S-Layers as a basic building block in a molecular construction kit

Crystalline arrays of protein or glycoprotein subunits forming surface layers (S-layers) are the most common outermost envelope components of prokaryotic organisms (archaea and bacteria). The wealth of information on the structure, chemistry, genetics, morphogenesis, and function of S-layers has revealed a broad application potential. As S-layers are periodic structures, they exhibit identical physicochemical properties for each molecular unit down to the subnanometer level and possess pores of identical size and morphology. Many applications of S-layers in nanobiotechnology depend on the ability of isolated subunits to recrystallize into monomolecular lattices in suspension or on suitable surfaces and interfaces. S-Layer lattices can be exploited as scaffolding and patterning elements for generating more complex supramolecular assemblies and structures, as required for life and nonlife science applications.     

Bacterial surface association of heat-labile enterotoxin through lipopolysaccharide after secretion via the general secretory pathway

Heat-labile enterotoxin (LT) is an important virulence factor expressed by enterotoxigenic Escherichia coli. The route of LT secretion through the outer membrane and the cellular and extracellular localization of secreted LT were examined. Using a fluorescently labeled receptor, LT was found to be specifically secreted onto the surface of wild type enterotoxigenic Escherichia coli. The main terminal branch of the general secretory pathway (GSP) was necessary and sufficient to localize LT to the bacterial surface in a K-12 strain. LT is a heteromeric toxin, and we determined that its cell surface localization was mediated by the its B subunit independent of an intact G(M1) ganglioside binding site and that LT binds lipopolysaccharide and G(M1) concurrently. The majority of LT secreted into the culture supernatant by the GSP in E. coli associated with vesicles. Only a mutation in hns, not overexpression of the GSP or LT, caused an increase in vesicle yield, supporting a specific vesicle formation machinery regulated by the nucleoid-associated protein HNS. We propose a model in which LT is secreted by the GSP across the outer membrane, secreted LT binds lipopolysaccharide via a G(M1)-independent binding region on its B subunit, and LT on the surface of released outer membrane vesicles interacts with host cell receptors, leading to intoxication. These data explain a novel mechanism of vesicle-mediated receptor-dependent delivery of a bacterial toxin into a host cell.     

Are S-layers exoskeletons? The basic function of protein surface layers revisited

Surface protein or glycoprotein layers (S-layers) are common structures of the prokaryotic cell envelope. They are either associated with the peptidoglycan or outer membrane of bacteria, and constitute the only cell wall component of many archaea. Despite their occurrence in most of the phylogenetic branches of microorganisms, the functional significance of S-layers is assumed to be specific for genera or groups of organisms in the same environment rather than common to all prokaryotes. Functional aspects have usually been investigated with isolated S-layer sheets or proteins, which disregards the interactions between S-layers and the underlying cell envelope components. This study discusses the synergistic effects in cell envelope assemblies, the hypothetical role of S-layers for cell shape formation, and the existence of a common function in view of new insights.     

Secretion of virulence determinants by the general secretory pathway in gram-negative pathogens: an evolving story

Secretion of proteins by the general secretory pathway (GSP) is a two-step process requiring the Sec translocase in the inner membrane and a separate substrate-specific secretion apparatus for translocation across the outer membrane. Gram-negative bacteria with pathogenic potential use the GSP to deliver virulence factors into the extracellular environment for interaction with the host. Well-studied examples of virulence determinants using the GSP for secretion include extracellular toxins, pili, curli, autotransporters, and crystaline S-layers. This article reviews our current understanding of the GSP and discusses examples of terminal branches of the GSP which are utilized by factors implicated in bacterial virulence.     

Bacterial and archaeal S-layers as object of bionanotechnology

Many species of Bacteria and Archaea posses a regularly structured surface layers (S-layers) as outermost cell envelope component. S-layers composed of a single protein or glycoprotein species. The individual subunits of S-layers interact with each other and with the supporting bacterial envelope component through non-covalent forces. Pores in the crystalline protein network are with mean diameter of 2-6 nm, the thickness of S-layer is 5-10 nm. The isolated S-layer subunits reassemble into two-dimensional crystalline arrays in solution, on solid supports, on planar lipid films. These unique features of S-layers have led to a broad spectrum applications. This review focuses on the structural properties S-layers and S-proteins and their applications with accent to using this structures in nanobiotechnology.     

Evidence for a repeating domain in type I restriction enzymes.

The primary structures of the recognition subunit (hsdS) in type I restriction enzymes from three isolates of Escherichia coli were compared and aligned by use of amino acid physical properties. A repeating domain was found in each of the subunits suggesting a pseudo-dimeric structure. Secondary structure predictions delineated two helical regions in each domain which suggested that the recognition subunits may act in a fashion similar to that proposed for repressor and activator molecules; namely, interaction with double-stranded DNA through helices and in two successive major grooves on the same DNA side. One helical motif could provide the specific recognition site and the other, the restriction site.     

Bacterial and Archaeal S-Layers as a Subject of Nanobiotechnology

Many bacteria and archaea have a crystalline surface layer (S-layer), which overlies the cell envelope. S-layers each consist of one protein or glycoprotein species. Protein subunits of the S-layer noncovalently interact with each other and with the underlying cell-envelope component. On average, the S-layer lattice has pores of 2–6 nm and is 5–10 nm high. Isolated S-layer proteins recrystallize to form two-dimensional crystalline structures in solution, on a solid support, and on planar lipid membranes. Owing to this unique property, S-layers have a broad range of applications. This review focuses on the structural features and applications of S-layers and their proteins, with special emphasis on their use in nanobiotechnology.     

Coiled-coil proteins associated with type III secretion systems: a versatile domain revisited

The pathogenic potential of many Gram-negative bacteria is indicated by the possession of a specialized type III secretion system that is used to deliver virulence effector proteins directly into the cellular environment of the eukaryotic host. Extracellular assemblies of secreted proteins contrive a physical link between the pathogen and host cytosol and enable the translocated effectors to bypass the bacterial and host membranes in a single step. Subsequent interactions of some effector proteins with host cytoskeletal and signalling proteins result in modulation of the cytoskeletal architecture of the aggressed cell and facilitate entry, survival and dissemination of the pathogen. Although the secreted components of type III secretion systems are diverse, many are predicted to share a common coiled-coil structural feature. Coiled-coils are ubiquitous and highly versatile assembly motifs found in a wide range of structural and regulatory proteins. The prevalence of these domains in secreted virulence effector proteins suggests a fundamental contribution to multiple aspects of their function, and evidence accumulating from functional studies suggests an intrinsic involvement of coiled-coils in subunit assembly, translocation and flexible interactions with multiple bacterial and host proteins. The known functional flexibility that coiled-coil domains confer upon proteins provides insights into some of the pathogenic mechanisms used during interaction with the host.     

S-layer-supported lipid membranes

Many prokaryotic organisms (archaea and bacteria) are covered by a regularly ordered surface layer (S-layer) as the outermost cell wall component. S-layers are built up of a single protein or glycoprotein species and represent the simplest biological membrane developed during evolution. Pores in S-layers are of regular size and morphology, and functional groups on the protein lattice are aligned in well-defined positions and orientations. Due to the high degree of structural regularity S-layers represent unique systems for studying the structure, morphogenesis, and function of layered supramolecular assemblies. Isolated S-layer subunits of numerous organisms are able to assemble into monomolecular arrays either in suspension, at air/water interfaces, on planar mono- and bilayer lipid films, on liposomes and on solid supports (e.g. silicon wafers). Detailed studies on composite S-layer/lipid structures have been performed with Langmuir films, freestanding bilayer lipid membranes, solid supported lipid membranes, and liposomes. Lipid molecules in planar films and liposomes interact via their head groups with defined domains on the S-layer lattice. Electrostatic interactions are the most prevalent forces. The hydrophobic chains of the lipid monolayers are almost unaffected by the attachment of the S-layer and no impact on the hydrophobic thickness of the membranes has been observed. Upon crystallization of a coherent S-layer lattice on planar and vesicular lipid membranes, an increase in molecular order is observed, which is reflected in a decrease of the membrane tension and an enhanced mobility of probe molecules within an S-layer-supported bilayer. Thus, the terminology 'semifluid membrane' has been introduced for describing S-layer-supported lipid membranes. The most important feature of composite S-layer/lipid membranes is an enhanced stability in comparison to unsupported membranes.     

Biology of type II secretion

The type II secretion pathway or the main terminal branch of the general secretion pathway, as it has also been referred to, is widely distributed among Proteobacteria, in which it is responsible for the extracellular secretion of toxins and hydrolytic enzymes, many of which contribute to pathogenesis in both plants and animals. Secretion through this pathway differs from most other membrane transport systems, in that its substrates consist of folded proteins. The type II secretion apparatus is composed of at least 12 different gene products that are thought to form a multiprotein complex, which spans the periplasmic compartment and is specifically required for translocation of the secreted proteins across the outer membrane. This pathway shares many features with the type IV pilus biogenesis system, including the ability to assemble a pilus-like structure. This review discusses recent findings on the organization of the secretion apparatus and the role of its various components in secretion. Different models for pilus-mediated secretion through the gated pore in the outer membrane are also presented, as are the possible properties that determine whether a protein is recognized and secreted by the type II pathway.     

A post-translational modification, unrelated to hydroxylation, in the collagenous domain of nonhelical pro-alpha 2(I) procollagen chains secreted by chemically transformed hamster fibroblasts.

Transformed Syrian hamster embryo (NQT-SHE) fibroblasts do not synthesize the pro-alpha 1 subunit of type I procollagen, but secrete two modified forms of the pro-alpha 2(I) subunit that migrate more slowly than the normal chain during gel electrophoresis (Peterkofsky, B., and Prather, W. (1986) J. Biol. Chem. 261, 16818-16826). By electrophoretic analysis of cyanogen bromide and V8 protease-derived peptides from the collagenous domains of intra- and extracellular pro-alpha 2(I) chains, we find that the modification occurs almost exclusively in secreted molecules, is located in the region spanned by the cyanogen bromide peptide CB3,5, and persists when hydroxylation is inhibited. Thus, modification is due to a post-translational reaction other than hydroxylation. The modified chains appear to be secreted in the denatured state since: 1) helical structures formed at 4 degrees C under acidic conditions were unstable under neutral conditions at 37 degrees C; 2) conditions that destabilize the type I procollagen helix and thus inhibit its secretion, i.e. inhibition of proline hydroxylation or incorporation of the proline analog cis-hydroxyproline, did not affect secretion of the modified chains. The time courses for secretion of nonhelical modified chains from NQT-SHE and of hydroxylated helical procollagen I from control cells, as a proportion of total collagen synthesized, were similar. Although cis-hydroxyproline did not inhibit the secretion of the modified chains, it induced their rapid intracellular degradation.     

Type IV pilin structures: insights on shared architecture, fiber assembly, receptor binding and type II secretion

Type IV pili are long, flexible filaments that extend from the surface of Gram-negative bacteria and are formed by the polymerization of pilin subunits. This review focuses on the structural information available for each pilin subclass, type IVa and type IVb, highlighting the contributions crystal and nuclear magnetic resonance structures have made in understanding pilus function and assembly. In addition, the type II secretion pseudopilus subunit structure and helical assembly is compared to that of the type IV pilus. The pilin subunits adopt an alphabeta-roll fold formed by the hydrophobic packing of the C-terminal half of a long alpha-helix against an antiparallel beta-sheet. The conserved N-terminal half of the same alpha-helix, as well as two sequence- and structurally-variable regions, protrude from this globular head domain. Filament models have a hydrophobic core formed by the signature long alpha-helices, with variable regions at the filament surface.     

Molecular organization of selected prokaryotic S-layer proteins

Regular crystalline surface layers (S-layers) are widespread among prokaryotes and probably represent the earliest cell wall structures. S-layer genes have been found in approximately 400 different species of the prokaryotic domains bacteria and archaea. S-layers usually consist of a single (glyco-)protein species with molecular masses ranging from about 40 to 200 kDa that form lattices of oblique, tetragonal, or hexagonal architecture. The primary sequences of hyperthermophilic archaeal species exhibit some characteristic signatures. Further adaptations to their specific environments occur by various post-translational modifications, such as linkage of glycans, lipids, phosphate, and sulfate groups to the protein or by proteolytic processing. Specific domains direct the anchoring of the S-layer to the underlying cell wall components and transport across the cytoplasma membrane. In addition to their presumptive original role as protective coats in archaea and bacteria, they have adapted new functions, e.g., as molecular sieves, attachment sites for extracellular enzymes, and virulence factors.     

Gonadotropin beta subunits determine the rate of assembly and the oligosaccharide processing of hormone dimer in transfected cells

The glycoprotein hormones lutropin (LH) and chorionic gonadotropin (CG) share a common structure consisting of an identical alpha subunit noncovalently linked to a hormone-specific beta subunit. While LH is produced in the anterior pituitary, CG is synthesized in placenta. To compare the assembly, processing, and secretion of human LH and CG in the same cell type, we have expressed their subunits, individually and together, in mouse C-127 mammary tumor cells. Analysis of transfected clones revealed an unexpected difference in the secretion of individually expressed subunits. Whereas alpha and CG beta subunits were rapidly and quantitatively secreted, only 10% of newly synthesized LH beta subunit reached the medium. The remaining subunit was found in an intracellular, endoglycosidase H (endo H)-sensitive pool that had a turnover rate of approximately 8 h. Coexpression with alpha subunit resulted in "rescue" of LH beta subunit by formation of LH dimer, which was efficiently secreted. However, combination of LH beta with alpha was slow, with an overall efficiency of only 50% despite the presence of excess alpha. In contrast, CG beta was rapidly assembled with the alpha subunit after synthesis. The two beta subunits also differed in their influence on the N-linked oligosaccharide processing of combined alpha. The oligosaccharides of LH dimer were endo H resistant, while those of CG dimer remained partially endo H sensitive. Thus, despite a high degree of homology between LH beta and CG beta, the two subunits differ in their secretion as free subunits, their rate of assembly with alpha subunit, and in their effect on the N-linked oligosaccharide processing of combined alpha.     

Chromatin remodeling: a complex affair

ATP‐dependent chromatin remodelers are multi‐subunit enzymes that catalyze nucleosome dynamics essential for chromosomal functions, and their inactivation or dysregulation can lead to numerous diseases, including neuro‐degenerative disorders and cancers. Each remodeler contains a conserved ATPase “motor” whose activity or targeting can be regulated by enzyme‐specific, accessory subunits. The human ISWI subfamily of remodelers has been defined as a group of more than six different enzyme complexes where one of two related ATPase subunits (Snf2L/SMARCA1 and Snf2H/SMARCA5) is paired with one of six different accessory subunits. In this issue of EMBO Reports, Oppikofer et al 1 find that the human ISWI subfamily is even more polymorphic in nature—every known accessory subunit can interact and function with both ATPase isoforms. This raises the complexity of the human ISWI subfamily to > 12 distinct enzymes, with the possibility for much higher levels of combinatorial assemblies, and has the potential to create enzymes with novel biochemical activities, as well as novel regulatory wiring through differential interactions with locus‐specific factors or histone modifications.     

EsxB,a secreted protein from Bacillus anthracis forms two distinct helical bundles

The EsxB protein from Bacillus anthracis belongs to the WXG100 family, a group of proteins secreted by a specialized secretion system. We have determined the crystal structures of recombinant EsxB and discovered that the small protein (∼10 kDa), comprised of a helix-loop-helix (HLH) hairpin, is capable of associating into two different helical bundles. The two basic quaternary assemblies of EsxB are an antiparallel (AP) dimer and a rarely observed bisecting U (BU) dimer. This structural duality of EsxB is believed to originate from the heptad repeat sequence diversity of the first helix of its HLH hairpin, which allows for two alternative helix packing. The flexibility of EsxB and the ability to form alternative helical bundles underscore the possibility that this protein can serve as an adaptor in secretion and can form hetero-oligomeric helix bundle(s) with other secreted members of the WXG100 family, such as EsxW. The highly conserved WXG motif is located within the loop of the HLH hairpin and is mostly buried within the helix bundle suggesting that its role is mainly structural. The exact functions of the motif, including a proposed role as a secretion signal, remain unknown.     

Secretion of killer toxin encoded on the linear DNA plasmid pGKL1 from Saccharomyces cerevisiae

By the kar1-mediated cytoduction, linear double-stranded DNA plasmids pGKL1 and pGKL2, encoding killer toxin complex, have been successfully transferred to the recipient strains with about 30% frequency. The killer toxin was found to be secreted through the normal yeast secretory pathway by introducing pGKL plasmids into the several Saccharomyces cerevisiae sec mutants and examining the secretion of killer toxin. S. cerevisiae cells, harboring newly isolated deletion plasmid pGKL1D, expressed only the 28K protein among three killer subunits, and secreted the 28K subunit at a level of zero to 20% efficiency of the cells containing intact pGKL1 plasmid. These data indicated that subunit interaction (cosecretion) of killer proteins is required for the efficient secretion of 28K subunit. The 28K precursor protein was found to translocate across the canine pancreatic endoplasmic reticulum membrane under the direction of its own signal peptide in vitro without any other subunits. From kex2 mutant cells harboring pGKL1 plasmid, the 97K subunit, and its precursor 128K protein were not secreted, however, the 28K subunit was secreted in the same amount as that secreted from KEX2 cells. These lines of evidence suggest that the final assembly of killer toxin complex after KEX2 site of Golgi apparatus is not essential for the secretion of 28K subunit, and therefore, that putative interaction between 128K protein and 28K subunit for the transport between endoplasmic reticulum and Golgi apparatus may be required for the efficient secretion of 28K subunit.     

Helical structure of the needle of the type III secretion system of Shigella flexneri

Gram-negative bacteria commonly interact with animal and plant hosts using type III secretion systems (TTSSs) for translocation of proteins into eukaryotic cells during infection. 10 of the 25 TTSS-encoding genes are homologous to components of the bacterial flagellar basal body, which the TTSS needle complex morphologically resembles. This indicates a common ancestry, although no TTSS sequence homologues for the genes encoding the flagellum are found. We here present an approximately 16-A structure of the central component, the needle, of the TTSS. Although the needle subunit is significantly smaller and shares no sequence homology with the flagellar hook and filament, it shares a common helical architecture ( approximately 5.6 subunits/turn, 24-A helical pitch). This common architecture implies that there will be further mechanistic analogies in the functioning of these two bacterial systems.     

Structural and functional aspects of unique type IV secretory components in the Helicobacter pylori cag-pathogenicity island

Helicobacter pylori cytotoxin-associated gene-pathogenicity island (cagPAI) is responsible for the secretion of the CagA effector through a type IV secretion system (T4SS) apparatus, as well as of peptidoglycan and possibly other not yet identified factors. Twenty-nine different polypeptide chains are encoded by this cluster of genes, although only some of them show a significant similarity with the constitutive elements of well characterized secretion systems from other bacteria. The other cagPAI components represent almost unique proteins in this scenario. The majority of the T4SS include approximately fifteen components, taking into account either the transmembrane complex subunits, ATPases or substrate factors. The composition of the cagPAI is very complex: it includes proteins most likely involved at different levels in the pilus assembly, stabilization and processing of secreted substrate, as well as regulatory particles possibly involved in the control of the entire apparatus. Despite recent findings with respect to components that play a role in the interaction with the host cell, the function of several cagPAI proteins remains unclear or unknown. This is particularly true for those that represent unique members with no clear similarity to those of other T4SS and no obvious evidence of involvement in the secretion of CagA or induction of pro-inflammatory responses. We summarize what is known about these accessory components, both from a molecular and structural point of view, as well as their putative physiological role.