The structure of CcmP, a tandem bacterial microcompartment domain protein from the ß-carboxysome, forms a subcompartment within a microcompartment.

The carboxysome is a bacterial organelle found in all cyanobacteria; it encapsulates CO2 fixation enzymes within a protein shell. The most abundant carboxysome shell protein contains a single bacterial microcompartment (BMC) domain. We present in vivo evidence that a hypothetical protein (dubbed CcmP) encoded in all ß-cyanobacterial genomes is part of the carboxysome. We show that CcmP is a tandem BMC domain protein, the first to be structurally characterized from a ß-carboxysome. CcmP forms a dimer of tightly stacked trimers, resulting in a nanocompartment-containing shell protein that may weakly bind 3-phosphoglycerate, the product of CO2 fixation. The trimers have a large central pore through which metabolites presumably pass into the carboxysome. Conserved residues surrounding the pore have alternate side-chain conformations suggesting that it can be open or closed. Furthermore, CcmP and its orthologs in α-cyanobacterial genomes form a distinct clade of shell proteins. Members of this subgroup are also found in numerous heterotrophic BMC-associated gene clusters encoding functionally diverse bacterial organelles, suggesting that the potential to form a nanocompartment within a microcompartment shell is widespread. Given that carboxysomes and architecturally related bacterial organelles are the subject of intense interest for applications in synthetic biology/metabolic engineering, our results describe a new type of building block with which to functionalize BMC shells.

Elucidating essential role of conserved carboxysomal protein CcmN reveals common feature of bacterial microcompartment assembly.

Bacterial microcompartments are organelles composed of a protein shell that surrounds functionally related proteins. Bioinformatic analysis of sequenced genomes indicates that homologs to shell protein genes are widespread among bacteria and suggests that the shell proteins are capable of encapsulating diverse enzymes. The carboxysome is a bacterial microcompartment that enhances CO(2) fixation in cyanobacteria and some chemoautotrophs by sequestering ribulose-1,5-bisphosphate carboxylase/oxygenase and carbonic anhydrase in the microcompartment shell. Here, we report the in vitro and in vivo characterization of CcmN, a protein of previously unknown function that is absolutely conserved in ß-carboxysomal gene clusters. We show that CcmN localizes to the carboxysome and is essential for carboxysome biogenesis. CcmN has two functionally distinct regions separated by a poorly conserved linker. The N-terminal portion of the protein is important for interaction with CcmM and, by extension, ribulose-1,5-bisphosphate carboxylase/oxygenase and the carbonic anhydrase CcaA, whereas the C-terminal peptide is essential for interaction with the carboxysome shell. Deletion of the peptide abolishes carboxysome formation, indicating that its interaction with the shell is an essential step in microcompartment formation. Peptides with similar length and sequence properties to those in CcmN can be bioinformatically detected in a large number of diverse proteins proposed to be encapsulated in functionally distinct microcompartments, suggesting that this peptide and its interaction with its cognate shell proteins are common features of microcompartment assembly.

Cyanobacterial-based approaches to improving photosynthesis in plants.

Plants rely on the Calvin-Benson (CB) cycle for CO(2) fixation. The key carboxylase of the CB cycle is ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). Efforts to enhance carbon fixation in plants have traditionally focused on RubisCO or on approaches that can help to remedy RubisCO's undesirable traits: its low catalytic efficiency and photorespiration. Towards reaching the goal of improving plant photosynthesis, cyanobacteria may be instrumental. Because of their evolutionary relationship to chloroplasts, they represent ideal model organisms for photosynthesis research. Furthermore, the molecular understanding of cyanobacterial carbon fixation provides a rich source of strategies that can be exploited for the bioengineering of chloroplasts. These strategies include the cyanobacterial carbon concentrating mechanism (CCM), which consists of active and passive transporter systems for inorganic carbon and a specialized organelle, the carboxysome. The carboxysome encapsulates RubisCO together with carbonic anhydrase in a protein shell, resulting in an elevated CO(2) concentration around RubisCO. Moreover, cyanobacteria differ from plants in the isoenzymes involved in the CB cycle and the photorespiratory pathways as well as in mechanisms that can affect the activity of RubisCO. In addition, newly available cyanobacterial genome sequence data from the CyanoGEBA project, which has more than doubled the amount of genomic information available for cyanobacteria, increases our knowledge on the CCM and the occurrence and distribution of genes of interest.

Structural determinants underlying photoprotection in the photoactive orange carotenoid protein of cyanobacteria.

The photoprotective processes of photosynthetic organisms involve the dissipation of excess absorbed light energy as heat. Photoprotection in cyanobacteria is mechanistically distinct from that in plants; it involves the orange carotenoid protein (OCP), a water-soluble protein containing a single carotenoid. The OCP is a new member of the family of blue light-photoactive proteins; blue-green light triggers the OCP-mediated photoprotective response. Here we report structural and functional characterization of the wild type and two mutant forms of the OCP, from the model organism Synechocystis PCC6803. The structural analysis provides high resolution detail of the carotenoid-protein interactions that underlie the optical properties of the OCP, unique among carotenoid-proteins in binding a single pigment per polypeptide chain. Collectively, these data implicate several key amino acids in the function of the OCP and reveal that the photoconversion and photoprotective responses of the OCP to blue-green light can be decoupled.

Comparative analysis of carboxysome shell proteins.

Carboxysomes are metabolic modules for CO(2) 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 CO(2) fixation in other organisms or creating novel biological nanostructures.

Engineering bacterial microcompartment shells: chimeric shell proteins and chimeric carboxysome shells.

Bacterial microcompartments (BMCs) are self-assembling organelles composed entirely of protein. Depending on the enzymes they encapsulate, BMCs function in either inorganic carbon fixation (carboxysomes) or organic carbon utilization (metabolosomes). The hallmark feature of all BMCs is a selectively permeable shell formed by multiple paralogous proteins, each proposed to confer specific flux characteristics. Gene clusters encoding diverse BMCs are distributed broadly across bacterial phyla, providing a rich variety of building blocks with a predicted range of permeability properties. In theory, shell permeability can be engineered by modifying residues flanking the pores (symmetry axes) of hexameric shell proteins or by combining shell proteins from different types of BMCs into chimeric shells. We undertook both approaches to altering shell properties using the carboxysome as a model system. There are two types of carboxysomes, α and ß. In both, the predominant shell protein(s) contain a single copy of the BMC domain (pfam00936), but they are significantly different in primary structure. Indeed, phylogenetic analysis shows that the two types of carboxysome shell proteins are more similar to their counterparts in metabolosomes than to each other. We solved high resolution crystal structures of the major shell proteins, CsoS1 and CcmK2, and the presumed minor shell protein CcmK4, representing both types of cyanobacterial carboxysomes and then tested the interchangeability. The in vivo study presented here confirms that both engineering pores to mimic those of other shell proteins and the construction of chimeric shells is feasible.

Assembly of robust bacterial microcompartment shells using building blocks from an organelle of unknown function.

Bacterial microcompartments (BMCs) sequester enzymes from the cytoplasmic environment by encapsulation inside a selectively permeable protein shell. Bioinformatic analyses indicate that many bacteria encode BMC clusters of unknown function and with diverse combinations of shell proteins. The genome of the halophilic myxobacterium Haliangium ochraceum encodes one of the most atypical sets of shell proteins in terms of composition and primary structure. We found that microcompartment shells could be purified in high yield when all seven H. ochraceum BMC shell genes were expressed from a synthetic operon in Escherichia coli. These shells differ substantially from previously isolated shell systems in that they are considerably smaller and more homogeneous, with measured diameters of 39±2nm. The size and nearly uniform geometry allowed the development of a structural model for the shells composed of 260 hexagonal units and 13 hexagons per icosahedral face. We found that new proteins could be recruited to the shells by fusion to a predicted targeting peptide sequence, setting the stage for the use of these remarkably homogeneous shells for applications such as three-dimensional scaffolding and the construction of synthetic BMCs. Our results demonstrate the value of selecting from the diversity of BMC shell building blocks found in genomic sequence data for the construction of novel compartments.