Crystallographic insights into the pore structures and mechanisms of the EutL and EutM shell proteins of the ethanolamine-utilizing microcompartment of Escherichia coli

Abstract

The ethanolamine-utilizing bacterial microcompartment (Eut-BMC) of Escherichia coli is a polyhedral organelle that harbors specific enzymes for the catabolic degradation of ethanolamine. The compartment is composed of a proteinaceous shell structure that maintains a highly specialized environment for the biochemical reactions inside. Recent structural investigations have revealed hexagonal assemblies of shell proteins that form a tightly packed two-dimensional lattice that is likely to function as a selectively permeable protein membrane, wherein small channels are thought to permit controlled exchange of specific solutes. Here, we show with two nonisomorphous crystal structures that EutM also forms a two-dimensional protein membrane. As its architecture is highly similar to the membrane structure of EutL, it is likely that the structure represents a physiologically relevant form. Thus far, of all Eut proteins, only EutM and EutL have been shown to form such proteinaceous membranes. Despite their similar architectures, however, both proteins exhibit dramatically different pore structures. In contrast to EutL, the pore of EutM appears to be positively charged, indicating specificity for different solutes. Furthermore, we also show that the central pore structure of the EutL shell protein can be triggered to open specifically upon exposure to zinc ions, suggesting a specific gating mechanism.

FIG. 1.

(A to C) Full-atom representation of a EutL trimer (tile) superimposed onto a ribbon diagram. (A) “Closed” conformation; (b) “open” conformation. The backbone atoms of individual monomers are colored in blue, green, and yellow, respectively. The solvent channel of the zinc-free protein is highlighted by the blue arrow. The positions of the zinc atoms are shown in red (red arrow). (C) Ribbon diagram of EutL closed (light gray) and EutL open (rainbow colored with N terminus in blue and C terminus in green) superimposed. The two loop structures that underwent dramatic conformational changes are highlighted in red. In this crystal form, two molecules are contained within the asymmetric unit. The F_o-F_c electron density of the zinc was calculated at a resolution of 2.7 Å (6.0 σ above the mean) and is shown in red.

FIG. 2.

Stereo diagram of the structural environment of the zinc binding site at the interface between two adjacent monomers (highlighted in blue and in yellow). Superimposed onto this structure is the loop conformation (residues 78 to 82 (purple)) of EutL in the absence of zinc. The F_o-F_c electron density was calculated same way as in Fig. 1C. The density was observed at the N-terminal site of helix 4 involving the carboxylate of Glu157 and the backbone oxygen of Pro155. Systematic soaks of the crystals with either water, buffers at various pH values, or other metals revealed that only the addition of zinc chloride resulted in a dramatic opening of the channel. F_{o (zinc)}-F_{o (native)} electron densities calculated in the absence of any solvent molecules resulted in similarly strong positive densities at this location.

FIG. 3.

Crystal packing of the EutM monomers into a 2D proteinaceous membrane. Shown is the crystal lattice of crystal form I. Despite the different crystallization conditions and packing interactions, the lateral packing interactions of the two-dimensional lattice of crystal forms I and II remained the same. Also visible is the central phosphate ion trapped in the pore of each tile. The honeycomb-patterned tile packing is thereby similar to the one observed in EutL and other carboxysomal crystal structures, suggesting a common assembly principle. The corresponding interactions between the tiles (within the red rectangle) are highlighted in Fig. 6.

FIG. 4.

Ribbon diagram of the respective monomers of EutM in crystal forms I and II. The rainbow coloring is shown with blue at the N terminus and with red at the C terminus. In both cases, the last seven residues adopt a fundamentally different conformation. In crystal form I, however, its structure resembled the corresponding peptide conformation of CcmK2. In all cases, the structure of the His tag was not visible.

FIG. 5.

(A to D) Surface representation of the electrostatic charge distribution of a EutM tile of crystal form II. The convex site of the tile (A) exhibits a moderately negatively charged crown (red) with a strongly positively charge in the center leading to the channel (blue). A somewhat stronger negative charge distribution was evident on the concave site of the tile (B), which is largely caused by the C-terminal extension of this crystal form. The strongest charge distribution was observed within the channel (C). The high concentration of positive field lines within the channel is shown in panel D. The positive charge of the channel is likely to provide access for negatively charged solutes to enter or leave the BMC. Electrostatic calculations and surface representations were performed with APBS (2) and VMD (12).

FIG. 6.

Congruent tiling of the protein hexagons within each two-dimensional lattice. The corresponding regions of the proteins CcmK2, EutM (form I and form II) and EutL are shown, with the contacting residues highlighted as a “stick” representation instead of as a “wire.” The respective anions are also shown as “sticks.” The crystal contacts of both CcmK2 and EutM (both crystal forms) are maintained by specific residues that are arranged in a 2-fold symmetrical manner (with the 2-fold axis shown as a blue oval). In crystal form II, a sulfate ion sits on the 2-fold axis. EutL also exhibits large residue side chains such as arginines and lysines in the interface, but at different relative positions, suggesting that the crystal contacts of EutL and EutM may be incompatible for mixed tiling. Furthermore, due to the imperfect sequence repeat of both domains within EutL, only a pseudo-2-fold rotation of the contacting residues is observed (the pseudo-axis is shown as a red oval).