J. Plegaria | Michigan State University (original) (raw)
Papers by J. Plegaria
Bacterial microcompartments (BMCs) are organelles that encapsulate enzymes involved in CO 2 fixat... more Bacterial microcompartments (BMCs) are organelles that encapsulate enzymes involved in CO 2 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.
Activating mutations in the anaplastic lymphoma kinase (ALK) gene were recently discovered in neu... more 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.
De novo protein design is a biologically relevant approach used to study the active centers of na... more 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 a 3 D, a de novo designed three-helix bundle peptide with a well-defined fold. We will then cover our recent work in functionalizing the a 3 D 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 a 3 D framework.
Keywords: De novo design Cupredoxin Three-helix bundle Photoinduced electron transfer Laser-flash... more Keywords: De novo design Cupredoxin Three-helix bundle Photoinduced electron transfer Laser-flash photolysis In this work, we characterized the intermolecular electron transfer (ET) properties of a de novo designed metallopeptide using laser-flash photolysis. α 3 D-CH3 is three helix bundle peptide that was designed to contain a copper ET site that is found in the β-barrel fold of native cupredoxins. The ET activity of Cuα 3 D–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)α 3 D-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 α 3 D requires further structural optimization to efficiently perform ET.
![Research paper thumbnail of Structure and Function of a Bacterial Microcompartment Shell Protein Engineered to Bind a [4Fe-4S] Cluster](https://attachments.academia-assets.com/44144144/thumbnails/1.jpg)
](Bacterial microcompartments (BMCs) are self-assembling organelles composed of a selectively perme... more 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.
De novo protein design is a biologically relevant approach that provides a novel process in eluci... more 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 α 3 DIV 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 χ 1). 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 α 3 D, the substitution of Leu residues to Cys enhanced the α-helical content of α 3 DIV 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 α 3 DIV by 18−24 °C compared to its apo counterpart. Further, the extended X-ray absorption fine structure analysis on Hg(II)-α 3 DIV produced an average Hg(II)−S bond length at 2.36 Å, indicating a trigonal T-shaped coordination environment. Overall, the structure of apo α 3 DIV 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.
Using de novo protein design, we incorporated a copper metal binding site within the three-helix ... more Using de novo protein design, we incorporated a copper metal binding site within the three-helix bundle α 3 D (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 β-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 (ε 600 nm = 5000 M −1 cm −1) of such centers. We incorporated 2HisCys(Met) residues in three distinct α 3 D 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 α 3 D.
Science Translational Medicine, 2011
Activating mutations in the anaplastic lymphoma kinase (ALK) gene were recently discovered in neu... more 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 9 Correspondence should be addressed to Y.P.M. or M.A.L. at: Yaël P. Mossé, Division
Bacterial microcompartments (BMCs) are organelles that encapsulate enzymes involved in CO 2 fixat... more Bacterial microcompartments (BMCs) are organelles that encapsulate enzymes involved in CO 2 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.
Activating mutations in the anaplastic lymphoma kinase (ALK) gene were recently discovered in neu... more 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.
De novo protein design is a biologically relevant approach used to study the active centers of na... more 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 a 3 D, a de novo designed three-helix bundle peptide with a well-defined fold. We will then cover our recent work in functionalizing the a 3 D 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 a 3 D framework.
Keywords: De novo design Cupredoxin Three-helix bundle Photoinduced electron transfer Laser-flash... more Keywords: De novo design Cupredoxin Three-helix bundle Photoinduced electron transfer Laser-flash photolysis In this work, we characterized the intermolecular electron transfer (ET) properties of a de novo designed metallopeptide using laser-flash photolysis. α 3 D-CH3 is three helix bundle peptide that was designed to contain a copper ET site that is found in the β-barrel fold of native cupredoxins. The ET activity of Cuα 3 D–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)α 3 D-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 α 3 D requires further structural optimization to efficiently perform ET.
Bacterial microcompartments (BMCs) are self-assembling organelles composed of a selectively perme... more 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.
De novo protein design is a biologically relevant approach that provides a novel process in eluci... more 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 α 3 DIV 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 χ 1). 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 α 3 D, the substitution of Leu residues to Cys enhanced the α-helical content of α 3 DIV 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 α 3 DIV by 18−24 °C compared to its apo counterpart. Further, the extended X-ray absorption fine structure analysis on Hg(II)-α 3 DIV produced an average Hg(II)−S bond length at 2.36 Å, indicating a trigonal T-shaped coordination environment. Overall, the structure of apo α 3 DIV 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.
Using de novo protein design, we incorporated a copper metal binding site within the three-helix ... more Using de novo protein design, we incorporated a copper metal binding site within the three-helix bundle α 3 D (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 β-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 (ε 600 nm = 5000 M −1 cm −1) of such centers. We incorporated 2HisCys(Met) residues in three distinct α 3 D 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 α 3 D.
Science Translational Medicine, 2011
Activating mutations in the anaplastic lymphoma kinase (ALK) gene were recently discovered in neu... more 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 9 Correspondence should be addressed to Y.P.M. or M.A.L. at: Yaël P. Mossé, Division