Structure of the surface layer of the methanogenic archaean Methanosarcina acetivorans.

Archaea have a self-assembling proteinaceous surface (S-) layer as the primary and outermost boundary of their cell envelopes. The S-layer maintains structural rigidity, protects the organism from adverse environmental elements, and yet provides access to all essential nutrients. We have determined the crystal structure of one of the two "homologous" tandem polypeptide repeats that comprise the Methanosarcina acetivorans S-layer protein and propose a high-resolution model for a microbial S-layer. The molecular features of our hexameric S-layer model recapitulate those visualized by medium resolution electron microscopy studies of microbial S-layers and greatly expand our molecular view of S-layer dimensions, porosity, and symmetry. The S-layer model reveals a negatively charged molecular sieve that presents both a charge and size barrier to restrict access to the cell periplasmic-like space. The ß-sandwich folds of the S-layer protein are structurally homologous to eukaryotic virus envelope proteins, suggesting that Archaea and viruses have arrived at a common solution for protective envelope structures. These results provide insight into the evolutionary origins of primitive cell envelope structures, of which the S-layer is considered to be among the most primitive: it also provides a platform for the development of self-assembling nanomaterials with diverse functional and structural properties.

Comparative proteomics identifies the cell-associated lethality of M. tuberculosis RelBE-like toxin-antitoxin complexes.

The Mycobacterium tuberculosis (Mtb) genome encodes approximately 90 toxin-antitoxin protein complexes, including three RelBE family members, which are believed to play a major role in bacterial fitness and pathogenicity. We have determined the crystal structures of Mtb RelBE-2 and RelBE-3, and the structures reveal homologous heterotetramers. Our structures suggest RelE-2, and by extension the closely related RelE-1, use a different catalytic mechanism than RelE-3, because our analysis of the RelE-2 structure predicts additional amino acid residues that are likely to be functionally significant and are missing from analogous positions in the RelE-3 structure. Toxicity assays corroborate our structural findings; overexpression of RelE-3, whose active site is more similar to Escherichia coli YoeB, has limited consequences on bacterial growth, whereas RelE-1 and RelE-2 overexpression results in acute toxicity. Moreover, RelE-2 overexpression results in an elongated cell phenotype in Mycobacterium smegmatis and protects M. tuberculosis against antibiotics, suggesting a different functional role for RelE-2.

Heterologous expression of mycobacterial Esx complexes in Escherichia coli for structural studies is facilitated by the use of maltose binding protein fusions.

The expression of heteroligomeric protein complexes for structural studies often requires a special coexpression strategy. The reason is that the solubility and proper folding of each subunit of the complex requires physical association with other subunits of the complex. The genomes of pathogenic mycobacteria encode many small protein complexes, implicated in bacterial fitness and pathogenicity, whose characterization may be further complicated by insolubility upon expression in Escherichia coli, the most common heterologous protein expression host. As protein fusions have been shown to dramatically affect the solubility of the proteins to which they are fused, we evaluated the ability of maltose binding protein fusions to produce mycobacterial Esx protein complexes. A single plasmid expression strategy using an N-terminal maltose binding protein fusion to the CFP-10 homolog proved effective in producing soluble Esx protein complexes, as determined by a small-scale expression and affinity purification screen, and coupled with intracellular proteolytic cleavage of the maltose binding protein moiety produced protein complexes of sufficient purity for structural studies. In comparison, the expression of complexes with hexahistidine affinity tags alone on the CFP-10 subunits failed to express in amounts sufficient for biochemical characterization. Using this strategy, six mycobacterial Esx complexes were expressed, purified to homogeneity, and subjected to crystallization screening and the crystal structures of the Mycobacterium abscessus EsxEF, M. smegmatis EsxGH, and M. tuberculosis EsxOP complexes were determined. Maltose binding protein fusions are thus an effective method for production of Esx complexes and this strategy may be applicable for production of other protein complexes.