Crystal structure of the EutL shell protein of the ethanolamine ammonia lyase microcompartment

Abstract

Bacterial microcompartments (BMCs) are specialized organelles that use proteinaceous membranes to confine chemical reaction spaces. The ethanolamine ammonialyase microcompartment of Escherichia coli represents such a class of cytosolic organelles that enables bacteria to survive on small organic molecules such as ethanolamine as the sole source for carbon and nitrogen. We present here the crystal structure of the shell protein EutL at 2.2-Å resolution. With 219 residues, it is the largest representative of this BMC's shell proteins. In the crystal, EutL forms a trimer that exhibits a hexagonally shaped tile structure. The tiles arrange into a tightly packed 2D array that is likely to resemble the proteinaceous membrane of the intact BMC. In contrast to other BMC shell proteins, which have only 1 pore per tile, EutL exhibits 3 pores per tile, thereby significantly increasing the overall porosity of this protein membrane. Each of the individual pores is lined with negatively charged residues and aromatic residues that are proposed to facilitate passive transport of specific solutes. The characteristic shape of the hexagonal tile, which is also found in the microcompartments of carbon-fixating bacteria, may present an inherent and fundamental building unit that may provide a general explanation for the formation of differently sized microcompartments.metabolosome ͉ carboxysome ͉ bacterial organelle

Short N-terminal sequences package proteins into bacterial microcompartments

“…-GFP and the MCP, a competition experiment was performed. PduP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] -GFP was produced via an isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible promoter in a WT background that also produced PduP. MCPs were purified, and Western blotting was used to detect the PduP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] -GFP and PduP (Fig.…”

“…A similar experiment was performed by producing PduP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] -MBP in place of PduP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] -GFP. Western blots showed that PduP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]…”

“…To test whether the fusion proteins were encapsulated within the shell, MCPs were purified from a strain producing PduP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] -eGFP and a portion was broken by dialysis and sonication. Intact and broken MCPs were incubated with anti-GFP antibody.…”

“…Recent crystallography has shown that BMC-domain polypeptides assemble into extended protein sheets that are perforated by discrete pores thought to mediate transport of enzyme substrates, products, and cofactors between the lumen of the MCP and the cytoplasm of the cell (12)(13)(14)(15)(16). Different shell proteins have pores that differ in size and charge, and some may have gated pores tentatively suggesting substrate selectively and regulated movement of metabolites across the shell (12)(13)(14)(15)(16)(17)(18). In the literature, MCPs have also been referred to as polyhedral bodies/organelles, enterosomes, and metabolosomes (1, 3, 10).…”

Bacterial Microcompartments

“…A second observation for a putative two-state pore in a tandem BMC protein was described for EutL (58,68), in which the open form has a negatively charged pore of 11 Å in diameter, and the relatively closed form has three small perforations (of 2.2 Å diameter at their narrowest point), each formed within a subunit (Figure 3). An additional observation of pores formed within a tandem BMC protein, similar to those in the relatively closed form of EutL, was reported for the newly described Etu BMC (33).…”