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Structural analysis of CsoS1A and the protein shell of the Halothiobacillus neapolitanus carboxysome - PubMed

Structural analysis of CsoS1A and the protein shell of the Halothiobacillus neapolitanus carboxysome

Yingssu Tsai et al. PLoS Biol. 2007 Jun.

Abstract

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.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Electron Micrographs of H. neapolitanus Carboxysomes

(A) A thin-section electron micrograph of H. neapolitanus cells with carboxysomes inside. In one of the cells shown, arrows highlight the visible carboxysomes. (B) A negatively stained image of intact carboxysomes isolated from H. neapolitanus. The features visualized arise from the distribution of stain around proteins forming the shell as well as around the RuBisCO molecules that fill the carboxysome interior. Scale bars indicate 100 nm.

Figure 2
Figure 2. Structure of the CsoS1A Hexamer That Forms the Building Block of the Alpha Type Carboxysome Shell

(A) A ribbon diagram with the monomeric units colored alternately in purple and teal. (B) A close-up of the CsoS1A monomer in the hexamer. Secondary structures are labeled and numbered. (C) An illustration of the differing C-terminal configurations of carboxysome shell proteins: CsoS1A (blue), CcmK2 (pink), and CcmK4 (green). The differences between the structures are apparent after residue 91, with the C-terminus of CsoS1A intermediate in position compared to the termini of CcmK2 and CcmK4. (D) An illustration of the geometric and electrostatic differences between the concave (top) and convex (bottom) sides of the CsoS1A hexamer. The concave side of CsoS1A is composed of mostly positive electrostatic potential (blue), whereas the convex side is composed of mostly negative electrostatic potential (red).

Figure 3
Figure 3. Packing of CsoS1A Hexamers in a Layer

(A) Individual CsoS1A molecules are colored differently in each hexamer. CsoS1A molecules in the same hexameric position are colored the same. (B) Backbone alignment of adjacent CsoS1A hexamers and adjacent CcmK2 hexamers [7] from the beta-type cyanobacterial carboxysome shell. CsoS1A is indicated in blue, and CcmK2 is shown in orange. The shift between adjacent hexamers in CsoS1A compared to CcmK2 leads to the tighter packing. (C) A close-up view of the interaction between two adjacent CsoS1A hexamers. The two hexamers are colored separately in yellow and gray. Arg83 from each hexamer is shown surrounded by residues belonging to the adjacent hexamer: Val33 (pink), Ala31 (green), and Thr28 (light blue).

Figure 4
Figure 4. Alignment of the Amino Acid Sequence of H. neapolitanus CsoS1A to Its Paralogs and to the Two Syn. 6803 Orthologs of Known Structure

Accessible residues on the concave side are in cyan, whereas those on the convex side are in magenta. Accessible residues that are conserved between all three CsoS1 paralogs are also colored in the other species.

Figure 5
Figure 5. A Sulfate Ion Found in the Pore of the CsoS1A Hexamer

The (Fobs-Fcalc) difference electron density map shown was calculated using diffraction data from a crystal soaked in sodium sulfate. The map is contoured at 3σ. The view is with the pore (arrow) running vertically in the plane of the paper. The pore is lined by the polypeptide backbone from all six proteins in a hexamer (two copies are shown). The identity of the sulfate ion was supported by an anomalous difference map (Figure S3).

Figure 6
Figure 6. Calculated Radius of the Central Pore through CsoS1A, Shown in Comparison to the Pores through Transmembrane Channel Proteins

In each case, the radius of the pore is plotted as a function of the vertical position in the layer. CsoS1A is indicated in black and compared to the acetylcholine receptor pore (1OED), the aquaporin1 water channel (1J4N), the cytoplasmic domain of the inward rectifier potassium channel 1 (1N9P), and the human potassium channel Kv β-subunit (1ZSX). The CsoS1A pore is much shorter in length and more conical in shape than the pores through transmembrane channel proteins. The position of the CsoS1A curve along the horizontal axis was chosen arbitrarily.

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