Design and Construction of Functional Supramolecular Metalloprotein

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Design and Construction of Functional Supramolecular Metalloprotein Assemblies Published as part of the Accounts of Chemical Research special issue “Artificial Metalloenzymes and Abiological Catalysis of Metalloenzymes”. Lewis A. Churchfield and F. Akif Tezcan*

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Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0356, United States CONSPECTUS: 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.

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INTRODUCTION

their robust structures and well-defined active site pockets, can readily accommodate alternative and frequently non-natural metal centers and withstand extensive modifications.3,5 Such protein reengineering efforts have produced artificial metalloenzymes that not only emulate the evolved activities of natural enzymes but are also capable of non-natural reactions.8−13 Protein repurposing is akin to divergent evolutionary processes in which certain protein folds are repeatedly used for alternative tasks like recognition and binding of different molecules or catalysis of different reactions. Thus, functional diversification is achieved without having to invent a new protein structure.

Over the last three decades, great progress has been made in the design of metalloproteins, whereby the targets have evolved from small metal-binding peptides with stable secondary structures to novel membrane-spanning proteins and artificial enzymes with well-defined active sites.1−7 Notwithstanding these advances, the design of an amino acid sequence to fold into a target 3D structure with the desired metal-based function still remains a daunting task. This issue is exacerbated by the fact that the proper functioning of any metalloprotein depends not only on its 3D architecture but also on other factors (e.g., local and global dynamics, solvation, and local dielectrics) that are often difficult to discern, let alone design. To circumvent these complications, researchers have taken advantage of pre-existing protein scaffolds, which, owing to © XXXX American Chemical Society

Received: December 4, 2018

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DOI: 10.1021/acs.accounts.8b00617 Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Figure 1. A retrosynthetic deconstruction of nitrogenase yields eight Rossman domains surrounding three metallocofactors.

Figure 2. (a) Schematic illustration of metal-directed protein self-assembly (MDPSA) and metal-templated interface redesign (MeTIR). (b) Applying MDPSA to the monomeric cyt cb562 affords M1, which, via a pair of surface-exposed bis-His clamps, self-assembles into oligomeric complexes driven by the coordination preferences of NiII, CuII, and ZnII. (c) Applying MeTIR to the i1 interface Zn4:M14 yields the homotetrameric Zn4:R14, which contains hydrophobic residues that comprise a self-associating interface.

In our work, we sought to mimic a different pathway of natural protein evolution whereby functional complexity is generated through the self-assembly of individual protein domains into supramolecular architectures. Multimeric metalloprotein assemblies are the ultimate drivers of biogeochemical cycles and major contributors to the chemical complexity of living systems.14 Whereas the functional scope of small, singledomain metalloproteins is typically restricted to elementary catalytic reactions or biochemical functions such as electron transfer and binding/recognition, protein complexes can perform multistep reactions and processes that involve a cooperative action of multiple components. From an engineering perspective, self-assembly of proteins into larger complexes (a) increases their stability, (b) brings them into a size regime (tens to hundreds of nanometers) that is necessary for their functional specificity in the complex cellular environment that cannot be achieved with single-chain peptide polymers, and (c) creates noncovalent interfaces, which themselves can be used as new interiors for building active sites or as mechanical conduits for intersubunit communication.15

A great case in point is provided by nitrogenase, the only known enzyme to catalyze the reduction of N2 into NH3 and one of our group’s favorites. Nitrogenase comprises two large proteins (Fe-protein and MoFe-protein, Figure 1), which themselves are constructed from multiple domains with complex Fe−S clusters positioned in their interfaces.16 Like an automobile with many moving parts and appropriately connected components, the supramolecular mode of construction of nitrogenase enables the formation and incorporation of the large metal clusters in the protein interior, leads to the creation of a deeply buried active site connected to the outside by proton and substrate channels, and allows the domains to transduce ATP-mediated conformational changes into electron and proton transfer reactions over long distances, ultimately giving rise to the unique catalytic chemistry.17 Clearly, the construction of such a biological machine has to occur through self-assembly from smaller components, as it would be quite challenging to build such complexity (i.e., moving parts, internalized metal clusters, channels, etc.) into a pre-existing, monolithic protein scaffold. Indeed, a retrosynB

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stabilization of the Zn4:M14 assembly through MeTIR. In that study, one (i1) of the three pairs of C2 symmetric interfaces of Zn4:M14 (i1, i2, and i3) was tailored with computationally prescribed hydrophobic interactions, requiring six mutations (R34A/L38A/Q41W/K42S/D66W/V69I) per protein monomer (Figure 2c).24 As planned, the resulting tetramer Zn4:R14 was isostructural with the parent architecture Zn4:M14 and possessed an identical tetrahedral coordination sphere composed of His63, His73, His77, and Asp74 but was able form at >100-fold lower protein monomer and Zn II concentrations, attesting to its increased stability.24

thetic deconstruction of nitrogenase reveals that it consists of multiple homologous domains that possess a Rossmann fold (Figure 1), a ubiquitous tertiary structural motif typically associated with nucleotide binding. Given the early emergence of nitrogenase in evolution, one can speculate on how it might have been constructed:18 first, the self-assembly of Rossmann modules into symmetrical superstructures, perhaps nucleated by interfacial Fe−S cluster formation, followed by the covalent attachment of these modules through gene fusion to produce the α- and β-subunits, and finally insertions and point mutations to yield the contemporary nitrogenase(s). Indeed, an analogous sequence of events has been proposed for the evolution of ancient proteins such as ferredoxins19 and other metalloprotein families20 and, in fact, can be envisioned for many proteins with supertertiary architectures.

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SELF-STANDING PROTEIN ASSEMBLIES FOR STABLE AND SELECTIVE METAL COORDINATION The primary prerequisite for any protein-associated, metalbased activity is the binding of the cognate metal ion(s) by the protein scaffold with high affinity and selectivity. In this regard, we were inspired by the seminal studies by Busch and Sargeson,25,26 who exploited metal coordination to spatially organize small ligands and then covalently link these ligands to construct macrocyclic ligands with enforced metal coordination geometries. Although the aforementioned Zn4:R14 complex was stabilized with respect to its parent, Zn4:M14, it remained a dynamically exchanging assembly that did not stay intact upon ZnII removal. As the D2 symmetry of Zn4:R14 dictates, the concurrent stabilization of any two of the three protein−protein interfaces should lead to the formation of a persistent tetramer whose formation is uncoupled from metal binding. Noting that the symmetrically related residues in position 96 are positioned within distance for disulfide (SS) cross-linking across i2, we combined the six hydrophobic mutations in i1 with the T96C mutation to generate C96R1.27 Through analytical ultracentrifugation experiments, we found that C96R1 indeed formed a stable tetramer in the absence or presence of ZnII ions (Figure 3a). Interestingly, the crystal structures of the corresponding species, C96R14 and Zn4:C96R14, showed that C96R14 possessed an open topology that underwent a large-scale motion upon ZnII coordination to adopt the intended holo structure (Figure 3b).27 This conformational change was enabled by the simultaneous flexibility and stability of the hydrophobic i1 and the SSlinked i2 interfaces. Despite the lack of a preorganized architecture for metal binding, C96R14 displayed uniformly high affinities for CoII, NiII, CuII, and ZnII with dissociation constants (Kd) in the nanomolar to femtomolar range. As intended by the template-and-stabilize strategy, it also bound ZnII with high selectivity over other metal ions including CuII (Figure 3c).27 In certain regards, C96R14 resembles a metalloregulatory protein that couples selective metal binding to conformational switching. In contrast, metalloenzymes typically are characterized by an invariant structure that can hold metal ions within a well-defined ligand sphere. We reasoned that C96R14 could be converted into such a preorganized architecture if i3, which undergoes a substantial change upon Zn coordination (Figure 3b), was also stabilized by a SS bond between residues in position 81. Thus, we prepared the third-generation variant, C81/C96 R1, which self-assembled with high selectivity into the tetramer C81/C96R14 through the simultaneous formation of hydrophobic contacts across i1 and two pairs of SS bonds across i2 and i3 (Figure 4a).28 The crystal structures of apo-C81/C96R14 and its Zn-bound form (Zn4:C81/C96R14)

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METAL-TEMPLATED FORMATION AND REDESIGN OF PROTEIN COMPLEXES As we introduced in an earlier Account,21 we set out to mimic such an evolutionary progression as a blueprint for rational protein design (Figure 2a). We surmised that a rate-limiting step in this progression would be the generation of associative surfaces on protein synthons to enable their self-assembly into supramolecular complexes. The reasoning behind this argument was that such associative surfaces would have to be quite extensive to provide the requisite driving force for selfassembly via noncovalent interactions, therefore requiring many simultaneous mutations. We envisioned that this ratelimiting step could be bypassed by exploiting metal coordination chemistry. As bonds between first-row transition metal ions and protein side chains typically are considerably stronger than noncovalent interactions but still reversible, the formation of well-defined protein−protein interfaces would be realized on a small design footprint and proceed under thermodynamic control. We termed this strategy, MetalDirected Protein Self-Assembly (MDPSA). As a model building block, we used a stable monomeric protein, cytochrome cb562, which has a four-helix bundle topology. Initially, cyt cb562 was tailored on the surface of Helix3 with two pairs of His residues (positions 59/63 and 73/77) to generate bidentate metal-chelating motifs. We found that the resulting variant MBPC1 (or M1) indeed self-assembled into discrete oligomers dictated by the stereochemical preferences of the added metal ions: C3 symmetric, NiII-mediated trimer Ni2:M13 imposed by octahedral coordination geometry; C2 symmetric, CuII-mediated dimer Cu 2:M12 imposed by tetragonal coordination geometry; and D2 symmetric, ZnIImediated tetramer Zn 4 :M1 4 (Figure 2b) imposed by tetrahedral coordination geometry.22,23 This was a simple but remarkable result−with only four mutations, a native monomeric protein was converted into a self-assembling synthon that formed three distinct oligomers with entirely different protein−protein interfaces, guided by metal ions that only differ by two electrons and two protons. A structural consequence of MDPSA is that new, evolutionarily naive protein−protein interfaces are generated around the now-buried metal centers. Using the crystal structures of the assemblies, these interfaces can be redesigned for stabilization through complementary noncovalent or covalent interactions, an approach termed Metal-Templated Interface Redesign (MeTIR; Figure 2a).24 During the writing of the previous Account, we had just conducted an initial proof-of-principle study that demonstrated the thermodynamic C

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ions. Importantly, it set the stage for engineering in vivo catalytic functions onto the R1 chassis as discussed below.

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DESIGN OF SUPRAMOLECULAR PROTEIN COMPLEXES WITH IN VIVO CATALYTIC ACTIVITIES Once a stable supramolecular scaffold with preorganized metal coordination sites was obtained, we started considering the possibility of engineering metal-based reactivities. Compared to designing a protein assembly for binding to a target of interest (a molecule or a metal ion), the de novo construction of a catalytic system is a tall order. Catalysis not only requires a reactive center but also a scaffold that can interact with, orient, and shuttle substrates, intermediates, and products, an optimized solvation environment for the reaction in question, and local and global protein dynamics to facilitate the process. Because such attributes, particularly the latter two factors, are very challenging in terms of de novo design, we first focused on deriving reactivity from the tetrahedral ZnII sites within Zn4:C81/C96R14. To this end, the D74 ligand was mutated into Ala with the idea of obtaining coordinatively unsaturated Zn centers with a 3His-1H2O ligand set resembling the active sites of carbonic anhydrases and matrix metalloproteinases. The resulting variant, A74/C81/C96R1, robustly formed a tetramer and anchored Zn ions in precisely the desired coordination geometry, with the Zn−H2O moiety directed into the central cavity of the assembly (Figure 4d). However, Zn4:A74/C81/C96R14 did not display any observable hydrolytic reactivity, even when treated with p-nitrophenyl acetate (pNPA) bearing a highly activated ester group.28 Initially, we attributed this lack of reactivity to inaccessibility of the central cavity. But given the potential flexibility of Zn4:A74/C81/C96R14 and its relatives, the actual reason is likely more complex than we believed we could address rationally by modifying this scaffold. One clear lesson that we learned was that simply replicating the inner-coordination sphere of a natural metalloenzyme does not guarantee even baseline catalytic activity. This led us to consider alternative locations on the tetrameric scaffold that might be more conducive to building catalytic Zn sites. We thus returned to the C96R14 assembly and, in particular, to the pair of i2 interfaces cross-linked by C96−C96 SS bonds. These C2 symmetric interfaces present several triads of amino acid positions across two protein monomers that could be used to build the triangular base of a tetrahedral Zn II−H2 O coordination motif (Figure 5a). Accordingly, we prepared four variants of C96R1 (termed AB1−AB4) with combinations of three coordinating residues (His or Glu) appropriately placed such that they would self-assemble into tetramers that possessed four structural Zn sites in their interiors and four potentially catalytic Zn sites in i2 (Figure 5b).29 Of these four variants, only AB3 with E86, H89, and H100 residues selfassembled into a tetramer in >90% yield and contained two ZnII ions per protein monomer as desired. The crystal structure of Zn8:AB34 confirmed formation of the targeted tetrameric architecture with the two types of Zn coordination geometries: core/structural Zn sites with the H63/H73/H77/D74 coordination sphere and peripheral Zn sites anchored by E86/H89 side chains from one monomer and H100 from another (Figure 5c). Unexpectedly, the K104 amine group also coordinated to the peripheral Zn sites, precluding any Znbased hydrolytic activity. Upon the elimination of this ligand via an Ala substitution, we obtained our first construct,

Figure 3. (a) Sedimentation coefficient distributions of various cyt cb562 constructs in the presence or absence of equimolar ZnII. (b) Crystal structures of Zn4:C96R14 (left) and C96R14 (right). (c) Divalent metal ion binding to C96R14 in competition experiments, illustrating the Zn binding selectivity of this complex.

showed that they were structurally similar (rmsd = 2.2 Å) and possessed a cryptand-like topology with a fully enclosed Znbinding core (Figure 4b). Like its more flexible predecessor C96 R14, C81/C96R14 also possessed a high ZnII affinity (Kd ≈ 10 nM) afforded by the templated 3His−1Asp coordination sphere. The stability and Zn-binding ability of C81/C96R14 led us to wonder if it might also properly self-assemble in bacterial cells and compete for ZnII ions in vivo. Conveniently, cyt cb562 and its variants had been engineered with a leader sequence for their transportation into the periplasmic space, which is an oxidizing environment and contains the biological machinery for SS bond exchange. Indeed, SDS-PAGE and ICP-OES analyses of the periplasmic extracts of C81/C96R1-expressing E. coli cells indicated that C81/C96R1 existed primarily as a tetramer (>60% abundance) inside the cells and retained a significant amount of Zn (>1.5 equiv per tetramer) and no other transition metal ions (Figure 4c).28 In contrast, the non-selfassembling variants M1 and R1 that lacked Cys residues for interfacial SS cross-linking did not form tetramers in vivo or bind any detectable Zn. These results established C81/C96R1 as a unique artificial protein scaffold with the ability to correctly self-assemble inside living cells and bind the cognate metal D

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Figure 4. (a) Crystal structures of Zn4:C81/C96R14 (left) and C81/C96R14 (right). (b) Cryptand-like cores of apo- (cyan) and Zn-bound (dark gray) C81/C96 R14 (residues 60−96) compared to a synthetic cryptand. (c) Nonreducing SDS-PAGE analysis of periplasmic extracts of E. coli expressing various cyt cb562 constructs. ICP-OES analysis of periplasmic extracts of E. coli expressing self-assembled C81/C96R14. (d) One of four identical 3HisZn coordination sites of Zn4:A74/C81/C96R14.

Figure 5. (a) Structure of C96R14 viewed along one of three 2-fold rational symmetry axes. (B) List of select cyt cb562 variants containing triads of Zn-binding residues. (C) Crystal structure of Zn8:AB34 and close-up views of different Zn coordination sites.

Building on our earlier findings about the in vivo self-assembly and Zn binding ability of C81/C96R1, we asked whether the βlactamase activity of Zn8:A104AB34 could also be operative within E. coli cells. As in the case of C81/C96R1, an analysis of the periplasmic extracts of A104AB3-expressing E. coli cells showed that this variant existed primarily as a tetramer and associated with two equivalents of Zn ions per monomer as desired. To test in vivo activity, A104AB3 was expressed in E. coli cells devoid of inherent β-lactamases, which were cultured in media containing various amounts of ampicillin. A104AB3expressing cells were found to grow in the presence of up to 1.1 mg/L ampicillin, whereas those expressing noncatalytic C96 R1 variant did not survive even smaller amounts, establish-

Zn8:A104AB34, that was catalytically competent for pNPA hydrolysis (kcat = 0.20 s−1 and kcat/Km = 120 s−1 M−1) (Figure 6a).29 Encouraged by this result, we tested the ability of Zn8:A104AB34 to catalyze the hydrolysis of β-lactam antibiotics, a more challenging hydrolytic reaction that, when catalyzed inside bacteria, could provide the benefit of survival to the organism. Indeed, Zn8:A104AB34 hydrolyzed ampicillin with a second-order rate constant of k = 115 min−1 M−1 at pH 9, an activity that was not observed with the control variants C96R14, Zn4:C96R14 and apo-A104AB34 (Figure 6b).29 Yet, this reaction did not display saturation behavior even at high substrate concentrations (≤20 mM), indicating that Zn8:A104AB34 did not have substantial binding interactions with the antibiotic. E

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Figure 6. (a) Michaelis−Menten kinetics of Zn8:A104AB34 for p-nitrophenyl acetate (pNPA) hydrolysis. (b) Ampicillin hydrolysis activities of cyt cb562 variants. (c). Representative LB/agar plates in the absence and presence of 0.8 mg/L ampicillin streaked with cells expressing C96R1 and A104 AB3.

ing that Zn8:A104AB34 has sufficient activity in bacterial cells to serve as a basis for in vivo selection (Figure 6c).29 To optimize the β-lactamase activity of Zn8:A104AB34 via directed evolution, we targeted four amino acid positions (E57, D60, A104, Y105) that surrounded the catalytic Zn sites (Figure 5c).29 A mid-throughput selection process was carried out using AB3 mutant libraries in which these four positions were individually randomized by saturation mutagenesis. This procedure revealed one particular variant carrying the E57G mutation as the clear winner that gave rise to >20-fold in vivo survival frequencies above the parent A104AB3 species (Figure 7a). The improved in vivo viability was reflected by increased in vitro activities: Zn8:G57/A104AB34 variant hydrolyzed ampicillin (kcat = 3.5 min−1, kcat/Km = 350 min−1 M−1; kcat/kuncat = 23 000) more than three times as efficiently as A104AB3 and displayed saturation behavior at increased ampicillin concentrations (Figure 7b), with a Km of 10−2 M, consistent with the emergence of a substrate binding site. Surprisingly, Zn8:G57/A104AB34 displayed a substantially more open architecture compared to Zn8:AB34 (Figure 7c) and the structured loop near the Zn-E86/H89/H100 active site (starting from Ala43 and terminating in residue 57) was found to be completely disordered because of the elimination of the side-

chain interactions between E57, K51 and S54 that stabilize this loop. Molecular docking simulations with ampicillin suggested that the E57G mutation might open up sufficient room for substrate binding and alleviate charge repulsion with the ampicillin carboxylate group to position the β-lactam ring directly above the ZnII−OH2 moiety for hydrolysis.29 These findings were noteworthy in several regards. First, Zn8:AB34 and its variants represent the first de novo constructed protein assemblies with in vivo catalytic activities that could be subjected to functional optimization via a selection strategy. Second, through MeTIR, a simple electron-transfer protein was converted into a metallo-β-lactamase despite having no sequence or structural homology to any β-lactamase or hydrolase. Finally, a highly mobile loop near the active site emerged as an unforeseen consequence of the selection process coupled to directed evolution. Coincidentally, such flexible loops are a key feature of natural β-lactamases that enable adaptable substrate binding and rapid diversification.30 The fact that the E57G substitution would not have been an obvious choice as a targeted mutation illustrated the utility of combining rational design with directed evolution to capture structural nuances that may have substantial effects on catalysis. F

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Figure 7. (a) In vivo survival frequency of A104AB3 active-site variants subjected to saturation mutagenesis. Original residues are marked with asterisks. (b) Michaelis−Menten kinetics of Zn8:A104/G57AB34 for ampicillin hydrolysis. (c) Ribbon and surface representations of the crystal structure of Zn8:A104/G57AB34 with catalytic and structural Zn ions (gray and navy spheres, respectively) and mobile surface loop (yellow) depicted.

Having gained a first-hand appreciation of the importance of local structure and conformational flexibility in enzymatic activity, we next asked (a) if Zn4:C96R14, the parent of Zn8:AB34, could harbor alternative active sites in its interfaces and (b) if these active sites could display different extents of evolvability due to their positioning. We engineered a variant, AB5, with a new triad of Zn-coordinating amino acids (E89/ H93/H100) in i2 (Figure 5a) and validated its proper assembly into a tetramer by crystallography (Figure 8a).31 Although the active site triad in Zn8:AB54 is positioned immediately adjacent to that in Zn8:G57AB34 and has the same inner-sphere coordination motif, it can be viewed as completely different in terms of its local microenvironment, secondary coordination sphere, and proximity to the C96− C96 SS linkage. Indeed, the active site Zn−OH2 moiety in Zn8:AB54 was found to be directed toward the exterior of the tetramer rather than to the interior as observed in Zn8:G57/A104AB34 and away from the hydrophobic 43−57 loop (Figure 8b), which had emerged as a key element in the directed evolution of Zn8:G57/A104AB34. Interestingly, Zn8:AB54 had only a slightly lower β-lactamase activity (kcat = 1.03 min−1 and kcat/KM = 120 ± 10 min−1 M−1 at pH 9) than Zn8:G57/A104AB34 and displayed substrate-saturation behavior even in the absence of any optimization (Figure 8c).31 Yet, despite this advantageous starting point, Zn8:AB54 proved to be recalcitrant to optimization via directed evolution. None of the single site saturation mutants of the active site pocket

chosen through in vivo selection were found to be significantly more active in vitro for ampicillin hydrolysis. Remembering again the key involvement of a flexible loop in the directed evolution of Zn8:G57/A104AB34, we hypothesized that the evolvability of the active site of Zn8:AB54 might be limited due the local rigidity imposed by the adjacent SS linkage. Removal of this bond through the C96T mutation indeed led to a relaxation of tetrameric architecture (Zn8:T96AB54) and the Zn-E89/H93/H100 active site, which became more exposed to solvent (Figure 8b).31 This alteration, in turn, brought about a considerable improvement (∼40%) in the catalytic rate for ampicillin hydrolysis (kcat = 1.4 min−1 at pH 9) despite a concomitant increase in the pKa of the Zn−OH2 moiety from 8.4 to 9.1 (Figure 8c). At pH 9.5, where the Zn− OH2 species is predominantly deprotonated and activated for nucleophilic attack, we observed kcat = 1.8 min−1 and kcat/KM = 210 min−1 M−1.31 These values were second only to the evolved Zn8:G57/A104AB34 among all β-lactamase variants examined; notably, they were achieved without the benefit of a nearby loop to enhance ampicillin binding interactions. Our collective observations on AB3 and AB5 assemblies emphasized once more the importance of local and global protein flexibility in terms of enzyme design and evolvability.29,31 G

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Figure 8. (a) Design of Zn8:AB54 from Zn4:C96R14. (b). Enlarged peripheral (top) and catalytic (bottom) Zn-binding sites of Zn8:A104/G57AB34, Zn8:AB54, and Zn8:T96AB54. (c) In vitro β-lactamase activity of Zn8:AB54 and Zn8:T96AB54.

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recap, C81/C96R14 is stabilized by two sets of SS bonds across i2 and i3 (C96−C96 and C81−C81, respectively) and hydrophobic interactions across i1.28 Due to the interconnectivity of all three pairs of interfaces, C81/C96R14 undergoes only a small global structural perturbation upon Zn binding. Locally, however, i1 interfaces undergo a noticeable deformation, whereby the average α-C distances between the symmetrically related A38 residues decreased by nearly 3 Å (Figure 4a). Therefore, we hypothesized that if the i1 interfaces in C81/C96 R14 were also SS-cross-linked at position 38, this would generate a spring-loaded quaternary structure with strained SS bonds. This, in turn, would allow long-distance structural coupling between the equilibria for Zn binding and SS bond formation. Toward this end, we generated the triple Cys mutant C38/C81/C96R1 and isolated it in the form of the desired, hexa-SS-cross-linked tetramer under oxidizing conditions.34 Zn-binding titrations indicated that the affinity of C38/C81/C96 R14 for ZnII ions was reduced by >20 kJ/mol per tetramer compared to its less extensively SS-cross-linked relatives (C96R14 and C81/C96R14), suggestive of quaternary strain associated with metal coordination. This scenario was borne out by the crystal structures of Zn-bound and apo-C38/C81/C96R14, which showed that the former contained six intact SS-bonds whereas the latter had only five, with a single C38−C38 linkage broken (Figure 9a).34 Dissociation of

CONSTRUCTION OF ALLOSTERIC METALLOPROTEINS Allostery is generally defined as the control of activity at a certain protein site through a binding event or another physical or chemical transformation at a distant location. Like catalysis, it represents another type of functional sophistication that requires coupled motions (thus local and global flexibility) in proteins. Hemoglobin provides one of the most well-known examples of allostery: local structural changes that result from O2 coordination to one of the four heme cofactors are propagated onto the entire quaternary architecture, leading to progressively more favorable O2 binding (positive cooperativity) by the remaining heme cofactors.32 Like catalysis, de novo construction of allosteric systems like hemoglobin represents an outstanding challenge, as this task not only requires the design of at least two (re)active sites in a single protein but also the efficient coupling of these sites via a structural conduit. Both of these design criteria would be disfavored in a very rigid or in a very flexible scaffold. Not surprisingly, a large number of natural allosteric proteins like hemoglobin are oligomeric complexes that contain semirigid subunits linked by semirigid interfaces that allow propagation of mechanical strain over long distances.15,33 In the course of our studies described above, we noticed that C81/C96 R14 (Figure 4a) fit this structural description well. To H

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Figure 9. (a) Crystal structures and schematic representations of Zn- (left) and apo-C38/C81/C96R14 (right). (b) Close-up view of the C38−C38 SS bond hydrolyzed upon Zn-removal.

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CONCLUSIONS In this Account, we have described how cyt cb562, a simple, one-electron transfer protein, was converted into (1) a metalselective conformational switch, (2) an esterase and in vivo evolvable β-lactamase, and (3) an allosteric assembly that links metal binding to distant bond formation−breakage events. Importantly, all of these functional complexes derived from a parent Zn-mediated protein complex, Zn4:M14, that was obtained through only four point mutations on the cyt cb562 scaffold. The functional complexes themselves shared >85% sequence identity with cyt cb562. In normal circumstances, such high sequence identity between two proteins would indicate that they look and function identically. Thus, our findings illustrate the facility with which protein−protein interfaces nucleated by metal ions lend themselves to creation and diversification of new biological structures and functions not only in the lab but perhaps also naturally during the course of evolution. Since we originally introduced nitrogenase as an example for an evolved supramolecular metalloprotein, it would be fair to ask if one could construct such a sophisticated biological machine through a rational design strategy. The answer is “no, not even close”. Still, we can picture some milestones on the path that may get us closer to such a target. For example, the ability to design and construct two- or three-component, heteromeric protein assemblies that possess one or more asymmetric metal centers in their interfaces would represent a substantial increase in complexity. Another milestone would be to achieve cooperativity between protein components that have different inherent functions to create coupled systems (e.g., electron transfer + redox catalysis, stimuli-dependent conformational changes + catalysis, light absorption +

this SS linkage was accompanied by a considerable distortion of i1, whereby the distance between the α-carbons of C38 residues increased by 5 Å. These observations suggested that ZnII coordination spring-loads the quaternary architecture through the formation of strained SS bonds and that this strain is relieved through the dissociation of a single C38−C38 SS bond, which is >14 Å away from the nearest Zn2+ coordination site. Remarkably, SS-bond cleavage occurs through a mechanically activated hydrolytic mechanism (rather than reduction), resulting in the formation of a sulfenic acid/thiol pair (Figure 9b).34 To understand the energetic basis of allosteric coupling in C38/C81/C96 R14, we carried out extensive molecular dynamics calculations, which revealed that allostery was governed by an entropy/enthalpy trade-off.35 This trade-off primarily involves the hydrophobic i1 interfaces, which rearrange upon ZnII dissociation to form favorable packing interactions that overcome the energetic cost incurred by reduced configurational freedom of the assembly. We concluded that the direct embedding of the C38−C38 bonds in these hydrophobic interfaces must account for their selective cleavage over the C96−C96 or C81−C81 linkages, which are located in the nondesigned interfaces that are free of structural constraints.35 An analysis of several globular proteins revealed that allosteric motions largely arose from sterically constrained competition among different modes of hydrophobic burial,36 in analogy to what we have observed with C38/C81/C96R14. Thus, our study showed that a de novo designed model system such as C38/C81/C96 R14 could recapitulate the complex interplay of energetic factors and structural features that underlie allosteric behavior of many evolved proteins. I

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of a highly active and enantiospecific metalloenzyme from short peptides. Science 2018, 362, 1285−1288. (8) Jeschek, M.; Reuter, R.; Heinisch, T.; Trindler, C.; Klehr, J.; Panke, S.; Ward, T. R. Directed evolution of artificial metalloenzymes for in vivo metathesis. Nature 2016, 537, 661. (9) Key, H. M.; Dydio, P.; Clark, D. S.; Hartwig, J. F. Abiological catalysis by artificial haem proteins containing noble metals in place of iron. Nature 2016, 534, 534. (10) Kan, S. B. J.; Lewis, R. D.; Chen, K.; Arnold, F. H. Directed evolution of cytochrome c for carbon−silicon bond formation: Bringing silicon to life. Science 2016, 354, 1048−1051. (11) Yang, H.; Swartz, A. M.; Park, H. J.; Srivastava, P.; EllisGuardiola, K.; Upp, D. M.; Lee, G.; Belsare, K.; Gu, Y.; Zhang, C.; Moellering, R. E.; Lewis, J. C. Evolving artificial metalloenzymes via random mutagenesis. Nat. Chem. 2018, 10, 318. (12) Bordeaux, M.; Tyagi, V.; Fasan, R. Highly Diastereoselective and Enantioselective Olefin Cyclopropanation Using Engineered Myoglobin-Based Catalysts. Angew. Chem. 2015, 127, 1764−1768. (13) Bos, J.; Browne, W. R.; Driessen, A. J. M.; Roelfes, G. Supramolecular Assembly of Artificial Metalloenzymes Based on the Dimeric Protein LmrR as Promiscuous Scaffold. J. Am. Chem. Soc. 2015, 137, 9796−9799. (14) Bertini, I.; Gray, H. B.; Stiefel, E. I.; Valentine, J. S. Biological Inorganic Chemistry, Structure & Reactivity; University Science Books: Sausalito, CA, 2007. (15) Goodsell, D. S.; Olson, A. J. Structural symmetry and protein function. Annu. Rev. Biophys. Biomol. Struct. 2000, 29, 105−153. (16) Rees, D. C.; Tezcan, F. A.; Haynes, C. A.; Walton, M. Y.; Andrade, S.; Einsle, O.; Howard, J. B. Structural basis of biological nitrogen fixation. Philos. Trans. R. Soc., A 2005, 363, 971−984. (17) Hoffman, B. M.; Lukoyanov, D.; Yang, Z.-Y.; Dean, D. R.; Seefeldt, L. C. Mechanism of Nitrogen Fixation by Nitrogenase: The Next Stage. Chem. Rev. 2014, 114, 4041−4062. (18) Raymond, J.; Siefert, J. L.; Staples, C. R.; Blankenship, R. E. The Natural History of Nitrogen Fixation. Mol. Biol. Evol. 2004, 21, 541−554. (19) Eck, R. V.; Dayhoff, M. O. Evolution of the Structure of Ferredoxin Based on Living Relics of Primitive Amino Acid Sequences. Science 1966, 152, 363−366. (20) Armstrong, R. N. Mechanistic diversity in a metalloenzyme superfamily. Biochemistry 2000, 39, 13625−13632. (21) Salgado, E. N.; Radford, R. J.; Tezcan, F. A. Metal-Directed Protein Self-Assembly. Acc. Chem. Res. 2010, 43, 661−672. (22) Salgado, E. N.; Faraone-Mennella, J.; Tezcan, F. A. Controlling Protein-Protein Interactions through Metal Coordination: Assembly of a 16-Helix Bundle Protein. J. Am. Chem. Soc. 2007, 129, 13374− 13375. (23) Salgado, E. N.; Lewis, R. A.; Mossin, S.; Rheingold, A. L.; Tezcan, F. A. Control of Protein Oligomerization Symmetry by Metal Coordination: C2 and C3 Symmetrical Assemblies through CuII and NiII Coordination. Inorg. Chem. 2009, 48, 2726−2728. (24) Salgado, E. N.; Ambroggio, X. I.; Brodin, J. D.; Lewis, R. A.; Kuhlman, B.; Tezcan, F. A. Metal-Templated Design of Protein Interfaces. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 1827−1832. (25) Thompson, M. C.; Busch, D. H. Reactions of Coordinated Ligands. 9. Utilization of Template Hypothesis to Synthesize Macrocyclic Ligands in Situ. J. Am. Chem. Soc. 1964, 86, 3651−3656. (26) Creaser, I. I.; Geue, R. J.; Harrowfield, J. M.; Herlt, A. J.; Sargeson, A. M.; Snow, M. R.; Springborg, J. Synthesis and Reactivity of Aza-Capped Encapsulated Co(III) Ions. J. Am. Chem. Soc. 1982, 104, 6016−6025. (27) Brodin, J. D.; Medina-Morales, A.; Ni, T.; Salgado, E. N.; Ambroggio, X. I.; Tezcan, F. A. Evolution of Metal Selectivity in Templated Protein Interfaces. J. Am. Chem. Soc. 2010, 132, 8610− 8617. (28) Medina-Morales, A.; Perez, A.; Brodin, J. D.; Tezcan, F. A. In Vitro and Cellular Self-Assembly of a Zn-Binding Protein Cryptand via Templated Disulfide Bonds. J. Am. Chem. Soc. 2013, 135, 12013− 12022.

unidirectional transport) with built-in sensory and feedback mechanisms. These milestones certainly necessitate the advent of new protein design and self-assembly strategies, a better understanding of protein structure/function/dynamics relationships and an improved command of protein−metal interactions. Smaller, synthetic metalloproteins can undoubtedly provide useful guidance in this regard.37,38 Finally, we have learned in our studies that directed evolution and highthroughput selection strategies can lead to functional solutions and insights that would have been difficult to find through rational design. This emphasizes the important need for the proper design of functional screens or in vivo selection systems that could enable the evolution of multicomponent/multifunctional protein architectures.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

F. Akif Tezcan: 0000-0002-4733-6500 Notes

The authors declare no competing financial interest. Biographies Lewis Churchfield received his B.S. in Biochemistry at the University of Minnesota in 2012 and his Ph.D. at UCSD in 2018, working on the design of allosteric protein assemblies in the Tezcan Lab. He was an NIH Biophysics trainee and received a Distinguished Graduate Fellowship in 2017. Akif Tezcan completed his B.A. at Macalester College and his Ph.D. with Harry Gray at Caltech, where he was also a postdoctoral fellow in the group of Doug Rees. He has been at UCSD since 2005 where he is currently a Professor of Chemistry and Biochemistry.

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ACKNOWLEDGMENTS We are grateful to the former members of the Tezcan Lab who carried out the research described here. This work was supported by NSF (Grants CHE1306656 and CHE1607145), as well as by an NIH traineeship to L.A.C. (Grant T32GM008326).

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REFERENCES

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