Structural Analysis of CsoS1A and the Protein Shell of the Halothiobacillus neapolitanus Carboxysome

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.     

Insights from multiple structures of the shell proteins from the β‐carboxysome

Carboxysomes are primitive bacterial organelles that function as a part of a carbon concentrating mechanism (CCM) under conditions where inorganic carbon is limiting. The carboxysome enhances the efficiency of cellular carbon fixation by encapsulating together carbonic anhydrase and the CO₂-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). The carboxysome has a roughly icosahedral shape with an outer shell between 800 and 1500 Å in diameter, which is constructed from a few thousand small protein subunits. In the cyanobacterium Synechocystis sp. PCC 6803, the previous structure determination of two homologous shell protein subunits, CcmK2 and CcmK4, elucidated how the outer shell is formed by the tight packing of CcmK hexamers into a molecular layer. Here we describe the crystal structure of the hexameric shell protein CcmK1, along with structures of mutants of both CcmK1 and CcmK2 lacking their sometimes flexible C-terminal tails. Variations in the way hexamers pack into layers are noted, while sulfate ions bound in pores through the layer provide further support for the hypothesis that the pores serve for transport of substrates and products into and out of the carboxysome. One of the new structures provides a high-resolution (1.3 Å) framework for subsequent computational studies of molecular transport through the pores. Crystal and solution studies of the C-terminal deletion mutants demonstrate the tendency of the terminal segments to participate in protein—protein interactions, thereby providing a clue as to which side of the molecular layer of hexameric shell proteins is likely to face toward the carboxysome interior.     

Identification and Structural Analysis of a Novel Carboxysome Shell Protein with Implications for Metabolite Transport

Bacterial microcompartments (BMCs) are polyhedral bodies, composed entirely of proteins, that function as organelles in bacteria; they promote subcellular processes by encapsulating and co-localizing targeted enzymes with their substrates. The best-characterized BMC is the carboxysome, a central part of the carbon-concentrating mechanism that greatly enhances carbon fixation in cyanobacteria and some chemoautotrophs. Here we report the first structural insights into the carboxysome of Prochlorococcus, the numerically dominant cyanobacterium in the world's oligotrophic oceans. Bioinformatic methods, substantiated by analysis of gene expression data, were used to identify a new carboxysome shell component, CsoS1D, in the genome of Prochlorococcus strain MED4; orthologs were subsequently found in all cyanobacteria. Two independent crystal structures of Prochlorococcus MED4 CsoS1D reveal three features not seen in any BMC-domain protein structure solved to date. First, CsoS1D is composed of a fused pair of BMC domains. Second, this double-domain protein trimerizes to form a novel pseudohexameric building block for incorporation into the carboxysome shell, and the trimers further dimerize, forming a two-tiered shell building block. Third, and most strikingly, the large pore formed at the 3-fold axis of symmetry appears to be gated. Each dimer of trimers contains one trimer with an open pore and one whose pore is obstructed due to side-chain conformations of two residues that are invariant among all CsoS1D orthologs. This is the first evidence of the potential for gated transport across the carboxysome shell and reveals a new type of building block for BMC shells.     

Identification and localization of the carboxysome peptide Csos3 and its corresponding gene in Thiobacillus neapolitanus

Four genes encoding carboxysome shell peptides (csoS1A, csoS1B, csoS1C, csoS2), the genes encoding the large and small subunits of RuBisCO (cbbL, cbbS), and three unidentified ORFs constitute an operon in Thiobacillus neapolitanus. An unidentified ORF 1.54 kb in size is predicted from sequence analysis to encode a protein with a molecular mass of approximately 57 kDa. When this ORF was expressed in Escherichia coli under the control of its endogenous ribosome-binding site, no peptide product was observed. In order to correlate this ORF with a carboxysome peptide, the ORF was overexpressed in E. coli by cloning it into pProExHTb, a prokaryotic expression vector containing an E. coli ribosome binding site. When antibodies raised against the recombinant protein were used to probe an immunoblot containing carboxysome peptides, a 60-kDa peptide was recognized. The peptide was subsequently named CsoS3. CsoS3 is a minor component of the carboxysome; a peptide of this size is commonly not observed or is very faint on Coomassie blue-stained SDS-polyacrylamide gels of purified carboxysomes. Immunogold labeling established CsoS3 to be a component of the carboxysome shell.     

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 CO₂ 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 CO₂ 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.     

Isolation and characterization of the Prochlorococcus carboxysome reveal the presence of the novel shell protein CsoS1D

Cyanobacteria, including members of the genus Prochlorococcus, contain icosahedral protein microcompartments known as carboxysomes that encapsulate multiple copies of the CO(2)-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) in a thin protein shell that enhances the catalytic performance of the enzyme in part through the action of a shell-associated carbonic anhydrase. However, the exact mechanism by which compartmentation provides a catalytic advantage to the enzyme is not known. Complicating the study of cyanobacterial carboxysomes has been the inability to obtain homogeneous carboxysome preparations. This study describes the first successful purification and characterization of carboxysomes from the marine cyanobacterium Prochlorococcus marinus MED4. Because the isolated P. marinus MED4 carboxysomes were free from contaminating membrane proteins, their protein complement could be assessed. In addition to the expected shell proteins, the CsoS1D protein that is not encoded by the canonical cso gene clusters of α-cyanobacteria was found to be a low-abundance shell component. This finding and supporting comparative genomic evidence have important implications for carboxysome composition, structure, and function. Our study indicates that carboxysome composition is probably more complex than was previously assumed based on the gene complements of the classical cso gene clusters.     

Organization of Carboxysome Genes in the Thiobacilli

The order of genes in the carboxysome gene clusters of four thiobacilli was examined and the possibility of the cluster forming an operon evaluated. Furthermore, carboxysome peptide homologs were compared with respect to similarities in primary sequence, and the unique structural features of the shell protein CsoS2 were described. Received: 27 March 2002 / Accepted: 30 April 2002     

The correlation of the gene csoS2 of the carboxysome operon with two polypeptides of the carboxysome in Thiobacillus neapolitanus

The carboxysomal polypeptides of Thiobacillus neapolitanus with apparent molecular masses of 85 and 130 kDa were isolated and subjected to N-terminal sequencing. The first 17 amino acids of the two peptides were identical. The sequence perfectly matched the deduced amino acid sequence of an open reading frame in the carboxysome operon. The gene was subsequently named csoS2. Expression of the gene in Escherichia coli resulted in the production of two peptides with apparent molecular masses of 85 and 130 kDa. Immunospecific antibodies generated against the smaller peptide recognized both peptides; the peptides were named CsoS2A and CsoS2B, respectively. A digoxigenin-hydrazide glycosylation assay revealed that both CsoS2A and CsoS2B are post-translationally modified by glycosylation. CsoS2 was localized to the edges of purified carboxysomes by immunogold electron microscopy using the monospecific CsoS2A antibodies. The molecular mass of CsoS2A calculated from the nucleotide sequence was 92.3 kDa. Received: 1 March 1999 / Accepted: 29 June 1999     

Structure of the PduU shell protein from the Pdu microcompartment of Salmonella

The Pdu microcompartment is a proteinaceous, subcellular structure that serves as an organelle for the metabolism of 1,2-propanediol in Salmonella enterica. It encapsulates several related enzymes within a shell composed of a few thousand protein subunits. Recent structural studies on the carboxysome, a related microcompartment involved in CO(2) fixation, have concluded that the major shell proteins from that microcompartment form hexamers that pack into layers comprising the facets of the shell. Here we report the crystal structure of PduU, a protein from the Pdu microcompartment, representing the first structure of a shell protein from a noncarboxysome microcompartment. Though PduU is a hexamer like other characterized shell proteins, it has undergone a circular permutation leading to dramatic differences in the hexamer pore. In view of the hypothesis that microcompartment metabolites diffuse across the outer shell through these pores, the unique structure of PduU suggests the possibility of a special functional role.     

Two‐dimensional crystals of carboxysome shell proteins recapitulate the hexagonal packing of three‐dimensional crystals

Bacterial microcompartments (BMCs) are large intracellular bodies that serve as simple organelles in many bacteria. They are proteinaceous structures composed of key enzymes encapsulated by a polyhedral protein shell. In previous studies, the organization of these large shells has been inferred from the conserved packing of the component shell proteins in two‐dimensional (2D) layers within the context of three‐dimensional (3D) crystals. Here, we show that well‐ordered, 2D crystals of carboxysome shell proteins assemble spontaneously when His‐tagged proteins bind to a monolayer of nickelated lipid molecules at an air–water interface. The molecular packing within the 2D crystals recapitulates the layered hexagonal sheets observed in 3D crystals. The results reinforce current models for the molecular design of BMC shells.     

Structural Determinants of the Outer Shell of β-Carboxysomes in Synechococcus elongatus PCC 7942: Roles for CcmK2, K3-K4, CcmO, and CcmL

Cyanobacterial CO(2)-fixation is supported by a CO(2)-concentrating mechanism which improves photosynthesis by saturating the primary carboxylating enzyme, ribulose 1, 5-bisphosphate carboxylase/oxygenase (RuBisCO), with its preferred substrate CO(2). The site of CO(2)-concentration is a protein bound micro-compartment called the carboxysome which contains most, if not all, of the cellular RuBisCO. The shell of β-type carboxysomes is thought to be composed of two functional layers, with the inner layer involved in RuBisCO scaffolding and bicarbonate dehydration, and the outer layer in selective permeability to dissolved solutes. Here, four genes (ccmK2-4, ccmO), whose products were predicted to function in the outer shell layer of β-carboxysomes from Synechococcus elongatus PCC 7942, were investigated by analysis of defined genetic mutants. Deletion of the ccmK2 and ccmO genes resulted in severe high-CO(2)-requiring mutants with aberrant carboxysomes, whilst deletion of ccmK3 or ccmK4 resulted in cells with wild-type physiology and normal ultrastructure. However, a tandem deletion of ccmK3-4 resulted in cells with wild-type carboxysome structure, but physiologically deficient at low CO(2) conditions. These results revealed the minimum structural determinants of the outer shell of β-carboxysomes from this strain: CcmK2, CcmO and CcmL. An accessory set of proteins was required to refine the function of the pre-existing shell: CcmK3 and CcmK4. These data suggested a model for the facet structure of β-carboxysomes with CcmL forming the vertices, CcmK2 forming the bulk facet, and CcmO, a "zipper protein," interfacing the edges of carboxysome facets.     

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 β-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.     

A novel evolutionary lineage of carbonic anhydrase (epsilon class) is a component of the carboxysome shell

A significant portion of the total carbon fixed in the biosphere is attributed to the autotrophic metabolism of prokaryotes. In cyanobacteria and many chemolithoautotrophic bacteria, CO(2) fixation is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), most if not all of which is packaged in protein microcompartments called carboxysomes. These structures play an integral role in a cellular CO(2)-concentrating mechanism and are essential components for autotrophic growth. Here we report that the carboxysomal shell protein, CsoS3, from Halothiobacillus neapolitanus is a novel carbonic anhydrase (epsilon-class CA) that has an evolutionary lineage distinct from those previously recognized in animals, plants, and other prokaryotes. Functional CAs encoded by csoS3 homologues were also identified in the cyanobacteria Prochlorococcus sp. and Synechococcus sp., which dominate the oligotrophic oceans and are major contributors to primary productivity. The location of the carboxysomal CA in the shell suggests that it could supply the active sites of RuBisCO in the carboxysome with the high concentrations of CO(2) necessary for optimal RuBisCO activity and efficient carbon fixation in these prokaryotes, which are important contributors to the global carbon cycle.     

Bacterial microcompartment shells of diverse functional types possess pentameric vertex proteins

Bacterial microcompartments (MCPs) are large proteinaceous structures comprised of a roughly icosahedral shell and a series of encapsulated enzymes. MCPs carrying out three different metabolic functions have been characterized in some detail, while gene expression and bioinformatics studies have implicated other types, including one believed to perform g lycyl r adical‐based metabolism of 1,2‐p ropanediol (Grp). Here we report the crystal structure of a protein (GrpN), which is presumed to be part of the shell of a Grp‐type MCP in Rhodospirillum rubrum F11. GrpN is homologous to a family of proteins (EutN/PduN/CcmL/CsoS4) whose members have been implicated in forming the vertices of MCP shells. Consistent with that notion, the crystal structure of GrpN revealed a pentameric assembly. That observation revived an outstanding question about the oligomeric state of this protein family: pentameric forms (for CcmL and CsoS4A) and a hexameric form (for EutN) had both been observed in previous crystal structures. To clarify these confounding observations, we revisited the case of EutN. We developed a molecular biology‐based method for accurately determining the number of subunits in homo‐oligomeric proteins, and found unequivocally that EutN is a pentamer in solution. Based on these convergent findings, we propose the name bacterial microcompartment vertex for this special family of MCP shell proteins.     

A multiprotein bicarbonate dehydration complex essential to carboxysome function in cyanobacteria

Carboxysomes are proteinaceous biochemical compartments that constitute the enzymatic "back end" of the cyanobacterial CO2-concentrating mechanism. These protein-bound organelles catalyze HCO3- dehydration and photosynthetic CO2 fixation. In Synechocystis sp. strain PCC6803 these reactions involve the beta-class carbonic anhydrase (CA), CcaA, and Form 1B ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The surrounding shell is thought to be composed of proteins encoded by the ccmKLMN operon, although little is known about how structural and catalytic proteins integrate to form a functional carboxysome. Using biochemical activity assays and molecular approaches we have identified a catalytic, multiprotein HCO3- dehydration complex (BDC) associated with the protein shell of Synechocystis carboxysomes. The complex was minimally composed of a CcmM73 trimer, CcaA dimer, and CcmN. Larger native complexes also contained RbcL, RbcS, and two or three immunologically identified smaller forms of CcmM (62, 52, and 36 kDa). Yeast two-hybrid analyses indicated that the BDC was associated with the carboxysome shell through CcmM73-specific protein interactions with CcmK and CcmL. Protein interactions between CcmM73 and CcaA, CcmM73 and CcmN, or CcmM73 and itself required the N-terminal gamma-CA-like domain of CcmM73. The specificity of the CcmM73-CcaA interaction provided both a mechanism to integrate CcaA into the fabric of the carboxysome shell and a means to recruit this enzyme to the BDC during carboxysome biogenesis. Functionally, CcaA was the catalytic core of the BDC. CcmM73 bound H14CO3- but was unable to catalyze HCO3- dehydration, suggesting that it may potentially regulate BDC activity.     

The Structure of CcmP,a Tandem Bacterial Microcompartment Domain Protein from the β-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 β-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 β-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.     

Crystal structures of hydrophobin HFBII in the presence of detergent implicate the formation of fibrils and monolayer films

Hydrophobins are small, amphiphilic proteins secreted by filamentous fungi. Their functionality arises from a patch of hydrophobic residues on the protein surface. Spontaneous self-assembly of hydrophobins leads to the formation of an amphiphilic layer that remarkably reduces the surface tension of water. We have determined by x-ray diffraction two new crystal structures of Trichoderma reesei hydrophobin HFBII in the presence of a detergent. The monoclinic crystal structure (2.2A resolution, R = 22, R(free) = 28) is composed of layers of hydrophobin molecules where the hydrophobic surface areas of the molecules are aligned within the layer. Viewed perpendicular to the aligned hydrophobic surface areas, the molecules in the layer pack together to form six-membered rings, thus leaving small pores in the layer. Similar packing has been observed in the atomic force microscopy images of the self-assembled layers of class II hydrophobin, indicating that the crystal structure resembles that of natural hydrophobin film. The orthorhombic crystal structure (1.0 A resolution, R = 13, R(free) = 15) is composed of fiber-like arrays of protein molecules. Rodlet structures have been observed on amphiphilic layers formed by class I hydrophobins; fibrils of class II hydrophobins appear by vigorous shaking. We propose that the structure of the fibrils and/or rodlets is similar to that observed in the crystal structure.     

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.     

Ultrastructural studies on the nematode Xiphinema diversicaudatum: Egg shell formation

The ultrastructure of the formation of the egg shell in the longidorid nematode Xiphinema diversicaudatum is described. Upon fertilization a vitelline membrane, which constitutes the vitelline layer of the egg shell, is formed. The chitinous layer is secreted in the perivitelline space, between the vitelline layer and the egg cell membrane. On completion of the chitinous layer, the material of the lipid layer is extruded from the egg cytoplasm to the outer surface, through finger-like projections. Both chitinous and lipid layers are secreted by granules in the egg cytoplasm that disappear as the layers are completed. Chitinous and lipid layers are formed during the passage of the egg through the oviduct. The vitelline layer is enriched with secretions produced by the oviduct cells and then by phospholipids secreted by the cells of the pars dilatata oviductus. The inner uterine layer is also formed by deposition of secretory products apposed on the egg shell in the distal uterine region and Z-differentiation. In the proximal part of the uterus, the egg has a discontinuous electron-dense layer, the external uterine layer. Tangential sections between chitinous and uterine layers revealed the presence of holes, possibly egg pores, delimited by the two uterine layers.