The structure of the bacterial DNA segregation ATPase filament reveals the conformational plasticity of ParA upon DNA binding.

The efficient segregation of replicated genetic material is an essential step for cell division. Bacterial cells use several evolutionarily-distinct genome segregation systems, the most common of which is the type I Par system. It consists of an adapter protein, ParB, that binds to the DNA cargo via interaction with the parS DNA sequence; and an ATPase, ParA, that binds nonspecific DNA and mediates cargo transport. However, the molecular details of how this system functions are not well understood. Here, we report the cryo-EM structure of the Vibrio cholerae ParA2 filament bound to DNA, as well as the crystal structures of this protein in various nucleotide states. These structures show that ParA forms a left-handed filament on DNA, stabilized by nucleotide binding, and that ParA undergoes profound structural rearrangements upon DNA binding and filament assembly. Collectively, our data suggest the structural basis for ParA's cooperative binding to DNA and the formation of high ParA density regions on the nucleoid.

Structure and lipid dynamics in the maintenance of lipid asymmetry inner membrane complex of A. baumannii.

Multi-resistant bacteria are a major threat in modern medicine. The gram-negative coccobacillus Acinetobacter baumannii currently leads the WHO list of pathogens in critical need for new therapeutic development. The maintenance of lipid asymmetry (MLA) protein complex is one of the core machineries that transport lipids from/to the outer membrane in gram-negative bacteria. It also contributes to broad-range antibiotic resistance in several pathogens, most prominently in A. baumannii. Nonetheless, the molecular details of its role in lipid transport has remained largely elusive. Here, we report the cryo-EM maps of the core MLA complex, MlaBDEF, from the pathogen A. baumannii, in the apo-, ATP- and ADP-bound states, revealing multiple lipid binding sites in the cytosolic and periplasmic side of the complex. Molecular dynamics simulations suggest their potential trajectory across the membrane. Collectively with the recently-reported structures of the E. coli orthologue, this data also allows us to propose a molecular mechanism of lipid transport by the MLA system.

Oligomerization of the FliF Domains Suggests a Coordinated Assembly of the Bacterial Flagellum MS Ring.

The bacterial flagellum is a complex, self-assembling macromolecular machine that powers bacterial motility. It plays diverse roles in bacterial virulence, including aiding in colonization and dissemination during infection. The flagellum consists of a filamentous structure protruding from the cell, and of the basal body, a large assembly that spans the cell envelope. The basal body is comprised of over 20 different proteins forming several concentric ring structures, termed the M- S- L- P- and C-rings, respectively. In particular, the MS rings are formed by a single protein FliF, which consists of two trans-membrane helices anchoring it to the inner membrane and surrounding a large periplasmic domain. Assembly of the MS ring, through oligomerization of FliF, is one of the first steps of basal body assembly. Previous computational analysis had shown that the periplasmic region of FliF consists of three structurally similar domains, termed Ring-Building Motif (RBM)1, RBM2, and RBM3. The structure of the MS-ring has been reported recently, and unexpectedly shown that these three domains adopt different symmetries, with RBM3 having a 34-mer stoichiometry, while RBM2 adopts two distinct positions in the complex, including a 23-mer ring. This observation raises some important question on the assembly of the MS ring, and the formation of this symmetry mismatch within a single protein. In this study, we analyze the oligomerization of the individual RBM domains in isolation, in the Salmonella enterica serovar Typhimurium FliF ortholog. We demonstrate that the periplasmic domain of FliF assembles into the MS ring, in the absence of the trans-membrane helices. We also report that the RBM2 and RBM3 domains oligomerize into ring structures, but not RBM1. Intriguingly, we observe that a construct encompassing RBM1 and RBM2 is monomeric, suggesting that RBM1 interacts with RBM2, and inhibits its oligomerization. However, this inhibition is lifted by the addition of RBM3. Collectively, this data suggest a mechanism for the controlled assembly of the MS ring.

Architecture and Self-Assembly of Clostridium sporogenes and Clostridium botulinum Spore Surfaces Illustrate a General Protective Strategy across Spore Formers.

Spores, the infectious agents of many Firmicutes, are remarkably resilient cell forms. Even distant relatives can have similar spore architectures although some display unique features; they all incorporate protective proteinaceous envelopes. We previously found that Bacillus spores can achieve these protective properties through extensive disulfide cross-linking of self-assembled arrays of cysteine-rich proteins. We predicted that this could be a mechanism employed by spore formers in general, even those from other genera. Here, we tested this by revealing in nanometer detail how the outer envelope (exosporium) in Clostridium sporogenes (surrogate for C. botulinum group I), and in other clostridial relatives, forms a hexagonally symmetric semipermeable array. A cysteine-rich protein, CsxA, when expressed in Escherichia coli, self-assembles into a highly thermally stable structure identical to that of the native exosporium. Like the exosporium, CsxA arrays require harsh "reducing" conditions for disassembly. We conclude that in vivo, CsxA self-organizes into a highly resilient, disulfide cross-linked array decorated with additional protein appendages enveloping the forespore. This pattern is remarkably similar to that in Bacillus spores, despite a lack of protein homology. In both cases, intracellular disulfide formation is favored by the high lattice symmetry. We have identified cysteine-rich proteins in many distantly related spore formers and propose that they may adopt a similar strategy for intracellular assembly of robust protective structures.IMPORTANCE Bacteria such as those causing botulism and anthrax survive harsh conditions and spread disease as spores. Distantly related species have similar spore architectures with protective proteinaceous layers aiding adhesion and targeting. The structures that confer these common properties are largely unstudied, and the proteins involved can be very dissimilar in sequence. We identify CsxA as a cysteine-rich protein that self-assembles in a two-dimensional lattice enveloping the spores of several Clostridium species. We show that apparently unrelated cysteine-rich proteins from very different species can self-assemble to form remarkably similar and robust structures. We propose that diverse cysteine-rich proteins identified in the genomes of a broad range of spore formers may adopt a similar strategy for assembly.

The cryo-EM structure of the bacterial flagellum cap complex suggests a molecular mechanism for filament elongation.

The bacterial flagellum is a remarkable molecular motor, whose primary function in bacteria is to facilitate motility through the rotation of a filament protruding from the bacterial cell. A cap complex, consisting of an oligomer of the protein FliD, is localized at the tip of the flagellum, and is essential for filament assembly, as well as adherence to surfaces in some bacteria. However, the structure of the intact cap complex, and the molecular basis for its interaction with the filament, remains elusive. Here we report the cryo-EM structure of the Campylobacter jejuni cap complex, which reveals that FliD is pentameric, with the N-terminal region of the protomer forming an extensive set of contacts across several subunits, that contribute to FliD oligomerization. We also demonstrate that the native C. jejuni flagellum filament is 11-stranded, contrary to a previously published cryo-EM structure, and propose a molecular model for the filament-cap interaction.

The molecular basis of endolytic activity of a multidomain alginate lyase from Defluviitalea phaphyphila, a representative of a new lyase family, PL39.

Alginate is a polymer containing two uronic acid epimers, ß-d-mannuronate (M) and α-l-guluronate (G), and is a major component of brown seaweed that is depolymerized by alginate lyases. These enzymes have diverse specificity, cleaving the chain with endo- or exotype activity and with differential selectivity for the sequence of M or G at the cleavage site. Dp0100 is a 201-kDa multimodular, broad-specificity endotype alginate lyase from the marine thermophile Defluviitalea phaphyphila, which uses brown algae as a carbon source, converting it to ethanol, and bioinformatics analysis suggested that its catalytic domain represents a new polysaccharide lyase family, PL39. The structure of the Dp0100 catalytic domain, determined at 2.07 Å resolution, revealed that it comprises three regions strongly resembling those of the exotype lyase families PL15 and PL17. The conservation of key catalytic histidine and tyrosine residues belonging to the latter suggests these enzymes share mechanistic similarities. A complex of Dp0100 with a pentasaccharide, M5, showed that the oligosaccharide is located in subsites -2, -1, +1, +2, and +3 in a long, deep canyon open at both ends, explaining the endotype activity of this lyase. This contrasted with the hindered binding sites of the exotype enzymes, which are blocked such that only one sugar moiety can be accommodated at the -1 position in the catalytic site. The biochemical and structural analyses of Dp0100, the first for this new class of endotype alginate lyases, have furthered our understanding of the structure-function and evolutionary relationships within this important class of enzymes.

Identification and structural analysis of the tripartite α-pore forming toxin of Aeromonas hydrophila.

The alpha helical CytolysinA family of pore forming toxins (α-PFT) contains single, two, and three component members. Structures of the single component Eschericia coli ClyA and the two component Yersinia enterolytica YaxAB show both undergo conformational changes from soluble to pore forms, and oligomerization to produce the active pore. Here we identify tripartite α-PFTs in pathogenic Gram negative bacteria, including Aeromonas hydrophila (AhlABC). We show that the AhlABC toxin requires all three components for maximal cell lysis. We present structures of pore components which describe a bi-fold hinge mechanism for soluble to pore transition in AhlB and a contrasting tetrameric assembly employed by soluble AhlC to hide their hydrophobic membrane associated residues. We propose a model of pore assembly where the AhlC tetramer dissociates, binds a single membrane leaflet, recruits AhlB promoting soluble to pore transition, prior to AhlA binding to form the active hydrophilic lined pore.

Self-Assembling Proteins as High-Performance Substrates for Embryonic Stem Cell Self-Renewal.

The development of extracellular matrix mimetics that imitate niche stem cell microenvironments and support cell growth for technological applications is intensely pursued. Specifically, mimetics are sought that can enact control over the self-renewal and directed differentiation of human pluripotent stem cells (hPSCs) for clinical use. Despite considerable progress in the field, a major impediment to the clinical translation of hPSCs is the difficulty and high cost of large-scale cell production under xeno-free culture conditions using current matrices. Here, a bioactive, recombinant, protein-based polymer, termed ZTFn , is presented that closely mimics human plasma fibronectin and serves as an economical, xeno-free, biodegradable, and functionally adaptable cell substrate. The ZTFn substrate supports with high performance the propagation and long-term self-renewal of human embryonic stem cells while preserving their pluripotency. The ZTFn polymer can, therefore, be proposed as an efficient and affordable replacement for fibronectin in clinical grade cell culturing. Further, it can be postulated that the ZT polymer has significant engineering potential for further orthogonal functionalization in complex cell applications.

Structural insights into the function of type VI secretion system TssA subunits.

The type VI secretion system (T6SS) is a multi-protein complex that injects bacterial effector proteins into target cells. It is composed of a cell membrane complex anchored to a contractile bacteriophage tail-like apparatus consisting of a sharpened tube that is ejected by the contraction of a sheath against a baseplate. We present structural and biochemical studies on TssA subunits from two different T6SSs that reveal radically different quaternary structures in comparison to the dodecameric E. coli TssA that arise from differences in their C-terminal sequences. Despite this, the different TssAs retain equivalent interactions with other components of the complex and position their highly conserved N-terminal ImpA_N domain at the same radius from the centre of the sheath as a result of their distinct domain architectures, which includes additional spacer domains and highly mobile interdomain linkers. Together, these variations allow these distinct TssAs to perform a similar function in the complex.

Molecular tiling on the surface of a bacterial spore - the exosporium of the Bacillus anthracis/cereus/thuringiensis group.

Bacteria of the genera Bacillus and Clostridium form highly resistant spores, which in the case of some pathogens act as the infectious agents. An exosporium forms the outermost layer of some spores; it plays roles in protection, adhesion, dissemination, host targeting in pathogens and germination control. The exosporium of the Bacillus cereus group, including the anthrax pathogen, contains a 2D-crystalline basal layer, overlaid by a hairy nap. BclA and related proteins form the hairy nap, and require ExsFA (BxpB) for their localization on the basal layer. Until now, the identity of the main structural protein components of the basal layer was unknown. We demonstrate here that ExsY forms one of the essential components. Through heterologous expression in Escherichia coli, we also demonstrate that ExsY can self-assemble into ordered 2D arrays that mimic the structure of the exosporium basal layer. Self-assembly is likely to play an important role in the construction of the exosporium. The ExsY array is stable to heat and chemical denaturants, forming a robust layer that would contribute to overall spore resistance. Our structural analysis also provides novel insight into the location of other molecular components anchored onto the exosporium, such as BclA and ExsFA.

Characterization of the spore surface and exosporium proteins of Clostridium sporogenes; implications for Clostridium botulinum group I strains.

Clostridium sporogenes is a non-pathogenic close relative and surrogate for Group I (proteolytic) neurotoxin-producing Clostridium botulinum strains. The exosporium, the sac-like outermost layer of spores of these species, is likely to contribute to adhesion, dissemination, and virulence. A paracrystalline array, hairy nap, and several appendages were detected in the exosporium of C. sporogenes strain NCIMB 701792 by EM and AFM. The protein composition of purified exosporium was explored by LC-MS/MS of tryptic peptides from major individual SDS-PAGE-separated protein bands, and from bulk exosporium. Two high molecular weight protein bands both contained the same protein with a collagen-like repeat domain, the probable constituent of the hairy nap, as well as cysteine-rich proteins CsxA and CsxB. A third cysteine-rich protein (CsxC) was also identified. These three proteins are also encoded in C. botulinum Prevot 594, and homologues (75-100% amino acid identity) are encoded in many other Group I strains. This work provides the first insight into the likely composition and organization of the exosporium of Group I C. botulinum spores.

Diverse supramolecular structures formed by self-assembling proteins of the Bacillus subtilis spore coat.

Bacterial spores (endospores), such as those of the pathogens Clostridium difficile and Bacillus anthracis, are uniquely stable cell forms, highly resistant to harsh environmental insults. Bacillus subtilis is the best studied spore-former and we have used it to address the question of how the spore coat is assembled from multiple components to form a robust, protective superstructure. B. subtilis coat proteins (CotY, CotE, CotV and CotW) expressed in Escherichia coli can arrange intracellularly into highly stable macro-structures through processes of self-assembly. Using electron microscopy, we demonstrate the capacity of these proteins to generate ordered one-dimensional fibres, two-dimensional sheets and three-dimensional stacks. In one case (CotY), the high degree of order favours strong, cooperative intracellular disulfide cross-linking. Assemblies of this kind could form exquisitely adapted building blocks for higher-order assembly across all spore-formers. These physically robust arrayed units could also have novel applications in nano-biotechnology processes.

Structure and function of the bacterial heterodimeric ABC transporter CydDC: stimulation of ATPase activity by thiol and heme compounds.

In Escherichia coli, the biogenesis of both cytochrome bd-type quinol oxidases and periplasmic cytochromes requires the ATP-binding cassette-type cysteine/GSH transporter, CydDC. Recombinant CydDC was purified as a heterodimer and found to be an active ATPase both in soluble form with detergent and when reconstituted into a lipid environment. Two-dimensional crystals of CydDC were analyzed by electron cryomicroscopy, and the protein was shown to be made up of two non-identical domains corresponding to the putative CydD and CydC subunits, with dimensions characteristic of other ATP-binding cassette transporters. CydDC binds heme b. Detergent-solubilized CydDC appears to adopt at least two structural states, each associated with a characteristic level of bound heme. The purified protein in detergent showed a weak basal ATPase activity (approximately 100 nmol Pi/min/mg) that was stimulated ∼3-fold by various thiol compounds, suggesting that CydDC could act as a thiol transporter. The presence of heme (either intrinsic or added in the form of hemin) led to a further enhancement of thiol-stimulated ATPase activity, although a large excess of heme inhibited activity. Similar responses of the ATPase activity were observed with CydDC reconstituted into E. coli lipids. These results suggest that heme may have a regulatory role in CydDC-mediated transmembrane thiol transport.

A urea channel from Bacillus cereus reveals a novel hexameric structure.

Urea is exploited as a nitrogen source by bacteria, and its breakdown products, ammonia and bicarbonate, are employed to counteract stomach acidity in pathogens such as Helicobacter pylori. Uptake in the latter is mediated by UreI, a UAC (urea amide channel) family member. In the present paper, we describe the structure and function of UACBc, a homologue from Bacillus cereus. The purified channel was found to be permeable not only to urea, but also to other small amides. CD and IR spectroscopy revealed a structure comprising mainly α-helices, oriented approximately perpendicular to the membrane. Consistent with this finding, site-directed fluorescent labelling indicated the presence of seven TM (transmembrane) helices, with a cytoplasmic C-terminus. In detergent, UACBc exists largely as a hexamer, as demonstrated by both cross-linking and size-exclusion chromatography. A 9 Å (1 Å=0.1 nm) resolution projection map obtained by cryo-electron microscopy of two-dimensional crystals shows that the six protomers are arranged in a planar hexameric ring. Each exhibits six density features attributable to TM helices, surrounding a putative central channel, while an additional helix is peripherally located. Bioinformatic analyses allowed individual TM regions to be tentatively assigned to the density features, with the resultant model enabling identification of residues likely to contribute to channel function.

AcrB contamination in 2-D crystallization of membrane proteins: lessons from a sodium channel and a putative monovalent cation/proton antiporter.

Contamination with the multidrug transporter AcrB represents a potential pitfall in the structural analysis of recombinant membrane proteins expressed in Escherichia coli, especially when high-throughput approaches are adopted. This can be a particular problem in two-dimensional (2-D) crystallization for electron cryomicroscopy since individual crystals are too small for compositional analysis. Using a broad 'sparse matrix' of buffer conditions typically used in 2-D crystallization, we have identified at least eight unique crystal forms of AcrB. Reference to images and projection maps of these different forms can greatly facilitate the early identification of false leads in 2-D crystallization trials of other membrane proteins of interest. We illustrate the usefulness of such data by highlighting two studies of membrane proteins in our laboratories. We show in one case (a bacterial sodium channel, NaChBac) how early crystallization 'hits' could be attributed to contaminating AcrB by comparison against our AcrB crystal image database. In a second case, involving a member of the monovalent cation/proton antiporter-1 family (MPSIL0171), a comparison with the observed AcrB crystal forms allowed easy identification of reconstituted AcrB particles, greatly facilitating the eventual purification and crystallization of the correct protein in pure form as ordered helical arrays. Our database of AcrB crystal images will be of general use in assisting future 2-D crystallization studies of other membrane proteins.

Surface architecture of endospores of the Bacillus cereus/anthracis/thuringiensis family at the subnanometer scale.

Bacteria of the Bacillus cereus family form highly resistant spores, which in the case of the pathogen B. anthracis act as the agents of infection. The outermost layer, the exosporium, enveloping spores of the B. cereus family as well as a number of Clostridia, plays roles in spore adhesion, dissemination, targeting, and germination control. We have analyzed two naturally crystalline layers associated with the exosporium, one representing the "basal" layer to which the outermost spore layer ("hairy nap") is attached, and the other likely representing a subsurface ("parasporal") layer. We have used electron cryomicroscopy at a resolution of 0.8-0.6 nm and circular dichroism spectroscopic measurements to reveal a highly α-helical structure for both layers. The helices are assembled into 2D arrays of "cups" or "crowns." High-resolution atomic force microscopy of the outermost layer showed that the open ends of these cups face the external environment and the highly immunogenic collagen-like fibrils of the hairy nap (BclA) are attached to this surface. Based on our findings, we present a molecular model for the spore surface and propose how this surface can act as a semipermeable barrier and a matrix for binding of molecules involved in defense, germination control, and other interactions of the spore with the environment.

The formation and structure of Escherichia coli K-12 haemolysin E pores.

Some enteric bacteria synthesize a pore-forming toxin, HlyE, which is cytolytic and cytotoxic to host cells. Measurement of HlyE binding to erythrocyte ghosts and the kinetics of HlyE-mediated erythrocyte lysis suggests that interaction with target membranes is not the rate-limiting step in the formation of HlyE pores, but that there is a temperature-dependent lag phase before a functional pore is formed. Circular dichroism and fluorescence energy transfer analyses show that HlyE protomers retain an alpha-helical structure when oligomerized to form a pore consisting of parallel HlyE protomers. Comparison of the proteolytic sensitivities of the water-soluble and oligomeric forms of HlyE identifies inner and outer surfaces of the pore. This new information has been used to constrain a model of the HlyE pore, which allows a more detailed interpretation of previous low-resolution 3D reconstructions and suggests a novel mechanism for insertion of HlyE into target membranes.

Structure of the hemolysin E (HlyE, ClyA, and SheA) channel in its membrane-bound form.

Hemolysin E (HlyE, ClyA, SheA) is a pore-forming protein toxin isolated from Escherichia coli. The three-dimensional structure of its water-soluble form is known, but that of the membrane-bound HlyE complex is not. We have used electron microscopy and image processing to show that the pores are predominantly octameric. Three-dimensional reconstructions of HlyE pores assembled in lipid/detergent micelles suggest a degree of conformational variability in the octameric complexes. The reconstructed pores were significantly longer than the maximum dimension of the water-soluble molecule, indicating that conformational changes occur on pore formation.

The crystal structures of Lactococcus lactis MG1363 Dps proteins reveal the presence of an N-terminal helix that is required for DNA binding.

Dps proteins play a major role in the protection of bacterial DNA from damage by reactive oxygen species. Previous studies have implicated the extended lysine-containing N-terminal regions of Dps subunits in DNA binding, but this part of the structure has not previously been observed crystallographically. Here the structures of two Dps proteins (DpsA and DpsB) from Lactococcus lactis MG1363 reveal for the first time the presence of an N-terminal alpha helix that extends from the core of the Dps subunit. Consequently, the N-terminal helices are displayed in parallel pairs on the exterior of the dodecameric Dps assemblies. Both DpsA and DpsB bind DNA. Deletion of the DpsA N-terminal helix impaired DNA binding. The N-terminal Lys residues of Escherichia coli Dps have been implicated in DNA binding. Replacement of the lactococcal DpsA Lys residues 9, 15 and 16 by Glu did not inhibit DNA binding. However, DNA binding was inhibited by EDTA, suggesting a role for cations in DNA binding. In contrast to E. coli, Bacillus brevis and Mycobacterium smegmatis Dps:DNA complexes, in which DNA interacts with crystalline Dps phases, L. lactis DNA:Dps complexes appeared as non-crystalline aggregates of protein and DNA in electron micrographs.

The role of phosphate groups in the VS ribozyme-substrate interaction.

The VS ribozyme trans-cleavage substrate interacts with the catalytic RNA via tertiary interactions. To study the role of phosphate groups in the ribozyme-substrate interaction, 18 modified substrates were synthesized, where an epimeric phosphorothioate replaces one of the phosphate diester linkages. Sites in the stem-loop substrate where phosphorothioate substitution impaired reaction cluster in two regions. The first site is the scissile phosphate diester linkage and nucleotides downstream of this and the second site is within the loop region. The addition of manganese ions caused recovery of the rate of reaction for phosphorothioate substitutions between A621 and A622 and U631 and C632, suggesting that these two phosphate groups may serve as ligands for two metal ions. In contrast, significant manganese rescue was not observed for the scissile phosphate diester linkage implying that electrophilic catalysis by metal ions is unlikely to contribute to VS ribozyme catalysis. In addition, an increase in the reaction rate of the unmodified VS ribozyme was observed when a mixture of magnesium and manganese ions acted as the cofactor. One possible explanation for this effect is that the cleavage reaction of the VS ribozyme is rate limited by a metal dependent docking of the substrate on the ribozyme.