Natural proteins can be versatile building blocks for multimeric, self-assembling structures. Yet, creating protein-based assemblies with specific geometries and chemical properties remains challenging. Highly porous materials represent particularly interesting targets for designed assembly. Here we utilize a strategy of fusing two natural protein oligomers using a continuous alpha-helical linker to design a novel protein that self assembles into a 750 kDa, 225 Å diameter, cube-shaped cage with large openings into a 130 Å diameter inner cavity. A crystal structure of the cage showed atomic level agreement with the designed model, while electron microscopy, native mass spectrometry, and small angle x-ray scattering revealed alternate assembly forms in solution. These studies show that accurate design of large porous assemblies with specific shapes is feasible, while further specificity improvements will likely require limiting flexibility to select against alternative forms. These results provide a foundation for the design of advanced materials with applications in bionanotechnology, nanomedicine and material sciences.
Porous protein nanoparticles; protein design; self-assembly; symmetry; synthetic biology and bioengineering
Details are emerging on the structure and function of a remarkable class of capsid-like protein assemblies that serve as simple metabolic organelles in many bacteria. These bacterial microcompartments consist of a few thousand shell proteins, which encapsulate two or more sequentially acting enzymes in order to enhance or sequester certain metabolic pathways, particularly those involving toxic or volatile intermediates. Genomic data indicate that bacterial microcompartment shell proteins are present in a wide range of bacterial species, where they encapsulate varied reactions. Crystal structures of numerous shell proteins from distinct types of microcompartments have provided keys for understanding how the shells are assembled and how they conduct molecular transport into and out of microcompartments. The structural data emphasize a high level of mechanistic sophistication in the protein shell, and point the way for further studies on this fascinating but poorly appreciated class of subcellular structures.
Protein assembly; capsid; molecular transport; pore; BMC; carboxysome
Disulfide bonds are generally not used to stabilize proteins in the cytosolic compartments of bacteria or eukaryotic cells, owing to the chemically reducing nature of those environments. In contrast, certain thermophilic archaea use disulfide bonding as a major mechanism for protein stabilization. Here, we provide a current survey of completely sequenced genomes, applying computational methods to estimate the use of disulfide bonding across the Archaea. Microbes belonging to the Crenarchaeal branch, which are essentially all hyperthermophilic, are universally rich in disulfide bonding while lesser degrees of disulfide bonding are found among the thermophilic Euryarchaea, excluding those that are methanogenic. The results help clarify which parts of the archaeal lineage are likely to yield more examples and additional specific data on protein disulfide bonding, as increasing genomic sequencing efforts are brought to bear.
Enzymes from natural product biosynthetic pathways are attractive candidates for creating tailored biocatalysts to produce semisynthetic pharmaceutical compounds. LovD is an acyltransferase that converts the inactive monacolin J acid (MJA) into the cholesterol-lowering lovastatin. LovD can also synthesize the blockbuster drug simvastatin using MJA and a synthetic α-dimethylbutyryl thioester, albeit with suboptimal properties as a biocatalyst. Here we used directed evolution to improve the properties of LovD towards semisynthesis of simvastatin. Mutants with improved catalytic efficiency, solubility and thermal stability were obtained, with the best mutant displaying an ~11-fold increase in an Escherichia coli based biocatalytic platform. To understand the structural basis of LovD enzymology, seven X-ray crystal structures were determined, including the parent LovD, an improved mutant G5, and G5 co-crystallized with ligands. Comparisons between the structures reveal that beneficial mutations stabilize the structure of G5 in a more compact conformation that is favorable for catalysis.
Bacterial microcompartments (MCPs) are the simplest organelles known. They function to enhance metabolic pathways by confining several related enzymes inside an all protein envelope called the shell. In this study, we investigated the factors that govern MCP assembly by performing scanning mutagenesis on the surface residues of PduA, a major shell protein of the MCP used for 1,2-propanediol degradation. Biochemical, genetic and structural analysis of 20 mutants allowed us to determine that PduA K26, N29 and R79 are crucial residues that stabilize the shell of the 1,2-propanediol MCP. In addition, we identify two PduA mutants (K37A and K55A) that impair MCP function most likely by altering the permeability of its protein shell. These are the first studies to examine the phenotypic effects of shell protein structural mutations in a microcompartment system. The findings reported here may be applicable to engineering protein containers with improved stability for biotechnology applications.
microcompartment; carboxysome; B12; 1,2-propanediol; Salmonella
Cystathionine β-synthase (CBS) domains are found in myriad proteins from organisms across the tree of life, and have been hypothesized to function as regulatory modules that sense the energy charge of the cell. Here we characterize the structure and stability of PAE2072, a dimeric, tandem CBS domain protein from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum. Crystal structures of the protein in unliganded and adenosine monophosphate (AMP)-bound forms, determined at resolutions of 2.10 Å and 2.35 Å respectively, reveal a remarkable conservation of key functional features seen in the γ subunit of the eukaryotic AMP-activated protein kinase (AMPK). The structures also confirm the presence of a suspected intermolecular disulfide bond between the two subunits that is shown to stabilize the protein. Our AMP-bound structure represents a first step in investigating the function of a large class of uncharacterized prokaryotic proteins. In addition, this work extends previous studies that have suggested that, in certain thermophilic microbes, disulfide bonds play a key role in stabilizing intracellular proteins and protein-protein complexes.
Cystathionine β-synthase; AMP-activated protein kinase; disulfide bond; protein stabilization; hyperthermophile
Among proteins of known three dimensional structure, only a few possess complex topological features such as knotted or interlinked (catenated) protein backbones. Such unusual proteins offer potentially unique insights into folding pathways and stabilization mechanisms. They also present special challenges for both theorists and computational scientists interested in understanding and predicting protein folding behavior. Here we review complex topological features in proteins with a focus on recent progress on the identification and characterization of knotted and interlinked protein systems. Also, an approach is described for designing an expanded set of knotted proteins.
Protein knots; protein links; protein folding; protein stability; protein topology
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.
Many bacteria produce giant proteinaceous structures within their cells, which they use to carry out special metabolic reactions in their interior. Much has been learned recently about the individual components—shell proteins and encapsulated enzymes—that assemble together, thousands of subunits in all, to make these bacterial microcompartments or MCPs. However, in order to carry out their biological functions, these systems must be highly organized through specific protein-protein interactions, and such a higher level understanding of organization in MCP systems is lacking. In this study, we use genomic data and phylogenetic analysis to predict the network of interactions between the approximately 20 different kinds of proteins and enzymes present in the Pdu MCP. Then, we use computational docking to examine a subset of those that are predicted to involve enzymes bound to the interior surface of the shell proteins, and show that the results are consistent with recent experimental data. We further provide new experimental evidence for one of the predicted protein-protein interactions. This study expands our understanding of a complex system of proteins serving as a metabolic organelle in bacterial cells, and provides a foundation for further experimental investigations.
The authors describe the structure determination of a hexagonally layered protein structure that suffered from a complicated combination of translational non-crystallographic symmetry and hemihedral twinning. This case serves as a reminder that broken crystallographic symmetry resulting from doubling of a unit-cell axis often requires a new choice of origin.
The carboxysome is a giant protein complex that acts as a metabolic organelle in cyanobacteria and some chemoautotrophs. Its outer structure is formed by the assembly of thousands of copies of hexameric shell protein subunits into a molecular layer. The structure determination of a CcmK1 shell protein mutant (L11K) from the β-carboxysome of the cyanobacterium Synechocystis PCC6803 led to challenges in structure determination. Twinning, noncrystallographic symmetry and packing of hexameric units in a special arrangement led to initial difficulties in space-group assignment. The correct space group was clarified after initial model refinement revealed additional symmetry. This study provides an instructive example in which broken symmetry requires a new choice of unit-cell origin in order to identify the highest symmetry space group. An additional observation related to the packing arrangement of molecules in this crystal suggests that these hexameric shell proteins might have lower internal symmetry than previously believed.
CcmK1 shell protein; carboxysome; hexameric shell proteins
The self-assembly of proteins into highly ordered nanoscale architectures is a hallmark of biological systems. The sophisticated functions of these molecular machines inspire the development of methods to engineer novel self-assembling protein structures. Although there has been exciting recent progress in this area, designing multi-component protein nanomaterials with high accuracy remains an outstanding challenge. Here we address this challenge by developing a general computational method for designing protein nanomaterials in which two distinct types of subunits coassemble to a target symmetric architecture. We use the method to design five novel 24-subunit cage-like protein nanomaterials in two distinct symmetric architectures, and experimentally demonstrate that the structures of the materials are in close agreement with the computational design models. The accuracy of the method and the universe of two-component materials that it makes accessible pave the way for the construction of functional protein nanomaterials tailored to specific applications.
Natural enzymes have evolved to perform their cellular functions under complex selective pressures, which often require their catalytic activities to be regulated by other proteins. We contrasted a natural enzyme, LovD, which acts on a protein-bound (LovF) acyl substrate, with a laboratory-generated variant that was transformed by directed evolution to accept instead a small free acyl thioester, and no longer requires the acyl carrier protein. The resulting 29-mutant variant is 1000-fold more efficient in the synthesis of the drug simvastatin than the wild-type LovD. This is the first non-patent report of the enzyme currently used for the manufacture of simvastatin, as well as the intermediate evolved variants. Crystal structures and microsecond molecular dynamics simulations revealed the mechanism by which the laboratory-generated mutations free LovD from dependence on protein-protein interactions. Mutations dramatically altered conformational dynamics of the catalytic residues, obviating the need for allosteric modulation by the acyl carrier LovF.
We describe a general computational method for designing proteins that self-assemble to a desired symmetric architecture. Protein building blocks are docked together symmetrically to identify complementary packing arrangements, and low-energy protein-protein interfaces are then designed between the building blocks in order to drive self-assembly. Here we use trimeric protein building blocks to design a 24-subunit, 13 nm diameter complex with octahedral symmetry and two related variants of a 12-subunit, 11 nm diameter complex with tetrahedral symmetry. The designed proteins assembled to the desired oligomeric states in solution, and crystal structures of the complexes revealed that the resulting materials closely match the design models. The method can be used to design a wide variety of self-assembling protein nanomaterials.
The idea of attacking the phase problem by crowdsourcing is introduced. Using an interactive, multi-player, web-based system, participants work simultaneously to select phase sets that correspond to better electron-density maps in order to solve low-resolution phasing problems.
The human mind innately excels at some complex tasks that are difficult to solve using computers alone. For complex problems amenable to parallelization, strategies can be developed to exploit human intelligence in a collective form: such approaches are sometimes referred to as ‘crowdsourcing’. Here, a first attempt at a crowdsourced approach for low-resolution ab initio phasing in macromolecular crystallography is proposed. A collaborative online game named CrowdPhase was designed, which relies on a human-powered genetic algorithm, where players control the selection mechanism during the evolutionary process. The algorithm starts from a population of ‘individuals’, each with a random genetic makeup, in this case a map prepared from a random set of phases, and tries to cause the population to evolve towards individuals with better phases based on Darwinian survival of the fittest. Players apply their pattern-recognition capabilities to evaluate the electron-density maps generated from these sets of phases and to select the fittest individuals. A user-friendly interface, a training stage and a competitive scoring system foster a network of well trained players who can guide the genetic algorithm towards better solutions from generation to generation via gameplay. CrowdPhase was applied to two synthetic low-resolution phasing puzzles and it was shown that players could successfully obtain phase sets in the 30° phase error range and corresponding molecular envelopes showing agreement with the low-resolution models. The successful preliminary studies suggest that with further development the crowdsourcing approach could fill a gap in current crystallographic methods by making it possible to extract meaningful information in cases where limited resolution might otherwise prevent initial phasing.
CrowdPhase; crowdsourcing; phase problem
Designing protein molecules that self-assemble into complex architectures is an outstanding goal in the area of nanobiotechnology. One design strategy for doing this involves genetically fusing together two natural proteins, each of which is known to form a simple oligomer on its own (e.g. a dimer or trimer). If two such components can be fused in a geometrically predefined configuration, that designed subunit can, in principle, assemble into highly symmetric architectures. Initial experiments showed that a 12-subunit tetrahedral cage, 16 nm in diameter, could be constructed following such a procedure.1,2 Here we characterize multiple crystal structures of protein cages constructed in this way, including cages assembled from two mutant forms of the same basic protein subunit. The flexibilities of the designed assemblies and their deviations from the target model are described, along with implications for further design developments.
Simvastatin is the active pharmaceutical ingredient of the blockbuster cholesterol lowering drug Zocor. We have previously developed an Escherichia coli based whole-cell biocatalytic platform towards the synthesis of simvastatin sodium salt (SS) starting from the precursor monacolin J sodium salt (MJSS). The centerpiece of the biocatalytic approach is the simvastatin synthase LovD, which is highly prone to misfolding and aggregation when overexpressed from E. coli. Increasing the solubility of LovD without decreasing its catalytic activity can therefore elevate the performance of the whole-cell biocatalyst. Using a combination of homology structural prediction and site-directed mutagenesis, we identified two cysteine residues in LovD that are responsible for nonspecific intermolecular crosslinking, which leads to oligomer formation and protein aggregation. Replacement of Cys40 and Cys60 with alanine residues resulted in marked gain in both protein solubility and whole-cell biocatalytic activities. Further mutagenesis experiments converting these two residues to small or polar natural amino acids showed that C40A and C60N are the most beneficial, affording 27% and 26% increase in whole cell activities, respectively. The double mutant C40A/C60N combines the individual improvements and displayed ~50% increase in protein solubility and whole-cell activity. Optimized fed-batch high-cell-density fermentation of the double mutant in an E. coli strain engineered for simvastatin production quantitatively (>99%) converted 45 mM MJSS to SS within 18 hours, which represents a significant improvement over the performance of wild type LovD under identical conditions. The high efficiency of the improved whole-cell platform renders the biocatalytic synthesis of SS an attractive substitute over the existing semisynthetic routes.
Acyltransferase; simvastatin; site-directed mutagenesis; whole-cell biocatalyst; protein solubility
A strategy using a new split green fluorescent protein (GFP) as a modular binding partner to form stable protein complexes with a target protein is presented. The modular split GFP may open the way to rapidly creating crystallization variants.
A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP β-strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was tested by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10–11) hairpin in complex with GFP(1–9) was determined at a resolution of 2.6 Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10–11) hairpin with a variety of GFP(1–9) mutants engineered for favorable crystallization.
protein crystallization; synthetic symmetrization; protein tagging; split GFP; split protein; green fluorescent protein; protein expression; protein-fragment complementation; crystallization reagents
The polypeptide backbones of a few proteins are tied in a knot. The biophysical effects and potential biological roles of knots are not well understood. Here, we test the consequences of protein knotting by taking a monomeric protein, carbonic anhydrase II, whose native structure contains a shallow knot, and polymerizing it end-to-end to form a deeply and multiply knotted polymeric filament. Thermal stability experiments show that the polymer is stabilized against loss of structure and aggregation by the presence of deep knots.
biomaterials; disulfide; protein design; protein knot; topology
Many bacteria conditionally express proteinaceous organelles referred to here as microcompartments (Fig. 1). These microcompartments are thought to be involved in a least seven different metabolic processes and the number is growing. Microcompartments are very large and structurally sophisticated. They are usually about 100–150 nm in cross section and consist of 10,000–20,000 polypeptides of 10–20 types. Their unifying feature is a solid shell constructed from proteins having bacterial microcompartment (BMC) domains. In the examples that have been studied, the microcompartment shell encases sequentially acting metabolic enzymes that catalyze a reaction sequence having a toxic or volatile intermediate product. It is thought that the shell of the microcompartment confines such intermediates, thereby enhancing metabolic efficiency and/or protecting cytoplasmic components. Mechanistically, however, this creates a paradox. How do microcompartments allow enzyme substrates, products and cofactors to pass while confining metabolic intermediates in the absence of a selectively permeable membrane? We suggest that the answer to this paradox may have broad implications with respect to our understanding of the fundamental properties of biological protein sheets including microcompartment shells, S-layers and viral capsids.
Some bacteria contain organelles or microcompartments consisting of a large virion-like protein shell encapsulating sequentially acting enzymes. These organized microcompartments serve to enhance or protect key metabolic pathways inside the cell. The variety of bacterial microcompartments provide diverse metabolic functions, ranging from CO2 fixation to the degradation of small organic molecules. Yet they share an evolutionarily related shell, which is defined by a conserved protein domain that is widely distributed across the bacterial kingdom. Structural studies on a number of these bacterial microcompartment shell proteins are illuminating the architecture of the shell and highlighting its critical role in controlling molecular transport into and out of microcompartments. Current structural, evolutionary, and mechanistic ideas are discussed, along with genomic studies for exploring the function and diversity of this family of bacterial organelles.
carboxysome; molecular transport; protein assembly; nanocompartments; protein shell
The crystal structure of a putative NTP pyrophosphohydrolase, YP_001813558.1 from E. sibiricum, reveals a novel segment-swapped linked-dimer assembly.
The crystal structure of a putative NTPase, YP_001813558.1 from Exiguobacterium sibiricum 255-15 (PF09934, DUF2166) was determined to 1.78 Å resolution. YP_001813558.1 and its homologs (dimeric dUTPases, MazG proteins and HisE-encoded phosphoribosyl ATP pyrophosphohydrolases) form a superfamily of all-α-helical NTP pyrophosphatases. In dimeric dUTPase-like proteins, a central four-helix bundle forms the active site. However, in YP_001813558.1, an unexpected intertwined swapping of two of the helices that compose the conserved helix bundle results in a ‘linked dimer’ that has not previously been observed for this family. Interestingly, despite this novel mode of dimerization, the metal-binding site for divalent cations, such as magnesium, that are essential for NTPase activity is still conserved. Furthermore, the active-site residues that are involved in sugar binding of the NTPs are also conserved when compared with other α-helical NTPases, but those that recognize the nucleotide bases are not conserved, suggesting a different substrate specificity.
structural genomics; putative NTP pyrophosphohydrolase; MazG nucleotide pyrophosphohydrolase; dUTPases
Many of the functional units in cells are multi-protein complexes such as RNA polymerase, the ribosome, and the proteasome. For such units to work together, one might expect a high level of regulation to enable co-appearance or repression of sets of complexes at the required time. However, this type of coordinated regulation between whole complexes is difficult to detect by existing methods for analyzing mRNA co-expression. We propose a new methodology that is able to detect such higher order relationships.
We detect coordinated regulation of multiple protein complexes using logic analysis of gene expression data. Specifically, we identify gene triplets composed of genes whose expression profiles are found to be related by various types of logic functions. In order to focus on complexes, we associate the members of a gene triplet with the distinct protein complexes to which they belong. In this way, we identify complexes related by specific kinds of regulatory relationships. For example, we may find that the transcription of complex C is increased only if the transcription of both complex A AND complex B is repressed. We identify hundreds of examples of coordinated regulation among complexes under various stress conditions. Many of these examples involve the ribosome. Some of our examples have been previously identified in the literature, while others are novel. One notable example is the relationship between the transcription of the ribosome, RNA polymerase and mannosyltransferase II, which is involved in N-linked glycan processing in the Golgi.
The analysis proposed here focuses on relationships among triplets of genes that are not evident when genes are examined in a pairwise fashion as in typical clustering methods. By grouping gene triplets, we are able to decipher coordinated regulation among sets of three complexes. Moreover, using all triplets that involve coordinated regulation with the ribosome, we derive a large network involving this essential cellular complex. In this network we find that all multi-protein complexes that belong to the same functional class are regulated in the same direction as a group (either induced or repressed).
Multiple crystal structures are reported of cross-linked actin dimers. Interactions that are conserved across crystal structures suggest detailed interactions that are likely to be present in F-actin filaments.
The structure of actin in its monomeric form is known at high resolution, while the structure of filamentous F-actin is only understood at considerably lower resolution. Knowing precisely how the monomers of actin fit together would lead to a deeper understanding of the dynamic behavior of the actin filament. Here, a series of crystal structures of actin dimers are reported which were prepared by cross-linking in either the longitudinal or the lateral direction in the filament state. Laterally cross-linked dimers, comprised of monomers belonging to different protofilaments, are found to adopt configurations in crystals that are not related to the native structure of filamentous actin. In contrast, multiple structures of longitudinal dimers consistently reveal the same interface between monomers within a single protofilament. The reappearance of the same longitudinal interface in multiple crystal structures adds weight to arguments that the interface visualized is similar to that in actin filaments. Highly conserved atomic interactions involving residues 199–205 and 287–291 are highlighted.
F-actin; cross-linking; actin filaments
A growing number of organisms have been discovered inhabiting extreme environments, including temperatures in excess of 100 °C. How cellular proteins from such organisms retain their native folds under extreme conditions is still not fully understood. Recent computational and structural studies have identified disulfide bonding as an important mechanism for stabilizing intracellular proteins in certain thermophilic microbes. Here, we present the first proteomic analysis of intracellular disulfide bonding in the hyperthermophilic archaeon Pyrobaculum aerophilum. Our study reveals that the utilization of disulfide bonds extends beyond individual proteins to include many protein-protein complexes. We report the 1.6Å crystal structure of one such complex, a citrate synthase homodimer. The structure contains two intramolecular disulfide bonds, one per subunit, which result in the cyclization of each protein chain in such a way that the two chains are topologically interlinked, rendering them inseparable. This unusual feature emphasizes the variety and sophistication of the molecular mechanisms that can be achieved by evolution.
disulfide bond; protein stability; catenane; citrate synthase; thermophile
The carboxysome is a bacterial organelle that functions to enhance the efficiency of CO2 fixation by encapsulating the enzymes ribulose bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase. The outer shell of the carboxysome is reminiscent of a viral capsid, being constructed from many copies of a few small proteins. Here we describe the structure of the shell protein CsoS1A from the chemoautotrophic bacterium Halothiobacillus neapolitanus. The CsoS1A protein forms hexameric units that pack tightly together to form a molecular layer, which is perforated by narrow pores. Sulfate ions, soaked into crystals of CsoS1A, are observed in the pores of the molecular layer, supporting the idea that the pores could be the conduit for negatively charged metabolites such as bicarbonate, which must cross the shell. The problem of diffusion across a semiporous protein shell is discussed, with the conclusion that the shell is sufficiently porous to allow adequate transport of small molecules. The molecular layer formed by CsoS1A is similar to the recently observed layers formed by cyanobacterial carboxysome shell proteins. This similarity supports the argument that the layers observed represent the natural structure of the facets of the carboxysome shell. Insights into carboxysome function are provided by comparisons of the carboxysome shell to viral capsids, and a comparison of its pores to the pores of transmembrane protein channels.
Bacterial cells are generally viewed as being relatively simple because they lack the membrane-bound organelles that help organize the interiors of eukaryotic cells. However, many bacterial cells produce large, protein-based microcompartments that serve effectively as simple organelles. These microcompartments enclose specific cellular enzymes, thereby successfully sequestering particular reactions or pathways from the rest of the cytosol. The prototypical bacterial microcompartment is the carboxysome, which is found in many bacteria that fix CO2 into organic carbon. In these bacteria, the efficiency of CO2 fixation is enhanced by having the key enzymes in that pathway encapsulated together. Carboxysomes were discovered more than 40 years ago, but an understanding of their assembly and function is just beginning to emerge. Here we report new structures of the proteins that form the outer shell of the carboxysome. These structures provide further evidence that the carboxysome shell is constructed according to principles similar to those seen in icosahedral viral capsids. The structure of the carboxysome serves as a model for understanding a variety of primitive bacterial organelles that are coming to light.
The structure of the bacterial carboxysome shell protein consists of tightly packed hexameric units perforated by narrow pores that bind sulfate ions, enabling metabolites to be conducted across the shell.
A new method for predicting recoding by rare amino acids such as selenocysteine and pyrrolysine was used to survey a set of microbial genomes.
In several natural settings, the standard genetic code is expanded to incorporate two additional amino acids with distinct functionality, selenocysteine and pyrrolysine. These rare amino acids can be overlooked inadvertently, however, as they arise by recoding at certain stop codons. We report a method for such recoding prediction from genomic data, using read-through similarity evaluation. A survey across a set of microbial genomes identifies almost all the known cases as well as a number of novel candidate proteins.