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Structure and Mechanisms of a Protein-Based Organelle in Escherichia coli

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Science  01 Jan 2010:
Vol. 327, Issue 5961, pp. 81-84
DOI: 10.1126/science.1179513

Abstract

Many bacterial cells contain proteinaceous microcompartments that act as simple organelles by sequestering specific metabolic processes involving volatile or toxic metabolites. Here we report the three-dimensional (3D) crystal structures, with resolutions between 1.65 and 2.5 angstroms, of the four homologous proteins (EutS, EutL, EutK, and EutM) that are thought to be the major shell constituents of a functionally complex ethanolamine utilization (Eut) microcompartment. The Eut microcompartment is used to sequester the metabolism of ethanolamine in bacteria such as Escherichia coli and Salmonella enterica. The four Eut shell proteins share an overall similar 3D fold, but they have distinguishing structural features that help explain the specific roles they play in the microcompartment. For example, EutL undergoes a conformational change that is probably involved in gating molecular transport through shell protein pores, whereas structural evidence suggests that EutK might bind a nucleic acid component. Together these structures give mechanistic insight into bacterial microcompartments.

Bacterial microcompartments are present in diverse bacteria, where they function as protein-based organelles (16). They range in size from just under 1000 Å to around 1500 Å, and they typically have a polyhedral shape (Fig. 1A). Each type of microcompartment contains a few different enzymes that catalyze sequential metabolic reactions. The enzymes are encapsulated by a shell formed from a few thousand shell protein subunits. The simplest microcompartment is the carboxysome, which encapsulates the two enzymes carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) in order to enhance cellular CO2 fixation (1, 7, 8). Recent structural studies on the carboxysome and its shell proteins have provided a basic understanding of how that type of microcompartment is assembled and how it operates (914).

Fig. 1

A model for the Eut microcompartment and its metabolic pathway. (A) A hypothetical model of the Eut microcompartment emphasizing the construction of a semiregular polyhedron primarily from hexameric shell proteins. (B) A model for the metabolism of ethanolamine in the Eut microcompartment. Ethanolamine is converted into ethanol, acetyl-phosphate, and acetyl–coenzyme A (CoA), to be used in the tricarboxylic acid cycle for energy production. The volatile intermediate, acetaldehyde (boxed in orange), is consumed before it can escape the protein shell. The four homologous shell proteins belonging to the conserved BMC family (EutK, EutL, EutM, and EutS) are colored in light blue. The unrelated EutN protein (dark pink) may be a minor structural component of the shell, based on analogy to the carboxysome (11). Enzymes thought to be associated with the microcompartment are indicated. Protein names: EutBC, ethanolamine ammonia-lyase; EutD, phosphotransacetylase; EutE, aldehyde dehydrogenase; EutG, alcohol dehydrogenase. The eut operon (fig. S1) encodes other enzymes and proteins involved in ethanolamine utilization, which are not shown.

Other bacterial microcompartments with more complex metabolic functions have also been discovered (5, 15). One of these, which is dedicated to ethanolamine utilization (Eut), is present in several bacteria, including Salmonella enterica and Escherichia coli (4, 16). The Eut microcompartment shares a number of homologous enzymes with a propanediol utilization (Pdu) microcompartment; both metabolic pathways proceed via aldehyde intermediates, propionaldehyde in the case of Pdu and acetaldehyde in the case of Eut (3). Experiments in Salmonella have shown that the cellular function of the Eut microcompartment is to metabolize ethanolamine without allowing the release of acetaldehyde into the cytosol (Fig. 1B), thus mitigating the potentially toxic effects of excess aldehyde in the bacterial cytosol (1719) and also preventing the volatile acetaldehyde from diffusing across the cell membrane and leading to a loss of carbon (20).

The major shell proteins from known bacterial microcompartments belong to a family of homologous proteins that are typically just over 100 amino acids long, referred to here as bacterial microcompartment (BMC) proteins. The amino-acid sequences of BMC proteins were determined first from microcompartments of the carboxysome type (21), and can now be identified in some 40 genera across the bacterial kingdom (2, 3). In Salmonella and E. coli, the 17-gene eut operon codes for four homologous BMC proteins: EutK, EutL, EutM, and EutS [(4) and supporting online material (SOM) text]. Genetic studies in Salmonella have shown that EutL, EutM, and EutK are required for growth on ethanolamine when it is the sole carbon source at high pH, and their deletion leads to rapid loss of acetaldehyde (20). Likewise, deleting EutS led to a measurable loss of acetaldehyde but did not cause a growth phenotype under the conditions examined. A fifth protein, EutN, which is probably a minor shell component but not homologous to the BMC shell proteins, was also shown to be essential for growth on ethanolamine; its structure was reported previously (11). Here we report the crystal structures of the BMC shell proteins from E. coli: EutM, EutS (wild-type and mutant), EutL (in two crystal forms), and a domain of EutK. The Eut shell proteins from E. coli share between 79 and 96% sequence identity with their orthologs from Salmonella (fig. S1 and SOM text), where functional studies have been performed (17, 18, 20).

The crystal structure of EutM was determined to a resolution of 2.1 Å. The 97–amino acid EutM protein adopts a 3D fold with an α/β structure that matches closely those reported earlier for proteins forming the shell of the carboxysome (9, 10). Six EutM subunits self-assemble to make a flat cyclic hexamer with a bowl-shaped depression on one side, punctuated by a narrow central pore (Fig. 2A). Similar hexameric structures have been established as the basic building blocks from which microcompartment shells are assembled by the tight packing of many hexamers side by side into a molecular layer or sheet (Fig. 1A) (9, 10, 12). Electron density, interpreted to be a sulfate ion from the crystallization mixture, was visualized in the center of the EutM pore, in accordance with previous suggestions that small molecules and ions can pass through the centers of the hexameric shell proteins (fig. S2) (9, 10). The relative abundances of the various shell proteins in the Eut microcompartment have not been established, but the EutM protein hexamer, with its relatively typical features, could serve as the basic assembly component of the shell, whereas the other shell proteins, whose unusual features we describe below, could perform more-specialized roles.

Fig. 2

Structure of EutM and EutS shell protein hexamers. (A) Cartoon diagrams of the EutM (left), EutS (middle), and mutant EutS G39V (right) hexamers, shown in two views. The wild-type EutS hexamer is bent away from a flat configuration by approximately 40° (SOM text). This asymmetric configuration of wild-type EutS was converted into a flat symmetric configuration by the G39V mutation. (B) A hypothetical model showing how EutS (orange) might introduce curvature in an otherwise flat hexameric sheet of shell protein hexamers. Hexamer interfaces in the model are based on packing arrangements that are conserved in other crystal structures. From a row of EutS hexamers at an edge, flat hexamers could extend to form facets of the shell without major collisions.

The crystal structure of EutS was determined at 1.65 Å resolution. The 111–amino acid protein adopts the conserved α/β core structure expected for BMC proteins (Fig. 2A), but the secondary structure elements occur in nonsequential order relative to the typical BMC fold. Similar circular permutations of the BMC protein fold have been observed previously (2224). Unlike other BMC proteins that have formed flat hexameric structures, the EutS hexamer has a bend of approximately 40° (Fig. 2 and SOM text). As a result, the six chemically equivalent protein subunits exist in three distinct structural environments. The closest parallels to this asymmetric assembly are to the proteins of certain viral capsids in which equivalent protein subunits are present in quasiequivalent, but distinct, environments (25, 26). The observation could be helpful in explaining microcompartment architecture, because previously observed (flat) BMC hexamers have not clarified how the edges of polyhedral microcompartment shells might be formed.

The cause of the bent EutS structure was investigated by mutagenesis. A glycine residue (Gly39) involved in a crystal contact was mutated to a valine side chain (G39V). Valine is the corresponding amino acid in a homologous shell protein from the Pdu microcompartment, namely PduU, which forms a flat symmetric hexamer (22). The G39V mutant of EutS migrated more slowly in a native gel (despite an unaltered net charge), implicating a substantial conformational change (fig. S3). That observation was consistent with a crystal structure that demonstrated that the mutated EutS was flattened into a symmetric structure (Fig. 2A). Thus Gly39 appears to play an important role in bending EutS. Gly39 is conserved among EutS proteins from the eut operon of other bacteria but is absent from other BMC proteins.

EutL is the longest of the Eut shell proteins. A recent structure (24) showed that it contains within a single polypeptide chain two tandem domains (Fig. 3A), with each domain being a circular permutation of the typical BMC fold (22), similar to EutS. As such, three copies of EutL assemble to form a pseudohexameric structure. We determined the crystal structure of EutL from two distinct crystal forms, both at a resolution of 2.3 Å. A comparison of the two structures revealed that protein backbone movements of nearly 15 Å (in loops covering residues 65 to 83 and 174 to 185) interconvert the EutL trimer between forms in which the central pore is either open or closed (Fig. 3B). The conformation for EutL identified here as being the closed form matches the one described earlier (24). The central pore is very nearly occluded. Very small openings in this structure were noted earlier as potential routes for transport, but it is likely the open form observed here that is competent for transport. The pore in the open form is triangular in shape, with an edge of approximately 11 Å and a diameter at its narrowest point of about 8 Å. This large pore is salient, because it provides a potential route for transporting bulky molecules such as cobalamin and other nucleotide cofactors that are required by reactions inside the microcompartment (3) (Fig. 1B). Model building suggests that the open pore could accommodate transport of these cofactors with only modest side-chain movements (fig. S6). The existence of a closed form is also potentially important, because the possibility of a triggered or gated opening goes partway toward addressing the paradox of how relatively large molecules might penetrate the shell without allowing rapid loss of the small acetaldehyde intermediate (3). A potential mechanism for selective transport would involve a coupling between cofactor binding and pore opening. A similar conformational change was reported recently in a carboxysome shell protein, CsoS1D (23) (figs. S4 and S5), suggesting that gated transport could be a common mechanism in microcompartments.

Fig. 3

The structure of the EutL shell protein and its gated pore. (A) A cartoon ribbon diagram of a EutL monomer in its closed form. The first and second BMC domains are colored in blue and purple, respectively. (B) The open (left) and closed (right) configurations of EutL trimers are shown in both ribbon diagram and surface representations. In both configurations, EutL trimers (or pseudohexamers) were observed to pack into tight molecular layers within their respective crystal forms (bottom). The conformational change between forms involves 15 Å movements of the protein backbone (fig. S4).

Like some other BMC proteins, EutL was found to associate into tightly packed molecular layers within both of the crystal forms characterized here (Fig. 3B). This observation lends further support to models for microcompartment architecture in which the shell is formed by the tight side-by-side packing of hexameric (or pseudohexameric) protein building blocks into a molecular layer or sheet (911). The appearance of the EutL protein layer, composed of subunits in either the open or closed configuration, illustrates how strongly the porosity of the microcompartment would be affected by the conformational change in EutL (Fig. 3B).

The fourth shell protein, EutK, is distinct among the shell proteins in the Eut microcompartment. First, although all other BMC proteins studied to date form hexamers (or pseudohexamers, like EutL), EutK is a monomer in solution (fig. S8). The apparent inability of EutK to assemble into a hexamer by itself suggests that different BMC paralogs might form mixed hexamers during assembly of the shell. Second, EutK has an extra protein domain of about 60 amino acids following the conserved BMC-type domain (Fig. 4A). Numerous instances of BMC-type proteins fused to other uncharacterized protein domains are evident in the protein sequence databases, but structural and functional data are limited (27). Based on sequence similarity of marginal statistical certainty (for example, a 40% probability of similarity due to random chance), the extra domain of EutK could be only tentatively assigned to a broad family of proteins bearing a well-known helix-turn-helix motif, which is common among nucleic acid–binding proteins.

Fig. 4

The structure of the EutK Ctail. (A) A schematic diagram of the BMC domain and the crystal structure of the C-terminal helix-turn-helix domain of EutK, interpreted as a monomer (fig. S7). (B) Superimpositions of the structure of the EutK-Ctail on four representative DNA/RNA binding domains (SOM text, colored according to fig. S9). (C) An electrostatically colored surface diagram of the EutK-Ctail domain emphasizing the positively charged (blue) patch that is characteristic of DNA binding domains.

Whereas crystals could not be grown using the full-length EutK protein, the crystal structure of the C-terminal domain was elucidated at a resolution of 2.1 Å. The crystal structure of this 59–amino acid fragment, referred to hereafter as EutK-Ctail, demonstrates that it is indeed a helix-turn-helix domain (Fig. 4B). A computational search for similar structures in the protein structure database identified many known helix-turn-helix domains as close matches, with coordinate differences as low as 1.0 Å. About 90% of the helix-turn-helix domain structures retrieved from the search bind nucleic acids, whereas a minority perform other varied cellular functions (SOM text). A comparison of the surface features of EutK-Ctail with previously characterized helix-turn-helix domains, including those that bind nucleic acids and those that do not, suggests strongly that EutK is a nucleic acid–binding protein. A prominent, positively charged surface is conserved in EutK-Ctail and in those domains that bind nucleic acids (Fig. 4C). Interestingly, on the basis of structure-guided alignments, EutK-Ctail shows the highest sequence similarity to the helix-turn-helix domains of the Ic1R family of transcription factors, which regulate, among other things, carbon metabolism in enterobacteria (28, 29) (fig. S9). The specific function of EutK and the identity of its cognate nucleic acid are unknown. Nonetheless, the key prediction that the Eut microcompartment shell binds a nucleic acid points up the possibility of unexpected parallels to viral capsids, which bind to and encapsulate their viral genomes.

Taken together, the structures of the shell proteins from the Eut microcompartment paint a picture of a complex, highly evolved organelle. The structural features and conformational changes observed illustrate how these evolutionarily related proteins have diverged to fulfill specialized architectural and biochemical roles in a shell that participates actively in the function of the microcompartment.

Supporting Online Material

www.sciencemag.org/cgi/content/full/327/5961/81/DC1

Materials and Methods

SOM Text

Figs. S1 to S9

Tables S1 to S3

References

References and Notes

  1. We thank T. Bobik, members of the Yeates lab, J. Escalante-Semerena, F. Guo, M. Faller, and Y. Chen for helpful discussions and M. Phillips for equilibrium sedimentation experiments. This work was supported by the Biological and Environmental Research program of the Department of Energy Office of Science and by NSF grant MCB-0843065. Coordinates and structure factors of the EutK-Ctail, EutL open form, EutL closed form, EutM, EutS, and EutS-G39V have been deposited in the Protein Data Bank under accession numbers 3I71, 3I87, 3I82, 3I6P, 3I96, and 3IA0, respectively.
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