Comparative analysis of carboxysome shell proteins

Carboxysomes are metabolic modules for CO2 fixation that are found in all cyanobacteria and some chemoautotrophic bacteria. They comprise a semi-permeable proteinaceous shell that encapsulates ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase. Structural studies are revealing the integral role of the shell protein paralogs to carboxysome form and function. The shell proteins are composed of two domain classes: those with the bacterial microcompartment (BMC; Pfam00936) domain, which oligomerize to form (pseudo)hexamers, and those with the CcmL/EutN (Pfam03319) domain which form pentamers in carboxysomes. These two shell protein types are proposed to be the basis for the carboxysome’s icosahedral geometry. The shell proteins are also thought to allow the flux of metabolites across the shell through the presence of the small pore formed by their hexameric/pentameric symmetry axes. In this review, we describe bioinformatic and structural analyses that highlight the important primary, tertiary, and quaternary structural features of these conserved shell subunits. In the future, further understanding of these molecular building blocks may provide the basis for enhancing CO2 fixation in other organisms or creating novel biological nanostructures

Effect of bio-engineering on size, shape, composition and rigidity of bacterial microcompartments

Bacterial microcompartments (BMCs) are proteinaceous organelles that are found in a broad range of bacteria and are composed of an outer shell that encases an enzyme cargo representing a specific metabolic process. The outer shell is made from a number of different proteins that form hexameric and pentameric tiles, which interact to allow the formation of a polyhedral edifice. We have previously shown that the Citrobacter freundii BMC associated with 1,2-propanediol utilization can be transferred into Escherichia coli to generate a recombinant BMC and that empty BMCs can be formed from just the shell proteins alone. Herein, a detailed structural and proteomic characterization of the wild type BMC is compared to the recombinant BMC and a number of empty BMC variants by 2D-gel electrophoresis, mass spectrometry, transmission electron microscopy (TEM) and atomic force microscopy (AFM). Specifically, it is shown that the wild type BMC and the recombinant BMC are similar in terms of composition, size, shape and mechanical properties, whereas the empty BMC variants are shown to be smaller, hollow and less malleable

Giant capsids from lattice self-assembly of cyclodextrin complexes

Proteins can readily assemble into rigid, crystalline and functional structures such as viral capsids and bacterial compartments. Despite ongoing advances, it is still a fundamental challenge to design and synthesize protein-mimetic molecules to form crystalline structures. Here we report the lattice self-assembly of cyclodextrin complexes into a variety of capsidlike structures such as lamellae, helical tubes and hollow rhombic dodecahedra. The dodecahedral morphology has not hitherto been observed in self-assembly systems. The tubes can spontaneously encapsulate colloidal particles and liposomes. The dodecahedra and tubes are respectively comparable to and much larger than the largest known virus. In particular, the resemblance to protein assemblies is not limited to morphology but extends to structural rigidity and crystallinity-a well-defined, 2D rhombic lattice of molecular arrangement is strikingly universal for all the observed structures. We propose a simple design rule for the current lattice self-assembly, potentially opening doors for new protein-mimetic materials

The Pentameric Vertex Proteins Are Necessary for the Icosahedral Carboxysome Shell to Function as a CO2 Leakage Barrier

BACKGROUND: Carboxysomes are polyhedral protein microcompartments found in many autotrophic bacteria; they encapsulate the CO(2) fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) within a thin protein shell and provide an environment that enhances the catalytic capabilities of the enzyme. Two types of shell protein constituents are common to carboxysomes and related microcompartments of heterotrophic bacteria, and the genes for these proteins are found in a large variety of bacteria. METHODOLOGY/PRINCIPAL FINDINGS: We have created a Halothiobacillus neapolitanus knockout mutant that does not produce the two paralogous CsoS4 proteins thought to occupy the vertices of the icosahedral carboxysomes and related microcompartments. Biochemical and ultrastructural analyses indicated that the mutant predominantly forms carboxysomes of normal appearance, in addition to some elongated microcompartments. Despite their normal shape, purified mutant carboxysomes are functionally impaired, although the activities of the encapsulated enzymes are not negatively affected. CONCLUSIONS/SIGNIFICANCE: In the absence of the CsoS4 proteins the carboxysome shell loses its limited permeability to CO(2) and is no longer able to provide the catalytic advantage RubisCO derives from microcompartmentalization. This study presents direct evidence that the diffusion barrier property of the carboxysome shell contributes significantly to the biological function of the carboxysome

Engineered Protein Nano-Compartments for Targeted Enzyme Localization

Compartmentalized co-localization of enzymes and their substrates represents an attractive approach for multi-enzymatic synthesis in engineered cells and biocatalysis. Sequestration of enzymes and substrates would greatly increase reaction efficiency while also protecting engineered host cells from potentially toxic reaction intermediates. Several bacteria form protein-based polyhedral microcompartments which sequester functionally related enzymes and regulate their access to substrates and other small metabolites. Such bacterial microcompartments may be engineered into protein-based nano-bioreactors, provided that they can be assembled in a non-native host cell, and that heterologous enzymes and substrates can be targeted into the engineered compartments. Here, we report that recombinant expression of Salmonella enterica ethanolamine utilization (eut) bacterial microcompartment shell proteins in E. coli results in the formation of polyhedral protein shells. Purified recombinant shells are morphologically similar to the native Eut microcompartments purified from S. enterica. Surprisingly, recombinant expression of only one of the shell proteins (EutS) is sufficient and necessary for creating properly delimited compartments. Co-expression with EutS also facilitates the encapsulation of EGFP fused with a putative Eut shell-targeting signal sequence. We also demonstrate the functional localization of a heterologous enzyme (β-galactosidase) targeted to the recombinant shells. Together our results provide proof-of-concept for the engineering of protein nano-compartments for biosynthesis and biocatalysis

Varieties of living things: Life at the intersection of lineage and metabolism

publication-status: Publishedtypes: Articl

Comparative analysis of 126 cyanobacterial genomes reveals evidence of functional diversity among homologs of the redox-regulated CP12 protein

CP12 is found almost universally among photosynthetic organisms, where it plays a key role in regulation of the Calvin cycle by forming a ternary complex with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase. Newly available genomic sequence data for the phylum Cyanobacteria reveals a heretofore unobserved diversity in cyanobacterial CP12 proteins. Cyanobacterial CP12 proteins can be classified into eight different types based on primary structure features. Among these are CP12-CBS (for cystathionine-β-synthase) domain fusions. CBS domains are regulatory modules for a wide range of cellular activities; many of these bind adenine nucleotides through a conserved motif that is also present in the CBS domains fused to CP12. In addition, a survey of expression data sets shows that the CP12 paralogs are differentially regulated. Furthermore, modeling of the cyanobacterial CP12 protein variants based on the recently available three-dimensional structure of the canonical cyanobacterial CP12 in complex with GAPDH suggests that some of the newly identified cyanobacterial CP12 types are unlikely to bind to GAPDH. Collectively these data show that, as is becoming increasingly apparent for plant CP12 proteins, the role of CP12 in cyanobacteria is likely more complex than previously appreciated, possibly involving other signals in addition to light.Moreover, our findings substantiate the proposal that this small protein may have multiple roles in photosynthetic organisms