Structure of the surface layer of the methanogenic archaean Methanosarcina acetivorans

Abstract

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 β-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.

Fig. 1.

The M. acetivorans cell envelope. (A) Electron micrograph of the cell envelope of Methanosarcina barkeri, a representative species of the order Methanosarcinales. Cytoplasmic membrane (CM), S-layer (SL), and metachondroitin (MC) are indicated. Reprinted from ref. . Copyright American Society for Microbiology. (B) Cartoon of the cell envelope indicating relative positions of cell envelope structures including the periplasmic-like space (PLS) contained between the S-layer and cytoplasmic membrane. (C) Linear cartoon representation of the domain organization of the M. acetivorans MA0829 S-layer protein including: N-terminal signal sequence (S), N- and C-terminal DUF1608 domains (NTR and CTR), tether region (T), and membrane anchor (M).

Fig. 2.

The crystal structure of the S-layer protein of M. acetivorans. (A) The MA0829 CTR shown in ribbon representation. Domains I and II are colored red and blue, respectively, and the connector subdomain is colored yellow. (B) Crystallographic CTR dimer that likely represents the physiological MA0829 NTR-CTR protein. Domains I and II of the symmetry-related CTR molecule are colored orange and cyan, respectively, and the connector subdomain is colored dark green. (C) Sequence alignment of the sequences for the MA0829 NTR and CTR DUF1608 domains with identical residues in gray shading. Secondary structure elements above the alignment are based on the MA0829 CTR structure and are colored according to the CTR domains in A. Residues predicted to be involved in the NTR-CTR interface based on the CTR dimer structure are indicated by green circles. Numbering is according to the mature processed sequence upon removal of the signal peptide.

Fig. 3.

Comparison of the interfaces of the crystallographic CTR dimer and the predicted NTR-CTR protein. (A) Crystallographic CTR dimer with CTR domains labeled and colored as in Fig. 2A. (B) Model of the NTR-CTR tandem DUF1608 protein. The DUF1608 domains are labeled and colored as in Fig. 2B. The position of the 12-aa residues inserted near the N terminus of the NTR model is indicated by an arrow. (C) Crystallographic CTR dimer with residues that contribute to the intermolecular interface shown in space-filling representation. Interfacing amino acid residues from one CTR domain are colored dark gray, whereas those from the other CTR domain are colored light gray. (D) Predicted NTR-CTR tandem DUF1608 protein with residues that contribute to the predicted interdomain interface shown in space-filling representation. Interfacing amino acid residues from the different domains are colored as in C.

Fig. 4.

The putative M. acetivorans S-layer structure inferred from crystal packing in the hexagonal crystal lattice. (A) Five S-layer tiles from the 2D sheet in the hexagonal crystal form as viewed from the extracellular side. The six CTR crystallographic dimers that contribute to the primary pore are shown in colors of the spectrum. CTR dimers that span adjacent tiles are shown in gray. The positions of primary (P), trimer (T), and asymmetric (A) pores are indicated by arrows. The trimeric building block that is the basic repeating unit of protomers in the S-layer is comprised of the gray, green, and yellow crystallographic dimers surrounding the indicated trimer pore. (B) Cutaway side view of the S-layer with the extracellular surface at the top. The distance between the arrowheads is ∼240 Å. (C) A single tile as seen from the extracellular surface (Upper) and from the periplasmic-like space (Lower). A smoothed molecular surface is shown and, on the top, the tile is tilted 40° away from the viewer from the view seen in A. (Lower) The tile is rotated 180°. (D) A single tile colored by electrostatic surface potential with the extracellular surface (Upper) and the view from the periplasmic-like space (Lower). (E) Cutaway views of the primary (P), asymmetric (A), and trimer (T) pores, respectively.

Fig. 5.

Structural homology between the M. acetivorans S-layer protein and eukaryotic virus envelope proteins. Superposition of domain II of the MA0829 CTR (colored as in Fig. 2A) with domain I of the envelope proteins of West Nile virus (PDB ID code 3I50; colored orange), a member of the Flaviviridae family (A), and Sindbis virus (PDB ID code 1Z8Y; colored green), a member of the Togaviridae family, in the alphavirus subfamily (B). Domain II of the MA0829 CTR is rotated ∼210° on the y axis relative to the orientation shown in Fig. 2A, and the N and C termini of the domains are labeled. Multiple breaks are visible in domain I of the envelope proteins, as there are multiple connections between domain I and domains II and III.