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

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.

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

“…1,20 In the a-carboxysome from the chemoautotrophic bacterium Halothiobacillus neapolitanus [ Fig. 1(C)], which has been studied extensively, 1,7,21 the csoS1 genes code for three paralogs of this shell protein (CsoS1ABC). Similarly, the shell proteins in the b-carboxysome are encoded by a series of homologous ccm-type genes.…”

“…Recent cryo-electron tomography studies provided important insights into the architecture of carboxysomes by showing that this bacterial microcompartment has a nearly icosahedral shape. 23,24 In complementary work, X-ray crystallographic studies have revealed the atomic structures of homologous BMC-type shell proteins from both types of carboxysome, including CcmK2 and CcmK4 from the b-carboxysome 6,7 and CsoS1A from the a-carboxysome. 7 The various carboxysome shell BMC proteins all form hexamers having a narrow central pore bearing a positive electrostatic potential.…”

“…23,24 In complementary work, X-ray crystallographic studies have revealed the atomic structures of homologous BMC-type shell proteins from both types of carboxysome, including CcmK2 and CcmK4 from the b-carboxysome 6,7 and CsoS1A from the a-carboxysome. 7 The various carboxysome shell BMC proteins all form hexamers having a narrow central pore bearing a positive electrostatic potential. Based on the tendency of the hexamers to pack side by side into molecular layers, it has been surmised that these layers represent the flat facets of the shell, and that the narrow pores likely serve for molecular transport.…”

“…Based on the tendency of the hexamers to pack side by side into molecular layers, it has been surmised that these layers represent the flat facets of the shell, and that the narrow pores likely serve for molecular transport. 6,7,13 A crystal structure of a BMC-type hexamer from a noncarboxysomal microcompartment-the propanediol utilization or pdu microcompartment, which requires the transport of different molecules across its shell-has a different pore structure. 25 Crystal structures of minor carboxysome shell proteins CcmL and CsoS4A, unrelated to the BMC family, were recently elucidated and found to be pentameric.…”

“…The hexameric structures of the CcmK proteins have been elucidated, but which side faces toward the carboxysome interior-and would therefore be most likely to be involved in protein-protein interactions-is uncertain. 7,8 Here we present a series of crystal structures of the CcmK1 and CcmK2 proteins in full length forms and as C-terminal truncations. Comparisons illustrate the structural features that are most conserved, while highlighting potentially relevant structural variations and providing further insights into molecular transport across pores through the hexagonally packed protein layers.…”