Comparative analysis of carboxysome shell proteins
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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.
KeywordsCarboxysome CO2 fixation Bacterial microcompartment Dark reactions Cyanobacteria Calvin cycle Chemoautotroph
Ribulose bisphosphate carboxylase/oxygenase
Hidden Markov model
Protein Data Bank
Root mean square deviation
Carbon concentrating mechanism
Open reading frame
Structural and functional overview
There are several differences in the complement of genes that are necessary for carboxysome formation. In addition to encapsulating RuBisCO, the α-carboxysome contains an unusual β-CA (Sawaya et al. 2006) for the conversion of bicarbonate to carbon dioxide and yet to be characterized structural protein, CsoS2 (Baker et al. 1999). A β-CA is also encapsulated in the β-carboxysome of some cyanobacteria (So et al. 2002).
All β-carboxysome gene clusters encode two proteins, CcmM and CcmN (Ludwig et al. 2000), that are also thought to play a catalytic and/or organizational role in the carboxysome interior. CcmM contains 3–5 repeats of the RuBisCO small subunit domain in its C-terminus, while the N-terminal domain is homologous to a γ-type CA (Cot et al. 2008; Long et al. 2007). This domain has been shown to be catalytically active in an organism that lacks the β-CA ortholog (Peña et al. 2010). CcmM has also been shown to interact with the RuBisCO large subunit (RbcL), the proteins of the shell, CcmN, and the CA CcaA (Cot et al. 2008; Long et al. 2007, 2010).
The structures of the BMC domain: a key building block of the carboxysome shell
Crystal structures of the CsoS1A (Tsai et al. 2007) and CsoS1C (Tsai et al. 2009) proteins from the α-carboxysome of Halothiobacillus neapolitanus have also been determined. These displayed the same concave/convex sidedness and uniformly oriented layer formation as observed for CcmK2. Despite a high degree of sequence homology between CsoS1A and CsoS1C (97% identity), a comparison of the electrostatics of these structures shows a difference in the charge distribution on the concave faces (Fig. 6). There is a single amino acid substitution between CsoS1A and CsoS1C at position 97 (from Glu to Gln) that apparently accounts for this difference in electrostatic potential.
The primary structures of CsoS1B, CcmK1, and CcmK4 contain a C-terminal extension of ~10 residues compared to their paralogs. A comparison of the structures of CcmK2 and CcmK4 from Synechocystis sp. PCC6803 reveals that the additional C-terminal residues of CcmK4 form an α helix. In CcmK2 a short, five residue helix occludes the depression in the concave face of the hexamer; in CcmK4 the additional C-terminal residues form an extended helix that folds back on the edge of the hexamer, leaving the concave side unobstructed (Figs. 6, 7). The structure of CcmK1 is missing its C-terminal 17 residues (Tanaka et al. 2009), but based on sequence similarity to the C-terminus of CcmK4 it could likewise be helical. This C-terminal extension may offer clues to the as yet unknown orientation of the shell proteins with regard to which side faces the cytosol. If facing the interior of the carboxysome, the disposition of this helix may be important for interacting with encapsulated proteins. A second hypothesis is that the orientation of the helix might act as a switch that can change the propensity for incorporation of the shell protein into an assembling shell (Kerfeld et al. 2005).
Pentameric proteins of the carboxysome shell
Representative structures of proteins containing the Pfam03319 domain have been solved from both the α- and β-carboxysome (Tanaka et al. 2008). CsoS4A is one of two paralogs (CsoS4A and B) in the α-carboxysome, and CcmL is the only protein with a Pfam03319 domain found among the β-carboxysome genes (Fig. 3). CcmL and CsoS4A have been structurally characterized (Tanaka et al. 2008); both form pentamers and have a pronounced concave/convex sidedness similar to the hexamers. In contrast to the hexameric shell proteins, the electrostatic potential of these proteins is predominantly positive (Fig. 6). The structures of CcmL and CsoS4A can be superimposed with an RMSD of 0.74 Å over 58 C-α atoms. The largest difference between the primary structures of these two proteins is in the region corresponding to an 8–10 amino acid loop on the concave face of the pentamer that seems to influence the charge of the concave face. A similar difference is seen between the paralogs CsoS4A and CsoS4B. In this region CsoS4B has more positively charged residues than CsoS4A.
Based on the current models of carboxysome function and structure, pores in the shell protein hexamers provide conduits for the flux of metabolites; bicarbonate ions and RuBP diffuse in and 3PGA to diffuses out, while preventing the leakage of CO2 from the interior (Dou et al. 2008). The shell also prevents oxygen from diffusing in, reducing unwanted photorespiration by RuBisCO (Marcus et al. 1992). As the shell localizes CA and RuBisCO together, the overall rate of CO2 fixation by RuBisCO is enhanced; effectively, the carboxysome provides a focal point for the carbon concentrating mechanism (CCM) (Fig. 2).
List of structurally characterized BMC-domain proteins from the carboxysome and their dimensions
Hexamer diameterb (Å)
Hexamer edge lengthc (Å)
Pore diameter (Å)
List of structurally characterized pentameric Pfam03319 domain-containing proteins from the carboxysome and their dimensions
Pentamer diametera (Å)
Pentamer edge lengthb (Å)
Pore diameter (Å)
Tandem BMC proteins
Among the genes encoding components of both the α- and β-carboxysomes are some containing fusions of BMC domains (Fig. 3): CsoS1D in the α-carboxysome and CcmO and a CsoS1D ortholog (slr0169 in Synechocystis sp. PCC6803) in the β-carboxysome. In 2009, the first structure of a tandem BMC protein was determined, CsoS1D of Prochlorococcus marinus MED4 (Klein et al. 2009). This protein was not predicted to contain two BMC domains; the N-terminal domain lacks obvious sequence similarity to any other BMC domain. However, the α-carbon backbones of the two domains superimpose with an RMSD of 1.27 Å over 95 atoms; guided by a structure-based sequence alignment, the domains are 18% identical. CsoS1D forms trimers resulting in pseudohexamers that are similar in dimensions to hexameric shell proteins (Table 1), with pronounced concave and convex sides (Fig. 9). The edges of the pseudohexamers contain the conserved D-X-X-X-K edge motif and CsoS1D could be readily fitted into existing models of the facets of the α-carboxysome shell (Fig. 5) (Klein et al. 2009).
Interestingly, in two independent crystal structures, the CsoS1D trimers stacked to form a dimer of trimers (Fig. 10). The two trimers were rotated ~60° with respect to each other so that the C-terminal domain of a subunit in the upper trimer interacted with the N-terminal domain of a subunit in the lower trimer. The dimerization was across the concave face of each trimer, resulting in a large cavity of 13,613 Å3. Additional biophysical analyses that support the potential biological relevance for the dimer of trimers include a buried surface area of 6,573 Å2 and a shape correlation value of 0.70 (range of 0–1, 1 being a perfect fit and 0 being no interaction) between the two trimers (Klein et al. 2009). The cavity could, like the pore gating, influence the flux of larger metabolites (e.g., RuBP, 3PGA) into and out of the carboxysome in a manner analogous to an airlock. For example, the trimer facing the cytosol would open to accept a metabolite and then close; subsequently, the trimer facing the carboxysome interior would open to allow for release of the metabolite from the cavity (Fig. 10).
An ortholog to CsoS1D, with the locus tag slr0169 in Synechocystis sp. PCC6803, has also been identified in all β-carboxysome-containing cyanobacteria (Klein et al. 2009). It is ~200 amino acids in length and lacks ~50 N-terminal residues that are present in the α-cyanobacterial CsoS1D homologs. slr0169 contains the conserved Glu and Arg residues (Glu69, Arg70) responsible for gating the CsoS1D pore as well as the universally conserved edge Lys residues in the N- and C-terminal domains (Lys108, Lys212) for interacting with other hexamers to incorporate into the shell (Cai et al. in press).
A second ~200 amino acid BMC-domain protein is found only in low-light adapted strains of Prochlorococcus and some marine Synechococcus species. This protein, dubbed CsoS1E, is similar to CsoS1D in that it has a C-terminal BMC domain, but its N-terminus lacks homology to any other known domain. A remarkable feature of CsoS1E is its high-isoelectric point of 10.3, with positively charged residues concentrated in the N-terminal half of the protein. Further structural studies are needed to determine whether this N-terminus will form a BMC domain much like the cryptic N-terminal BMC domain of CsoS1D. Finally, CcmO represents another type of tandem BMC-domain protein that is present in all the β-cyanobacteria. It is ~260 amino acids in length and appears to be a fusion of two CcmK-like proteins. It is known to be essential for carboxysome formation in the β-cyanobacteria (Marco et al. 1994).
Variability of shell composition
Both the α- and β-carboxysome shells are composed of multiple paralogs of single BMC-domain proteins. The reason for this redundancy is unknown. One hypothesis is that the carboxysome shell composition might be altered by a change in the environment; this is consistent with the observation that the size of the pore varies among paralogs. Alternatively, hexamers could form from more than one paralog, resulting in hetero-hexamers. By modulating the shell protein composition, selectivity for metabolites may be increased or decreased based on the charge or size differences present at the pores of the shell subunits. This could help to increase the organism’s fitness in a wider variety of growth conditions.
Some evidence for modulation of shell protein expression under different conditions has come from transcriptome analysis of the β-cyanobacterium Synechocystis sp. PCC6803, where the expression of the CsoS1D ortholog, slr0169 is greater under high-light and low-carbon stresses and clusters with other carboxysome shell components (Cai et al. in press; Eisenhut et al. 2007).
Conclusions and future prospects
Structural information for the building blocks of the carboxysome shell is rapidly accumulating. With the current knowledge, several convincing models of the protein interactions involved in forming the carboxysome have been built (Cot et al. 2008; Iancu et al. 2007; Long et al. 2007; Tanaka et al. 2008) and attractive hypotheses regarding the metabolic flux and function of the shell have been posited (Dou et al. 2008; Fridlyand et al. 1996). An area that needs more attention is the structural characterization and analysis of the interactions among the encapsulated proteins (CsoS2, CcmN and CcmM and RuBisCO). Also, little is known about the assembly of the carboxysome and the dynamics of the shell. More sophisticated imaging methods and/or gene expression analysis under controlled growth conditions may give a better idea as to the composition of the carboxysome shell.
When the first structural characterization of carboxysome shell proteins was reported, it was pointed out that proteins with homology to carboxysome shell proteins are widespread among bacteria (Kerfeld et al. 2005). Collectively, these are known as BMCs. Our bioinformatic analyses of all sequenced bacterial genomes that have a BMC-domain homolog have yielded a phyletically and functionally diverse set of BMCs, a majority of which have yet to be characterized (Kerfeld et al. 2010). Approximately 20% of bacteria with genomic sequence data have open reading frames (ORFs) coding for BMC-domain proteins. The distribution of BMC shell proteins across the bacterial phyla has been suggested to be the product of horizontal gene transfer. Inferences can be made as to the function of unknown BMC operons using a “guilt-by-association” analysis of the putative operon, where the enzymes near known BMC-domain homologs and a Pfam03319 homolog are analyzed and an encapsulated metabolism proposed. Most of the functionally uncharacterized BMCs belong to heterotrophic organisms. An interesting observation from comparison of the genomes of Rhodopseudomonas palustris strains, which can grow autotrophically, is that only strain BisB18 contains a BMC gene cluster, and it is associated with a glycyl-radical enzyme but not RuBisCO.
Two types of heterotrophic BMCs are well characterized. Studies of the propanediol utilization (pdu) BMC and the ethanolamine utilization (eut) BMC mostly in Salmonella typhimurium LT2 have yielded other important clues involving the structure, function, and assembly of microcompartment shells (Crowley et al. 2008; Parsons et al. 2008; Sagermann et al. 2009). Surprisingly, several of the pdu single BMC-domain proteins and those of the β-carboxysome are very similar and share the same pore residues although they are encapsulating completely different enzymatic reactions. Another curious observation from the eut microcompartment is that the oligomeric state of the Pfam03319 homolog EutN (Tanaka et al. 2008; Wunderlich et al. 2004) is a hexamer and not a pentamer as in the CcmL and CsoS4A structures. Thus, the possibility that carboxysome shell proteins may display quasi-equivalency like viral capsid proteins, where the protein can be either a hexamer or a pentamer, cannot be ruled out. Since BMCs were first observed, their resemblance to viral capsids has been pointed out (Gantt and Conti 1969; Shively et al. 1973). Although microcompartments are larger than viral capsids, they can be modeled as icosahedra. However, an evolutionary link between microcompartments and viral capsids, from either sequence or structural data, has not been established.
We thank Fei Cai, Annette Salmeen, Gustaf Sandh, and William Greenleaf for helpful discussions. We also thank Patrick Shih for the transmission electron micrograph image. This work was supported by the U.S. Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357. The JGI is under the auspices of the US Department of Energy’s Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract number DE-AC02-05CH11231, Lawrence Livermore National Laboratory under contract number DE-AC52-07NA27344.
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