In Vitro Assembly of Diverse Bacterial Microcompartment Shell Architectures.

Bacterial microcompartments (BMCs) are organelles composed of a selectively permeable protein shell that encapsulates enzymes involved in CO2 fixation (carboxysomes) or carbon catabolism (metabolosomes). Confinement of sequential reactions by the BMC shell presumably increases the efficiency of the pathway by reducing the crosstalk of metabolites, release of toxic intermediates, and accumulation of inhibitory products. Because BMCs are composed entirely of protein and self-assemble, they are an emerging platform for engineering nanoreactors and molecular scaffolds. However, testing designs for assembly and function through in vivo expression is labor-intensive and has limited the potential of BMCs in bioengineering. Here, we developed a new method for in vitro assembly of defined nanoscale BMC architectures: shells and nanotubes. By inserting a "protecting group", a short ubiquitin-like modifier (SUMO) domain, self-assembly of shell proteins in vivo was thwarted, enabling preparation of concentrates of shell building blocks. Addition of the cognate protease removes the SUMO domain and subsequent mixing of the constituent shell proteins in vitro results in the self-assembly of three types of supramolecular architectures: a metabolosome shell, a carboxysome shell, and a BMC protein-based nanotube. We next applied our method to generate a metabolosome shell engineered with a hyper-basic luminal surface, allowing for the encapsulation of biotic or abiotic cargos functionalized with an acidic accessory group. This is the first demonstration of using charge complementarity to encapsulate diverse cargos in BMC shells. Collectively, our work provides a generally applicable method for in vitro assembly of natural and engineered BMC-based architectures.

Structural and Functional Characterization of a Short-Chain Flavodoxin Associated with a Noncanonical 1,2-Propanediol Utilization Bacterial Microcompartment.

Bacterial microcompartments (BMCs) are proteinaceous organelles that encapsulate enzymes involved in CO2 fixation (carboxysomes) or carbon catabolism (metabolosomes). Metabolosomes share a common core of enzymes and a distinct signature enzyme for substrate degradation that defines the function of the BMC (e.g., propanediol or ethanolamine utilization BMCs, or glycyl-radical enzyme microcompartments). Loci encoding metabolosomes also typically contain genes for proteins that support organelle function, such as regulation, transport of substrate, and cofactor (e.g., vitamin B12) synthesis and recycling. Flavoproteins are frequently among these ancillary gene products, suggesting that these redox active proteins play an undetermined function in many metabolosomes. Here, we report the first characterization of a BMC-associated flavodoxin (Fld1C), a small flavoprotein, derived from the noncanonical 1,2-propanediol utilization BMC locus (PDU1C) of Lactobacillus reuteri. The 2.0 Å X-ray structure of Fld1C displays the α/ß flavodoxin fold, which noncovalently binds a single flavin mononucleotide molecule. Fld1C is a short-chain flavodoxin with redox potentials of -240 ± 3 mV oxidized/semiquinone and -344 ± 1 mV semiquinone/hydroquinone versus the standard hydrogen electrode at pH 7.5. It can participate in an electron transfer reaction with a photoreductant to form a stable semiquinone species. Collectively, our structural and functional results suggest that PDU1C BMCs encapsulate Fld1C to store and transfer electrons for the reactivation and/or recycling of the B12 cofactor utilized by the signature enzyme.

Electron transfer activity of a de novo designed copper center in a three-helix bundle fold.

In this work, we characterized the intermolecular electron transfer (ET) properties of a de novo designed metallopeptide using laser-flash photolysis. α3D-CH3 is three helix bundle peptide that was designed to contain a copper ET site that is found in the ß-barrel fold of native cupredoxins. The ET activity of Cuα3D-CH3 was determined using five different photosensitizers. By exhibiting a complete depletion of the photo-oxidant and the successive formation of a Cu(II) species at 400 nm, the transient and generated spectra demonstrated an ET transfer reaction between the photo-oxidant and Cu(I)α3D-CH3. This observation illustrated our success in integrating an ET center within a de novo designed scaffold. From the kinetic traces at 400 nm, first-order and bimolecular rate constants of 10(5) s(-1) and 10(8) M(-1) s(-1) were derived. Moreover, a Marcus equation analysis on the rate versus driving force study produced a reorganization energy of 1.1 eV, demonstrating that the helical fold of α3D requires further structural optimization to efficiently perform ET. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

Structure and Function of a Bacterial Microcompartment Shell Protein Engineered to Bind a [4Fe-4S] Cluster.

Bacterial microcompartments (BMCs) are self-assembling organelles composed of a selectively permeable protein shell and encapsulated enzymes. They are considered promising templates for the engineering of designed bionanoreactors for biotechnology. In particular, encapsulation of oxidoreductive reactions requiring electron transfer between the lumen of the BMC and the cytosol relies on the ability to conduct electrons across the shell. We determined the crystal structure of a component protein of a synthetic BMC shell, which informed the rational design of a [4Fe-4S] cluster-binding site in its pore. We also solved the structure of the [4Fe-4S] cluster-bound, engineered protein to 1.8 Å resolution, providing the first structure of a BMC shell protein containing a metal center. The [4Fe-4S] cluster was characterized by optical and EPR spectroscopies; it has a reduction potential of -370 mV vs the standard hydrogen electrode (SHE) and is stable through redox cycling. This remarkable stability may be attributable to the hydrogen-bonding network provided by the main chain of the protein scaffold. The properties of the [4Fe-4S] cluster resemble those in low-potential bacterial ferredoxins, while its ligation to three cysteine residues is reminiscent of enzymes such as aconitase and radical S-adenosymethionine (SAM) enzymes. This engineered shell protein provides the foundation for conferring electron-transfer functionality to BMC shells.

Apoprotein Structure and Metal Binding Characterization of a de Novo Designed Peptide, α3DIV, that Sequesters Toxic Heavy Metals.

De novo protein design is a biologically relevant approach that provides a novel process in elucidating protein folding and modeling the metal centers of metalloproteins in a completely unrelated or simplified fold. An integral step in de novo protein design is the establishment of a well-folded scaffold with one conformation, which is a fundamental characteristic of many native proteins. Here, we report the NMR solution structure of apo α3DIV at pH 7.0, a de novo designed three-helix bundle peptide containing a triscysteine motif (Cys18, Cys28, and Cys67) that binds toxic heavy metals. The structure comprises 1067 NOE restraints derived from multinuclear multidimensional NOESY, as well as 138 dihedral angles (ψ, φ, and χ1). The backbone and heavy atoms of the 20 lowest energy structures have a root mean square deviation from the mean structure of 0.79 (0.16) Å and 1.31 (0.15) Å, respectively. When compared to the parent structure α3D, the substitution of Leu residues to Cys enhanced the α-helical content of α3DIV while maintaining the same overall topology and fold. In addition, solution studies on the metalated species illustrated metal-induced stability. An increase in the melting temperatures was observed for Hg(II), Pb(II), or Cd(II) bound α3DIV by 18-24 °C compared to its apo counterpart. Further, the extended X-ray absorption fine structure analysis on Hg(II)-α3DIV produced an average Hg(II)-S bond length at 2.36 Å, indicating a trigonal T-shaped coordination environment. Overall, the structure of apo α3DIV reveals an asymmetric distorted triscysteine metal binding site, which offers a model for native metalloregulatory proteins with thiol-rich ligands that function in regulating toxic heavy metals, such as ArsR, CadC, MerR, and PbrR.

De novo design and characterization of copper metallopeptides inspired by native cupredoxins.

Using de novo protein design, we incorporated a copper metal binding site within the three-helix bundle α3D (Walsh et al. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 5486-5491) to assess whether a cupredoxin center within an α-helical domain could mimic the spectroscopic, structural, and redox features of native type-1 copper (CuT1) proteins. We aimed to determine whether a CuT1 center could be realized in a markedly different scaffold rather than the native ß-barrel fold and whether the characteristic short Cu-S bond (2.1-2.2 Å) and positive reduction potentials could be decoupled from the spectroscopic properties (ε600 nm = 5000 M(-1) cm(-1)) of such centers. We incorporated 2HisCys(Met) residues in three distinct α3D designs designated core (CR), chelate (CH), and chelate-core (ChC). XAS analysis revealed a coordination environment similar to reduced CuT1 proteins, producing Cu-S(Cys) bonds ranging from 2.16 to 2.23 Å and Cu-N(His) bond distances of 1.92-1.99 Å. However, Cu(II) binding to the CR and CH constructs resulted in tetragonal type-2 copper-like species, displaying an intense absorption band between 380 and 400 nm (>1500 M(-1) cm(-1)) and A|| values of (150-185) × 10(-4) cm(-4). The ChC construct, which possesses a metal-binding site deeper in its helical bundle, yielded a CuT1-like brown copper species, with two absorption bands at 401 (4429 M(-1) cm(-1)) and 499 (2020 M(-1) cm(-1)) nm and an A|| value ∼30 × 10(-4) cm(-4) greater than its native counterparts. Electrochemical studies demonstrated reduction potentials of +360 to +460 mV (vs NHE), which are within the observed range for azurin and plastocyanin. These observations showed that the designed metal binding sites lacked the necessary rigidity to enforce the appropriate structural constraints for a Cu(II) chromophore (EPR and UV-vis); however, the Cu(I) structural environment and the high positive potential of CuT1 centers were recapitulated within the α-helical bundle of α3D.

Sculpting Metal-binding Environments in De Novo Designed Three-helix Bundles.

De novo protein design is a biologically relevant approach used to study the active centers of native metalloproteins. In this review, we will first discuss the design process in achieving α3D, a de novo designed three-helix bundle peptide with a well-defined fold. We will then cover our recent work in functionalizing the α3D framework by incorporating a tris(cysteine) and tris(histidine) motif. Our first design contains the thiol-rich sites found in metalloregulatory proteins that control the levels of toxic metal ions (Hg, Cd, and Pb). The latter design recapitulates the catalytic site and activity of a natural metalloenzyme carbonic anhydrase. The review will conclude with future design goals aimed at introducing an asymmetric metal-binding site in the α3D framework.

Redox Characterization of Electrode-Immobilized Bacterial Microcompartment Shell Proteins Engineered To Bind Metal Centers.

Bacterial microcompartment (BMC) shells are modular, selectively permeable, nanoscale protein shells that self-assemble from hexagonal and pentagonal building blocks in vivo or in vitro. Natural and engineered BMC shells colocalize and concentrate catalysts and metabolites in their lumen, increasing reaction kinetics. Here, we describe the design and characterization of a shell protein (pseudohexameric/trimeric BMC-T1HO protein) engineered to coordinate a Cu ion in its pore. Several designs, each varying the position of an introduced coordinating histidine residue, were shown to maintain their trimeric oligomerization state upon Cu coordination via chemical denaturation experiments. We measured reversible redox activity from electrode-bound Cu-3His BMC-T1HO variants, with formal potential(s) that were dependent on the Cu coordination site within the discoidal shaped trimer (E°' = +208 to +265 mV vs SHE). These results represent important steps toward expanding the functionality (Cu coordination) and applicability (redox activity on an electrode surface) of engineered BMC reactor architectures.

Engineering nanoreactors using bacterial microcompartment architectures.

Bacterial microcompartments (BMCs) are organelles that encapsulate enzymes involved in CO2 fixation or carbon catabolism in a selectively permeable protein shell. Here, we highlight recent advances in the bioengineering of these protein-based nanoreactors in heterologous systems, including transfer and expression of BMC gene clusters, the production of template empty shells, and the encapsulation of non-native enzymes.

De Novo Design of Metalloproteins and Metalloenzymes in a Three-Helix Bundle.

For more than two decades, de novo protein design has proven to be an effective methodology for modeling native proteins. De novo design involves the construction of metal-binding sites within simple and/or unrelated α-helical peptide structures. The preparation of α3D, a single polypeptide that folds into a native-like three-helix bundle structure, has significantly expanded available de novo designed scaffolds. Devoid of a metal-binding site (MBS), we incorporated a 3Cys and 3His motif in α3D to construct a heavy metal and a transition metal center, respectively. These efforts produced excellent functional models for native metalloproteins/metalloregulatory proteins and metalloenzymes. Morever, these α3D derivatives serve as a foundation for constructing redox active sites with either the same (e.g., 4Cys) or mixed (e.g., 2HisCys) ligands, a feat that could be achieved in this preassembled framework. Here, we describe the process of constructing MBSs in α3D and our expression techniques.

Differential inhibitor sensitivity of anaplastic lymphoma kinase variants found in neuroblastoma.

Activating mutations in the anaplastic lymphoma kinase (ALK) gene were recently discovered in neuroblastoma, a cancer of the developing autonomic nervous system that is the most commonly diagnosed malignancy in the first year of life. The most frequent ALK mutations in neuroblastoma cause amino acid substitutions (F1174L and R1275Q) in the intracellular tyrosine kinase domain of the intact ALK receptor. Identification of ALK as an oncogenic driver in neuroblastoma suggests that crizotinib (PF-02341066), a dual-specific inhibitor of the ALK and Met tyrosine kinases, will be useful in treating this malignancy. Here, we assessed the ability of crizotinib to inhibit proliferation of neuroblastoma cell lines and xenografts expressing mutated or wild-type ALK. Crizotinib inhibited proliferation of cell lines expressing either R1275Q-mutated ALK or amplified wild-type ALK. In contrast, cell lines harboring F1174L-mutated ALK were relatively resistant to crizotinib. Biochemical analyses revealed that this reduced susceptibility of F1174L-mutated ALK to crizotinib inhibition resulted from an increased adenosine triphosphate-binding affinity (as also seen in acquired resistance to epidermal growth factor receptor inhibitors). Thus, this effect should be surmountable with higher doses of crizotinib and/or with higher-affinity inhibitors.