The Experts below are selected from a list of 270 Experts worldwide ranked by ideXlab platform
Cheryl A Kerfeld - One of the best experts on this subject based on the ideXlab platform.
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redox characterization of electrode immobilized Bacterial Microcompartment shell proteins engineered to bind metal centers
ACS Applied Bio Materials, 2020Co-Authors: Jefferson S Plegaria, Matthew D Yates, Sarah M Glaven, Cheryl A KerfeldAbstract: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 co-localize 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 (Eo’ = +208 to +265 mV vs. SHE). These results represent important steps toward expanding the functionality (Cu coordination) and app...
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functionalization of Bacterial Microcompartment shell proteins with covalently attached heme
Frontiers in Bioengineering and Biotechnology, 2020Co-Authors: Jingcheng Huang, Cheryl A Kerfeld, Bryan Ferlez, Eric J Young, David M Kramer, Daniel C DucatAbstract:Author(s): Huang, Jingcheng; Ferlez, Bryan H; Young, Eric J; Kerfeld, Cheryl A; Kramer, David M; Ducat, Daniel C | Abstract: Heme is a versatile redox cofactor that has considerable potential for synthetic biology and bioelectronic applications. The capacity to functionalize non-heme-binding proteins with covalently bound heme moieties in vivo could expand the variety of bioelectronic materials, particularly if hemes could be attached at defined locations so as to facilitate position-sensitive processes like electron transfer. In this study, we utilized the cytochrome maturation system I to develop a simple approach that enables incorporation of hemes into the backbone of target proteins in vivo. We tested our methodology by targeting the self-assembling Bacterial Microcompartment shell proteins, and inserting functional hemes at multiple locations in the protein backbone. We found substitution of three amino acids on the target proteins promoted heme attachment with high occupancy. Spectroscopic measurements suggested these modified proteins covalently bind low-spin hemes, with relative low redox midpoint potentials (about -210 mV vs. SHE). Heme-modified shell proteins partially retained their self-assembly properties, including the capacity to hexamerize, and form inter-hexamer attachments. Heme-bound shell proteins demonstrated the capacity to integrate into higher-order shell assemblies, however, the structural features of these macromolecular complexes was sometimes altered. Altogether, we report a versatile strategy for generating electron-conductive cytochromes from structurally-defined proteins, and provide design considerations on how heme incorporation may interface with native assembly properties in engineered proteins.
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a designed Bacterial Microcompartment shell with tunable composition and precision cargo loading
Metabolic Engineering, 2019Co-Authors: Bryan Ferlez, Markus Sutter, Cheryl A KerfeldAbstract:Abstract Microbes often augment their metabolism by conditionally constructing proteinaceous organelles, known as Bacterial Microcompartments (BMCs), that encapsulate enzymes to degrade organic compounds or assimilate CO2. BMCs self-assemble and are spatially delimited by a semi-permeable shell made up of hexameric, trimeric, and pentameric shell proteins. Bioengineers aim to recapitulate the organization and efficiency of these complex biological architectures by redesigning the shell to incorporate non-native enzymes from biotechnologically relevant pathways. To meet this challenge, a diverse set of synthetic biology tools are required, including methods to manipulate the properties of the shell as well as target and organize cargo encapsulation. We designed and determined the crystal structure of a synthetic shell protein building block with an inverted sidedness of its N- and C-terminal residues relative to its natural counterpart; the inversion targets genetically fused protein cargo to the lumen of the shell. Moreover, the titer of fluorescent protein cargo encapsulated using this strategy is controllable using an inducible tetracycline promoter. These results expand the available set of building blocks for precision engineering of BMC-based nanoreactors and are compatible with orthogonal methods which will facilitate the installation and organization of multi-enzyme pathways.
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the plasticity of molecular interactions governs Bacterial Microcompartment shell assembly
Structure, 2019Co-Authors: Markus Sutter, Basil J Greber, Cheryl A KerfeldAbstract:Summary Bacterial Microcompartments (BMCs) are composed of an enzymatic core encapsulated by a selectively permeable protein shell that enhances catalytic efficiency. Many pathogenic bacteria derive competitive advantages from their BMC-based catabolism, implicating BMCs as drug targets. BMC shells are of interest for bioengineering due to their diverse and selective permeability properties and because they self-assemble. A complete understanding of shell composition and organization is a prerequisite for biotechnological applications. Here, we report the cryoelectron microscopy structure of a BMC shell at 3.0-A resolution, using an image-processing strategy that allowed us to determine the previously uncharacterized structural details of the interactions formed by the BMC-TS and BMC-TD shell subunits in the context of the assembled shell. We found unexpected structural plasticity among these interactions, resulting in distinct shell populations assembled from varying numbers of the BMC-TS and BMC-TD subunits. We discuss the implications of these findings on shell assembly and function.
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structural characterization of a synthetic tandem domain Bacterial Microcompartment shell protein capable of forming icosahedral shell assemblies
ACS Synthetic Biology, 2019Co-Authors: Markus Sutter, Cheryl A Kerfeld, Bryan Ferlez, Sean McguireAbstract:Bacterial Microcompartments are subcellular compartments found in many prokaryotes; they consist of a protein shell that encapsulates enzymes that perform a variety of functions. The shell protects the cell from potentially toxic intermediates and colocalizes enzymes for higher efficiency. Accordingly, it is of considerable interest for biotechnological applications. We have previously structurally characterized an intact 40 nm shell comprising three different types of proteins. One of those proteins, BMC-H, forms a cyclic hexamer; here we have engineered a synthetic protein that consists of a tandem duplication of BMC-H connected by a short linker. The synthetic protein forms cyclic trimers that self-assemble to form a smaller (25 nm) icosahedral shell with gaps at the pentamer positions. When coexpressed in vivo with the pentamer fused to an affinity tag we can purify complete icosahedral shells. This engineered shell protein constitutes a minimal shell system to study permeability; reducing symmetry fr...
Martin J Warren - One of the best experts on this subject based on the ideXlab platform.
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effect of metabolosome encapsulation peptides on enzyme activity coaggregation incorporation and Bacterial Microcompartment formation
MicrobiologyOpen, 2020Co-Authors: Rokas Juodeikis, Stefanie Frank, Michael B Prentice, Judith Mantell, Ian R Brown, Paul Verkade, Derek N Woolfson, Matthias Mayer, Martin J WarrenAbstract:Metabolosomes, catabolic Bacterial Microcompartments, are proteinaceous organelles that are associated with the breakdown of metabolites such as propanediol and ethanolamine. They are composed of an outer multi-component protein shell that encases a specific metabolic pathway. Protein cargo found within BMCs is directed by the presence of an encapsulation peptide that appears to trigger aggregation prior to the formation of the outer shell. We investigated the effect of three distinct encapsulation peptides on foreign cargo in a recombinant BMC system. Our data demonstrate that these peptides cause variation with respect to enzyme activity and protein aggregation. We observed that the level of protein aggregation generally correlates with the size of metabolosomes, while in the absence of cargo BMCs self-assemble into smaller compartments. The results agree with a flexible model for BMC formation based around the ability of the BMC shell to associate with an aggregate formed due to the interaction of encapsulation peptides.
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Bacterial Microcompartment mediated ethanolamine metabolism in escherichia coli urinary tract infection
Infection and Immunity, 2019Co-Authors: Katherine Dadswell, Martin J Warren, Mingzhi Liang, Ian R Brown, Sinead Creagh, Edward Mccullagh, Alan Mcnally, John Macsharry, Michael B PrenticeAbstract:ABSTRACT Urinary tract infections (UTIs) are common and in general are caused by intestinal uropathogenic Escherichia coli (UPEC) ascending via the urethra. Microcompartment-mediated catabolism of ethanolamine, a host cell breakdown product, fuels the competitive overgrowth of intestinal E. coli, both pathogenic enterohemorrhagic E. coli and commensal strains. During a UTI, urease-negative E. coli bacteria thrive, despite the comparative nutrient limitation in urine. The role of ethanolamine as a potential nutrient source during UTIs is understudied. We evaluated the role of the metabolism of ethanolamine as a potential nitrogen and carbon source for UPEC in the urinary tract. We analyzed infected urine samples by culture, high-performance liquid chromatography, reverse transcription-quantitative PCR, and genomic sequencing. The ethanolamine concentration in urine was comparable to the concentration of the most abundant reported urinary amino acid, d-serine. Transcription of the eut operon was detected in the majority of urine samples containing E. coli screened. All sequenced UPEC strains had conserved eut operons, while metabolic genotypes previously associated with UTI (dsdCXA, metE) were mainly limited to phylogroup B2. In vitro ethanolamine was found to be utilized as a sole source of nitrogen by UPEC strains. The metabolism of ethanolamine in artificial urine medium (AUM) induced metabolosome formation and provided a growth advantage at the physiological levels found in urine. Interestingly, eutE (which encodes acetaldehyde dehydrogenase) was required for UPEC strains to utilize ethanolamine to gain a growth advantage in AUM, suggesting that ethanolamine is also utilized as a carbon source. These data suggest that urinary ethanolamine is a significant additional carbon and nitrogen source for infecting E. coli strains.
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biotechnological advances in Bacterial Microcompartment technology
Trends in Biotechnology, 2019Co-Authors: David Palmer, Martin J WarrenAbstract:Bacterial Microcompartments (BMCs) represent proteinaceous macromolecular nanobioreactors that are found in a broad range of bacteria, and which are associated with either anabolic or catabolic processes. They consist of a semipermeable outer shell that packages a central metabolic enzyme or pathway, providing both enhanced flux and protection against toxic intermediates. Recombinant production of BMCs has led to their repurposing with the incorporation of altogether new pathways. Deconstructing BMCs into their component parts has shown that some individual shell proteins self-associate into filaments that can be further modified into a cytoplasmic scaffold, or cytoscaffold, to which enzymes/proteins can be targeted. BMCs therefore represent a modular system that is highly suited for engineering biological systems for useful purposes.
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A Generic Self-Assembly Process in Microcompartments and Synthetic Protein Nanotubes.
Small, 2018Co-Authors: Ismail Uddin, Stefanie Frank, Martin J Warren, Richard W PickersgillAbstract:: Bacterial Microcompartments enclose a biochemical pathway and reactive intermediate within a protein envelope formed by the shell proteins. Herein, the orientation of the propanediol-utilization (Pdu) Microcompartment shell protein PduA in Bacterial Microcompartments and in synthetic nanotubes, and the orientation of PduB in synthetic nanotubes are revealed. When produced individually, PduA hexamers and PduB trimers, tessellate to form flat sheets in the crystal, or they can self-assemble to form synthetic protein nanotubes in solution. Modelling the orientation of PduA in the 20 nm nanotube so as to preserve the shape complementarity and key interactions seen in the crystal structure suggests that the concave surface of the PduA hexamer faces out. This orientation is confirmed experimentally in synthetic nanotubes and in the Bacterial Microcompartment produced in vivo. The PduB nanotubes described here have a larger diameter, 63 nm, with the concave surface of the trimer again facing out. The conserved concave surface out characteristic of these nano-structures reveals a generic assembly process that causes the interface between adjacent subunits to bend in a common direction that optimizes shape complementarity and minimizes steric clashes. This understanding underpins engineering strategies for the biotechnological application of protein nanotubes.
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Engineered synthetic scaffolds for organizing proteins within the Bacterial cytoplasm
Nature Chemical Biology, 2018Co-Authors: Judith Mantell, Stefanie Frank, Lorna Hodgson, Dominic Alibhai, Jordan M Fletcher, Ian R Brown, Paul Verkade, Derek N Woolfson, Martin J WarrenAbstract:We have developed a system for producing a supramolecular scaffold that permeates the entire Escherichia coli cytoplasm. This cytoscaffold is constructed from a three-component system comprising a Bacterial Microcompartment shell protein and two complementary de novo coiled-coil peptides. We show that other proteins can be targeted to this intracellular filamentous arrangement. Specifically, the enzymes pyruvate decarboxylase and alcohol dehydrogenase have been directed to the filaments, leading to enhanced ethanol production in these engineered Bacterial cells compared to those that do not produce the scaffold. This is consistent with improved metabolic efficiency through enzyme colocation. Finally, the shell-protein scaffold can be directed to the inner membrane of the cell, demonstrating how synthetic cellular organization can be coupled with spatial optimization through in-cell protein design. The cytoscaffold has potential in the development of next-generation cell factories, wherein it could be used to organize enzyme pathways and metabolite transporters to enhance metabolic flux. Two complementary coiled-coil peptides and a Bacterial Microcompartment shell protein are combined to construct cytoscaffolds within Escherichia coli cells. Targeting enzymes to the cytoplasmic scaffold results in colocalization and improved metabolic flux.
Todd O Yeates - One of the best experts on this subject based on the ideXlab platform.
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molecular dynamics simulations of selective metabolite transport across the propanediol Bacterial Microcompartment shell
Journal of Physical Chemistry B, 2017Co-Authors: Jiyong Park, Thomas A Bobik, Sunny Chun, K N Houk, Todd O YeatesAbstract:Bacterial Microcompartments are giant protein-based organelles that encapsulate special metabolic pathways in diverse bacteria. Structural and genetic studies indicate that metabolic substrates enter these Microcompartments by passing through the central pores in hexameric assemblies of shell proteins. Limiting the escape of toxic metabolic intermediates created inside the Microcompartments would confer a selective advantage for the host organism. Here, we report the first molecular dynamics (MD) simulation studies to analyze small-molecule transport across a Microcompartment shell. PduA is a major shell protein in a Bacterial Microcompartment that metabolizes 1,2-propanediol via a toxic aldehyde intermediate, propionaldehyde. Using both metadynamics and replica-exchange umbrella sampling, we find that the pore of the PduA hexamer has a lower energy barrier for passage of the propanediol substrate compared to the toxic propionaldehyde generated within the Microcompartment. The energetic effect is consiste...
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structure of a novel 13 nm dodecahedral nanocage assembled from a redesigned Bacterial Microcompartment shell protein
Chemical Communications, 2016Co-Authors: Julien Jorda, M C Thompson, D J Leibly, Todd O YeatesAbstract:We report the crystal structure of a novel 60-subunit dodecahedral cage that results from self-assembly of a re-engineered version of a natural protein (PduA) from the Pdu Microcompartment shell. Biophysical data illustrate the dependence of assembly on solution conditions, opening up new applications in Microcompartment studies and nanotechnology.
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selective molecular transport through the protein shell of a Bacterial Microcompartment organelle
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Chiranjit Chowdhury, A H Pang, Todd O Yeates, Sharmistha Sinha, Sunny Chun, Michael R. Sawaya, Thomas A BobikAbstract:Bacterial Microcompartments are widespread prokaryotic organelles that have important and diverse roles ranging from carbon fixation to enteric pathogenesis. Current models for Microcompartment function propose that their outer protein shell is selectively permeable to small molecules, but whether a protein shell can mediate selective permeability and how this occurs are unresolved questions. Here, biochemical and physiological studies of structure-guided mutants are used to show that the hexameric PduA shell protein of the 1,2-propanediol utilization (Pdu) Microcompartment forms a selectively permeable pore tailored for the influx of 1,2-propanediol (the substrate of the Pdu Microcompartment) while restricting the efflux of propionaldehyde, a toxic intermediate of 1,2-propanediol catabolism. Crystal structures of various PduA mutants provide a foundation for interpreting the observed biochemical and phenotypic data in terms of molecular diffusion across the shell. Overall, these studies provide a basis for understanding a class of selectively permeable channels formed by nonmembrane proteins.
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exploring Bacterial organelle interactomes a model of the protein protein interaction network in the pdu Microcompartment
PLOS Computational Biology, 2015Co-Authors: Julien Jorda, Thomas A Bobik, Todd O YeatesAbstract:Bacterial Microcompartments (MCPs) are protein-bound organelles that carry out diverse metabolic pathways in a wide range of bacteria. These supramolecular assemblies consist of a thin outer protein shell, reminiscent of a viral capsid, which encapsulates sequentially acting enzymes. The most complex MCP elucidated so far is the propanediol utilizing (Pdu) Microcompartment. It contains the reactions for degrading 1,2-propanediol. While several experimental studies on the Pdu system have provided hints about its organization, a clear picture of how all the individual components interact has not emerged yet. Here we use co-evolution-based methods, involving pairwise comparisons of protein phylogenetic trees, to predict the protein-protein interaction (PPI) network governing the assembly of the Pdu MCP. We propose a model of the Pdu interactome, from which selected PPIs are further inspected via computational docking simulations. We find that shell protein PduA is able to serve as a “universal hub” for targeting an array of enzymes presenting special N-terminal extensions, namely PduC, D, E, L and P. The varied N-terminal peptides are predicted to bind in the same cleft on the presumptive luminal face of the PduA hexamer. We also propose that PduV, a protein of unknown function with remote homology to the Ras-like GTPase superfamily, is likely to localize outside the MCP, interacting with the protruding β-barrel of the hexameric PduU shell protein. Preliminary experiments involving a Bacterial two-hybrid assay are presented that corroborate the existence of a PduU-PduV interaction. This first systematic computational study aimed at characterizing the interactome of a Bacterial Microcompartment provides fresh insight into the organization of the Pdu MCP.
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structure of a Bacterial Microcompartment shell protein bound to a cobalamin cofactor
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2014Co-Authors: M C Thompson, Thomas A Bobik, Christopher S Crowley, J S Kopstein, Todd O YeatesAbstract:The EutL shell protein is a key component of the ethanolamine-utilization Microcompartment, which serves to compartmentalize ethanolamine degradation in diverse bacteria. The apparent function of this shell protein is to facilitate the selective diffusion of large cofactor molecules between the cytoplasm and the lumen of the Microcompartment. While EutL is implicated in molecular-transport phenomena, the details of its function, including the identity of its transport substrate, remain unknown. Here, the 2.1 A resolution X-ray crystal structure of a EutL shell protein bound to cobalamin (vitamin B12) is presented and the potential relevance of the observed protein–ligand interaction is briefly discussed. This work represents the first structure of a Bacterial Microcompartment shell protein bound to a potentially relevant cofactor molecule.
Richard W Pickersgill - One of the best experts on this subject based on the ideXlab platform.
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A Generic Self-Assembly Process in Microcompartments and Synthetic Protein Nanotubes.
Small, 2018Co-Authors: Ismail Uddin, Stefanie Frank, Martin J Warren, Richard W PickersgillAbstract:: Bacterial Microcompartments enclose a biochemical pathway and reactive intermediate within a protein envelope formed by the shell proteins. Herein, the orientation of the propanediol-utilization (Pdu) Microcompartment shell protein PduA in Bacterial Microcompartments and in synthetic nanotubes, and the orientation of PduB in synthetic nanotubes are revealed. When produced individually, PduA hexamers and PduB trimers, tessellate to form flat sheets in the crystal, or they can self-assemble to form synthetic protein nanotubes in solution. Modelling the orientation of PduA in the 20 nm nanotube so as to preserve the shape complementarity and key interactions seen in the crystal structure suggests that the concave surface of the PduA hexamer faces out. This orientation is confirmed experimentally in synthetic nanotubes and in the Bacterial Microcompartment produced in vivo. The PduB nanotubes described here have a larger diameter, 63 nm, with the concave surface of the trimer again facing out. The conserved concave surface out characteristic of these nano-structures reveals a generic assembly process that causes the interface between adjacent subunits to bend in a common direction that optimizes shape complementarity and minimizes steric clashes. This understanding underpins engineering strategies for the biotechnological application of protein nanotubes.
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structural insights into higher order assembly and function of the Bacterial Microcompartment protein pdua
Journal of Biological Chemistry, 2014Co-Authors: A H Pang, Martin J Warren, Ian R Brown, Stephanie Frank, Richard W PickersgillAbstract:Bacterial Microcompartments are large proteinaceous assemblies that are found in the cytoplasm of some bacteria. These structures consist of proteins constituting a shell that houses a number of enzymes involved in specific metabolic processes. The 1,2-propanediol-utilizing Microcompartment is assembled from seven different types of shell proteins, one of which is PduA. It is one of the more abundant components of the shell and intriguingly can form nanotubule-like structures when expressed on its own in the cytoplasm of Escherichia coli. We propose a model that accounts for the size and appearance of these PduA structures and underpin our model using a combinatorial approach. Making strategic mutations at Lys-26, Val-51, and Arg-79, we targeted residues predicted to be important for PduA assembly. We present the effect of the amino acid residue substitution on the phenotype of the PduA higher order assemblies (transmission electron microscopy) and the crystal structure of the K26D mutant with one glycerol molecule bound to the central pore. Our results support the view that the hexamer-hexamer interactions seen in PduA crystals persist in the cytoplasmic structures and reveal the profound influence of the two key amino acids, Lys-26 and Arg-79, on tiling, not only in the crystal lattice but also in the Bacterial cytoplasm. Understanding and controlling PduA assemblies is valuable in order to inform manipulation for synthetic biology and biotechnological applications.
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substrate channels revealed in the trimeric lactobacillus reuteri Bacterial Microcompartment shell protein pdub
Acta Crystallographica Section D-biological Crystallography, 2012Co-Authors: A H Pang, Mingzhi Liang, Michael B Prentice, Richard W PickersgillAbstract:Lactobacillus reuteri metabolizes two similar three-carbon molecules, 1,2-propanediol and glycerol, within closed polyhedral subcellular Bacterial organelles called Bacterial Microcompartments (metabolosomes). The outer shell of the propanediol-utilization (Pdu) metabolosome is composed of hundreds of mainly hexagonal protein complexes made from six types of protein subunits that share similar domain structures. The structure of the Bacterial Microcompartment protein PduB has a tandem structural repeat within the subunit and assembles into a trimer with pseudo-hexagonal symmetry. This trimeric structure forms sheets in the crystal lattice and is able to fit within a polymeric sheet of the major shell component PduA to assemble a facet of the polyhedron. There are three pores within the trimer and these are formed between the tandem repeats within the subunits. The structure shows that each of these pores contains three glycerol molecules that interact with conserved residues, strongly suggesting that these subunit pores channel glycerol substrate into the metabolosome. In addition to the observation of glycerol occupying the subunit channels, the presence of glycerol on the molecular threefold symmetry axis suggests a role in locking closed the central region.
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Structure of PduT, a trimeric Bacterial Microcompartment protein with a 4Fe–4S cluster-binding site
Acta Crystallographica Section D-biological Crystallography, 2011Co-Authors: A H Pang, Martin J Warren, Richard W PickersgillAbstract:: Propanediol metabolism in Citrobacter freundii occurs within a metabolosome, a subcellular proteinaceous Bacterial Microcompartment. The propanediol-utilization (Pdu) Microcompartment shell is constructed from thousands of hexagonal-shaped protein complexes made from seven different types of protein subunit. Here, the structure of the Bacterial Microcompartment protein PduT, which has a tandem structural repeat within the subunit and forms trimers with pseudo-hexagonal symmetry, is reported. This trimeric assembly forms a flat approximately hexagonally shaped disc with a central pore that is suitable for a 4Fe-4S cluster. The essentially cubic shaped 4Fe-4S cluster conforms to the threefold symmetry of the trimer with one free iron, the role of which could be to supply electrons to an associated Microcompartment enzyme, PduS.
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structure of pdut a trimeric Bacterial Microcompartment protein with a 4fe 4s cluster binding site
Acta Crystallographica Section D-biological Crystallography, 2010Co-Authors: A H Pang, Martin J Warren, Richard W PickersgillAbstract:: Propanediol metabolism in Citrobacter freundii occurs within a metabolosome, a subcellular proteinaceous Bacterial Microcompartment. The propanediol-utilization (Pdu) Microcompartment shell is constructed from thousands of hexagonal-shaped protein complexes made from seven different types of protein subunit. Here, the structure of the Bacterial Microcompartment protein PduT, which has a tandem structural repeat within the subunit and forms trimers with pseudo-hexagonal symmetry, is reported. This trimeric assembly forms a flat approximately hexagonally shaped disc with a central pore that is suitable for a 4Fe-4S cluster. The essentially cubic shaped 4Fe-4S cluster conforms to the threefold symmetry of the trimer with one free iron, the role of which could be to supply electrons to an associated Microcompartment enzyme, PduS.
Markus Sutter - One of the best experts on this subject based on the ideXlab platform.
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a designed Bacterial Microcompartment shell with tunable composition and precision cargo loading
Metabolic Engineering, 2019Co-Authors: Bryan Ferlez, Markus Sutter, Cheryl A KerfeldAbstract:Abstract Microbes often augment their metabolism by conditionally constructing proteinaceous organelles, known as Bacterial Microcompartments (BMCs), that encapsulate enzymes to degrade organic compounds or assimilate CO2. BMCs self-assemble and are spatially delimited by a semi-permeable shell made up of hexameric, trimeric, and pentameric shell proteins. Bioengineers aim to recapitulate the organization and efficiency of these complex biological architectures by redesigning the shell to incorporate non-native enzymes from biotechnologically relevant pathways. To meet this challenge, a diverse set of synthetic biology tools are required, including methods to manipulate the properties of the shell as well as target and organize cargo encapsulation. We designed and determined the crystal structure of a synthetic shell protein building block with an inverted sidedness of its N- and C-terminal residues relative to its natural counterpart; the inversion targets genetically fused protein cargo to the lumen of the shell. Moreover, the titer of fluorescent protein cargo encapsulated using this strategy is controllable using an inducible tetracycline promoter. These results expand the available set of building blocks for precision engineering of BMC-based nanoreactors and are compatible with orthogonal methods which will facilitate the installation and organization of multi-enzyme pathways.
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the plasticity of molecular interactions governs Bacterial Microcompartment shell assembly
Structure, 2019Co-Authors: Markus Sutter, Basil J Greber, Cheryl A KerfeldAbstract:Summary Bacterial Microcompartments (BMCs) are composed of an enzymatic core encapsulated by a selectively permeable protein shell that enhances catalytic efficiency. Many pathogenic bacteria derive competitive advantages from their BMC-based catabolism, implicating BMCs as drug targets. BMC shells are of interest for bioengineering due to their diverse and selective permeability properties and because they self-assemble. A complete understanding of shell composition and organization is a prerequisite for biotechnological applications. Here, we report the cryoelectron microscopy structure of a BMC shell at 3.0-A resolution, using an image-processing strategy that allowed us to determine the previously uncharacterized structural details of the interactions formed by the BMC-TS and BMC-TD shell subunits in the context of the assembled shell. We found unexpected structural plasticity among these interactions, resulting in distinct shell populations assembled from varying numbers of the BMC-TS and BMC-TD subunits. We discuss the implications of these findings on shell assembly and function.
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structural characterization of a synthetic tandem domain Bacterial Microcompartment shell protein capable of forming icosahedral shell assemblies
ACS Synthetic Biology, 2019Co-Authors: Markus Sutter, Cheryl A Kerfeld, Bryan Ferlez, Sean McguireAbstract:Bacterial Microcompartments are subcellular compartments found in many prokaryotes; they consist of a protein shell that encapsulates enzymes that perform a variety of functions. The shell protects the cell from potentially toxic intermediates and colocalizes enzymes for higher efficiency. Accordingly, it is of considerable interest for biotechnological applications. We have previously structurally characterized an intact 40 nm shell comprising three different types of proteins. One of those proteins, BMC-H, forms a cyclic hexamer; here we have engineered a synthetic protein that consists of a tandem duplication of BMC-H connected by a short linker. The synthetic protein forms cyclic trimers that self-assemble to form a smaller (25 nm) icosahedral shell with gaps at the pentamer positions. When coexpressed in vivo with the pentamer fused to an affinity tag we can purify complete icosahedral shells. This engineered shell protein constitutes a minimal shell system to study permeability; reducing symmetry fr...
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Programmed loading and rapid purification of engineered Bacterial Microcompartment shells
Nature Communications, 2018Co-Authors: Andrew Hagen, Markus Sutter, Nancy Sloan, Cheryl A KerfeldAbstract:Bacterial Microcompartments (BMCs) are selectively permeable proteinaceous organelles which encapsulate segments of metabolic pathways across Bacterial phyla. They consist of an enzymatic core surrounded by a protein shell composed of multiple distinct proteins. Despite great potential in varied biotechnological applications, engineering efforts have been stymied by difficulties in their isolation and characterization and a dearth of robust methods for programming cores and shell permeability. We address these challenges by functionalizing shell proteins with affinity handles, enabling facile complementation-based affinity purification (CAP) and specific cargo docking sites for efficient encapsulation via covalent-linkage (EnCo). These shell functionalizations extend our knowledge of BMC architectural principles and enable the development of minimal shell systems of precisely defined structure and composition. The generalizability of CAP and EnCo will enable their application to functionally diverse Microcompartment systems to facilitate both characterization of natural functions and the development of bespoke shells for selectively compartmentalizing proteins. Bacterial Microcompartments are protein-bound organelles encapsulating segments of metabolic pathways. Here the authors functionalise shell proteins to facilitate facile purification and enable cargo encapsulation via covalent linkage.
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Bacterial Microcompartments
Nature Reviews Microbiology, 2018Co-Authors: Cheryl A Kerfeld, Clement Aussignargues, Jan Zarzycki, Markus SutterAbstract:Bacterial Microcompartments are self-assembling organelles that consist of an enzymatic core that is encapsulated by a selectively permeable protein shell. In this Review, Kerfeld and colleagues discuss recent insights into the structure, assembly, diversity and function of Bacterial Microcompartments. Bacterial Microcompartments are functional analogues of the lipid-bound organelles of eukaryotes. They enclose chemical reactions that benefit from being separated from the cytosol. The delimiting membrane of Bacterial Microcompartments consists entirely of protein, and its components are highly conserved in sequence and structure. Bacterial Microcompartments are found in a wide variety of Bacterial species (at least 19 established phyla). They are easily identified in genomes by their tendency to colocalize the associated genes into a large gene cluster called a superlocus. Carboxysomes (CO_2-fixing organelles) were the first type of Bacterial Microcompartment to be identified, but recently, many more have been discovered and characterized; they are involved in catabolizing a variety of nutrients and enable cells to grow in otherwise unavailable niches. The shell and cargo of Bacterial Microcompartments self-assemble using different pathways; some build the shell around a cargo aggregate, whereas others assemble the shell and cargo concomitantly. There are proteins that facilitate cargo aggregation and small encapsulation peptides that specifically associate proteins to the lumen of the shell. Bacterial Microcompartments are linked to the pathogenesis of certain bacteria because they confer a growth advantage. For example, the human gut is enriched in propanediol and ethanolamine, initial substrates of specific Bacterial Microcompartments. The knowledge gained from understanding the native functions has led to substantial progress in modifying the shell for bioengineering purposes. Bacterial Microcompartment shells can be produced recombinantly, and shell proteins and cores have been engineered to adopt new functions. Bacterial Microcompartments (BMCs) are self-assembling organelles that consist of an enzymatic core that is encapsulated by a selectively permeable protein shell. The potential to form BMCs is widespread and found across the kingdom Bacteria. BMCs have crucial roles in carbon dioxide fixation in autotrophs and the catabolism of organic substrates in heterotrophs. They contribute to the metabolic versatility of bacteria, providing a competitive advantage in specific environmental niches. Although BMCs were first visualized more than 60 years ago, it is mainly in the past decade that progress has been made in understanding their metabolic diversity and the structural basis of their assembly and function. This progress has not only heightened our understanding of their role in microbial metabolism but is also beginning to enable their use in a variety of applications in synthetic biology. In this Review, we focus on recent insights into the structure, assembly, diversity and function of BMCs.
