordination motif in which His and Glu residues are rigidly placed in a tetrahedral geometry. These results highlight the utility of protein flexibility in the design and discovery of selective binding motifs.

Dynamic, Polymer-Integrated Crystals for Efficient, Reversible Protein Encapsulation.

Crystalline materials are increasingly being used as platforms for encapsulating proteins to create stable, functional materials. However, the uptake efficiencies and stimuli-responsiveness of crystalline frameworks are limited by their rigidities. We have recently reported a new form of materials, polymer-integrated crystals (PIX), which combine the structural order of protein crystals with the dynamic, stimuli-responsive properties of synthetic polymers. Here we show that the crystallinity, flexibility, and chemical tunability of PIX can be exploited to encapsulate guest proteins with high loading efficiencies (up to 46% w/w). The electrostatic host-guest interactions enable reversible, pH-controlled uptake/release of guest proteins as well as the mutual stabilization of the host and the guest, thus creating a uniquely synergistic platform toward the development of functional biomaterials and the controlled delivery of biological macromolecules.

Role of Serine Coordination in the Structural and Functional Protection of the Nitrogenase P-Cluster.

Nitrogenase catalyzes the multielectron reduction of dinitrogen to ammonia. Electron transfer in the catalytic protein (MoFeP) proceeds through a unique [8Fe-7S] cluster (P-cluster) to the active site (FeMoco). In the reduced, all-ferrous (PN) state, the P-cluster is coordinated by six cysteine residues. Upon two-electron oxidation to the P2+ state, the P-cluster undergoes conformational changes in which a highly conserved oxygen-based residue (a Ser or a Tyr) and a backbone amide additionally ligate the cluster. Previous studies of Azotobacter vinelandii (Av) MoFeP revealed that when the oxygen-based residue, ßSer188, was mutated to a noncoordinating residue, Ala, the P-cluster became redox-labile and reversibly lost two of its eight Fe centers. Surprisingly, the Av strain with a MoFeP variant that lacked the serine ligand (Av ßSer188Ala MoFeP) displayed the same diazotrophic growth and in vitro enzyme turnover rates as wild-type Av MoFeP, calling into question the necessity of this conserved ligand for nitrogenase function. Based on these observations, we hypothesized that ßSer188 plays a role in protecting the P-cluster under nonideal conditions. Here, we investigated the protective role of ßSer188 both in vivo and in vitro by characterizing the ability of Av ßSer188Ala cells to grow under suboptimal conditions (high oxidative stress or Fe limitation) and by determining the tendency of ßSer188Ala MoFeP to be mismetallated in vitro. Our results demonstrate that ßSer188 (1) increases Av cell survival upon exposure to oxidative stress in the form of hydrogen peroxide, (2) is necessary for efficient Av diazotrophic growth under Fe-limiting conditions, and (3) may protect the P-cluster from metal exchange in vitro. Taken together, our findings suggest a structural adaptation of nitrogenase to protect the P-cluster via Ser ligation, which is a previously unidentified functional role of the Ser residue in redox proteins and adds to the expanding functional roles of non-Cys ligands to FeS clusters.

Electron Transfer in Nitrogenase.

Nitrogenase is the only enzyme capable of reducing N2 to NH3. This challenging reaction requires the coordinated transfer of multiple electrons from the reductase, Fe-protein, to the catalytic component, MoFe-protein, in an ATP-dependent fashion. In the last two decades, there have been significant advances in our understanding of how nitrogenase orchestrates electron transfer (ET) from the Fe-protein to the catalytic site of MoFe-protein and how energy from ATP hydrolysis transduces the ET processes. In this review, we summarize these advances, with focus on the structural and thermodynamic redox properties of nitrogenase component proteins and their complexes, as well as on new insights regarding the mechanism of ET reactions during catalysis and how they are coupled to ATP hydrolysis. We also discuss recently developed chemical, photochemical, and electrochemical methods for uncoupling substrate reduction from ATP hydrolysis, which may provide new avenues for studying the catalytic mechanism of nitrogenase.

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 ((C98)RhuA) are essentially defect-free and undergo substantial, but fully correlated, changes in molecular arrangement, yielding coherently dynamic 2D molecular lattices. (C98)RhuA lattices display a Poisson's ratio of -1-the lowest thermodynamically possible value for an isotropic material-making them auxetic.

Enzyme-Directed Functionalization of Designed, Two-Dimensional Protein Lattices.

The design and construction of crystalline protein arrays to selectively assemble ordered nanoscale materials have potential applications in sensing, catalysis, and medicine. Whereas numerous designs have been implemented for the bottom-up construction of protein assemblies, the generation of artificial functional materials has been relatively unexplored. Enzyme-directed post-translational modifications are responsible for the functional diversity of the proteome and, thus, could be harnessed to selectively modify artificial protein assemblies. In this study, we describe the use of phosphopantetheinyl transferases (PPTases), a class of enzymes that covalently modify proteins using coenzyme A (CoA), to site-selectively tailor the surface of designed, two-dimensional (2D) protein crystals. We demonstrate that a short peptide (ybbR) or a molecular tag (CoA) can be covalently tethered to 2D arrays to enable enzymatic functionalization using Sfp PPTase. The site-specific modification of two different protein array platforms is facilitated by PPTases to afford both small molecule- and protein-functionalized surfaces with no loss of crystalline order. This work highlights the potential for chemoenzymatic modification of large protein surfaces toward the generation of sophisticated protein platforms reminiscent of the complex landscape of cell surfaces.

Tunable and Cooperative Thermomechanical Properties of Protein-Metal-Organic Frameworks.

We recently introduced protein-metal-organic frameworks (protein-MOFs) as chemically designed protein crystals, composed of ferritin nodes that predictably assemble into 3D lattices upon coordination of various metal ions and ditopic, hydroxamate-based linkers. Owing to their unique tripartite construction, protein-MOFs possess extremely sparse lattice connectivity, suggesting that they might display unusual thermomechanical properties. Leveraging the synthetic modularity of ferritin-MOFs, we investigated the temperature-dependent structural dynamics of six distinct frameworks. Our results show that the thermostabilities of ferritin-MOFs can be tuned through the metal component or the presence of crowding agents. Our studies also reveal a framework that undergoes a reversible and isotropic first-order phase transition near-room temperature, corresponding to a 4% volumetric change within 1 °C and a hysteresis window of ∼10 °C. This highly cooperative crystal-to-crystal transformation, which stems from the soft crystallinity of ferritin-MOFs, illustrates the advantage of modular construction strategies in discovering tunable-and unpredictable-material properties.

Anisotropic Dynamics and Mechanics of Macromolecular Crystals Containing Lattice-Patterned Polymer Networks.

The mechanical and functional properties of many crystalline materials depend on cooperative changes in lattice arrangements in response to external perturbations. However, the flexibility and adaptiveness of crystalline materials are limited. Additionally, the bottom-up, molecular-level design of crystals with desired dynamic and mechanical properties at the macroscopic level remains a considerable challenge. To address these challenges, we had previously integrated mesoporous, cubic ferritin crystals with hydrogel networks, resulting in hybrid materials (polymer-integrated crystals or PIX) which could undergo dramatic structural changes while maintaining crystalline periodicity and display efficient self-healing. The dynamics and mechanics of these ferritin-PIX were devoid of directionality, which is an important attribute of many molecular and macroscopic materials/devices. In this study, we report that such directionality can be achieved through the use of ferritin crystals with anisotropic symmetries (rhombohedral or trigonal), which enable the templated formation of patterned hydrogel networks in crystallo. The resulting PIX expand and contract anisotropically without losing crystallinity, undergo prompt bending motions in response to stimuli, and self-heal efficiently, capturing some of the essential features of sophisticated biological devices like skeletal muscles.

An Exceptionally Stable Metal-Organic Framework Constructed from Chelate-Based Metal-Organic Polyhedra.

We report the rational design and synthesis of a water-stable metal-organic framework (MOF), Fe-HAF-1, constructed from supramolecular, Fe3+-hydroxamate-based polyhedra with mononuclear metal nodes. Owing to its chelate-based construction, Fe-HAF-1 displays exceptional chemical stability in organic and aqueous solvents over a wide pH range (pH 1-14), including in the presence of 5 M NaOH. Despite the charge neutrality of the Fe3+-tris(hydroxamate) centers, Fe-HAF-1 crystals are negatively charged above pH 4. This unexpected property is attributed to the formation of defects during crystallization that results in uncoordinated hydroxamate ligands or hydroxide-coordinated Fe centers. The anionic nature of Fe-HAF-1 crystals enables selective adsorption of positively charged ions in aqueous solution, resulting in efficient separation of organic dyes and other charged species in a size-selective fashion. Fe-HAF-1 presents a new addition to a small group of chelate-based MOFs and provides a rare framework whose 3D connectivity is exclusively formed by metal-hydroxamate coordination.

Design and Construction of Functional Supramolecular Metalloprotein Assemblies.

Nature puts to use only a small fraction of metal ions in the periodic table. Yet, when incorporated into protein scaffolds, this limited set of metal ions carry out innumerable cellular functions and execute essential biochemical transformations such as photochemical H2O oxidation, O2 or CO2 reduction, and N2 fixation, highlighting the outsized importance of metalloproteins in biology. Not surprisingly, elucidating the intricate interplay between metal ions and protein structures has been the focus of extensive structural and mechanistic scrutiny over the last several decades. As a result of such top-down efforts, we have gained a reasonably detailed understanding of how metal ions shape protein structures and how protein structures in turn influence metal reactivity. It is fair to say that we now have some idea-and in some cases, a good idea-about how most known metalloproteins function and we possess enough insight to quickly assess the modus operandi of newly discovered ones. However, translating this knowledge into an ability to construct functional metalloproteins from scratch represents a challenge at a whole different level: it is one thing to know how an automobile works; it is another to build one. In our quest to build new metalloproteins, we have taken an original approach in which folded, monomeric proteins are used as ligands or synthons for building supramolecular complexes through metal-mediated self-assembly (MDPSA, Metal-Directed Protein Self-Assembly). The interfaces in the resulting protein superstructures are subsequently tailored with covalent, noncovalent, or additional metal-coordination interactions for stabilization and incorporation of new functionalities (MeTIR, Metal Templated Interface Redesign). In an earlier Account, we had described the proof-of-principle studies for MDPSA and MeTIR, using a four-helix bundle, heme protein cytochrome cb562 (cyt cb562), as a model building block. By the end of those studies, we were able to demonstrate that a tetrameric, Zn-directed cyt cb562 complex (Zn4:M14) could be stabilized through computationally prescribed noncovalent interactions inserted into the nascent protein-protein interfaces. In this Account, we first describe the rationale and motivation for our particular metalloprotein engineering strategy and a brief summary of our earlier work. We then describe the next steps in the "evolution" of bioinorganic complexity on the Zn4:M14 scaffold, namely, (a) the generation of a self-standing protein assembly that can stably and selectively bind metal ions, (b) the creation of reactive metal centers within the protein assembly, and (c) the coupling of metal coordination and reactivity to external stimuli through allosteric effects.