Assembly mechanism of a Tad secretion system secretin-pilotin complex.

The bacterial Tight adherence Secretion System (TadSS) assembles surface pili that drive cell adherence, biofilm formation and bacterial predation. The structure and mechanism of the TadSS is mostly unknown. This includes characterisation of the outer membrane secretin through which the pilus is channelled and recruitment of its pilotin. Here we investigate RcpA and TadD lipoprotein from Pseudomonas aeruginosa. Light microscopy reveals RcpA colocalising with TadD in P. aeruginosa and when heterologously expressed in Escherichia coli. We use cryogenic electron microscopy to determine how RcpA and TadD assemble a secretin channel with C13 and C14 symmetries. Despite low sequence homology, we show that TadD shares a similar fold to the type 4 pilus system pilotin PilF. We establish that the C-terminal four residues of RcpA bind TadD - an interaction essential for secretin formation. The binding mechanism between RcpA and TadD appears distinct from known secretin-pilotin pairings in other secretion systems.

Conserved structures of ESCRT-III superfamily members across domains of life.

Structural and evolutionary studies of cyanobacterial phage shock protein A (PspA) and inner membrane-associated protein of 30 kDa (IM30) have revealed that these proteins belong to the endosomal sorting complex required for transport-III (ESCRT-III) superfamily, which is conserved across all three domains of life. PspA and IM30 share secondary and tertiary structures with eukaryotic ESCRT-III proteins, whilst also oligomerizing via conserved interactions. Here, we examine the structures of bacterial ESCRT-III-like proteins and compare the monomeric and oligomerized forms with their eukaryotic counterparts. We discuss conserved interactions used for self-assembly and highlight key hinge regions that mediate oligomer ultrastructure versatility. Finally, we address the differences in nomenclature assigned to equivalent structural motifs in both the bacterial and eukaryotic fields and suggest a common nomenclature applicable across the ESCRT-III superfamily.

Bacterial Vipp1 and PspA are members of the ancient ESCRT-III membrane-remodeling superfamily.

Membrane remodeling and repair are essential for all cells. Proteins that perform these functions include Vipp1/IM30 in photosynthetic plastids, PspA in bacteria, and ESCRT-III in eukaryotes. Here, using a combination of evolutionary and structural analyses, we show that these protein families are homologous and share a common ancient evolutionary origin that likely predates the last universal common ancestor. This homology is evident in cryo-electron microscopy structures of Vipp1 rings from the cyanobacterium Nostoc punctiforme presented over a range of symmetries. Each ring is assembled from rungs that stack and progressively tilt to form dome-shaped curvature. Assembly is facilitated by hinges in the Vipp1 monomer, similar to those in ESCRT-III proteins, which allow the formation of flexible polymers. Rings have an inner lumen that is able to bind and deform membranes. Collectively, these data suggest conserved mechanistic principles that underlie Vipp1, PspA, and ESCRT-III-dependent membrane remodeling across all domains of life.

The structure and mechanism of the bacterial type II secretion system.

The type II secretion system (T2SS) is a multi-protein complex used by many bacteria to move substrates across their cell membrane. Substrates released into the environment serve as local and long-range effectors that promote nutrient acquisition, biofilm formation, and pathogenicity. In both animals and plants, the T2SS is increasingly recognized as a key driver of virulence. The T2SS spans the bacterial cell envelope and extrudes substrates through an outer membrane secretin channel using a pseudopilus. An inner membrane assembly platform and a cytoplasmic motor controls pseudopilus assembly. This microreview focuses on the structure and mechanism of the T2SS. Advances in cryo-electron microscopy are enabling increasingly elaborate sub-complexes to be resolved. However, key questions remain regarding the mechanism of pseudopilus extension and retraction, and how this is coupled with the choreography of the substrate moving through the secretion system. The T2SS is part of an ancient type IV filament superfamily that may have been present within the last universal common ancestor (LUCA). Overall, mechanistic principles that underlie T2SS function have implication for other closely related systems such as the type IV and tight adherence pilus systems.

Core architecture of a bacterial type II secretion system.

Bacterial type II secretion systems (T2SSs) translocate virulence factors, toxins and enzymes across the cell outer membrane. Here we use negative stain and cryo-electron microscopy to reveal the core architecture of an assembled T2SS from the pathogen Klebsiella pneumoniae. We show that 7 proteins form a ~2.4 MDa complex that spans the cell envelope. The outer membrane complex includes the secretin PulD, with all domains modelled, and the pilotin PulS. The inner membrane assembly platform components PulC, PulE, PulL, PulM and PulN have a relative stoichiometric ratio of 2:1:1:1:1. The PulE ATPase, PulL and PulM combine to form a flexible hexameric hub. Symmetry mismatch between the outer membrane complex and assembly platform is overcome by PulC linkers spanning the periplasm, with PulC HR domains binding independently at the secretin base. Our results show that the T2SS has a highly dynamic modular architecture, with implication for pseudo-pilus assembly and substrate loading.

Structure of a mitochondrial fission dynamin in the closed conformation.

Dynamin 1-like proteins (DNM1-L) are mechanochemical GTPases that induce membrane fission in mitochondria and peroxisomes. Their mechanism depends on conformational changes driven by nucleotide and lipid cycling. Here we show the crystal structure of a mitochondrial fission dynamin (CmDnm1) from the algae Cyanidioschyzon merolae. Unlike other eukaryotic dynamin structures, CmDnm1 is in a hinge 1 closed conformation, with the GTPase domain compacted against the stalk. Within the crystal, CmDnm1 packs as a diamond-shaped tetramer that is consistent with an inactive off-membrane state. Crosslinking, photoinduced electron transfer assays, and electron microscopy verify these structures. In vitro, CmDnm1 forms concentration-dependent rings and protein-lipid tubes reminiscent of DNM1-L and classical dynamin with hinge 1 open. Our data provides a mechanism for filament collapse and membrane release that may extend to other dynamin family members. Additionally, hinge 1 closing may represent a key conformational change that contributes to membrane fission.

Structural basis for membrane tethering by a bacterial dynamin-like pair.

Dynamin-like proteins (DLPs) are large GTPases that restructure membrane. DLPs such as the mitofusins form heterotypic oligomers between isoform pairs that bridge and fuse opposing membranes. In bacteria, heterotypic oligomerisation may also be important for membrane remodelling as most DLP genes are paired within operons. How DLPs tether opposing membranes is unknown. Here we show the crystal structure of a DLP heterotypic pair from the pathogen Campylobacter jejuni. A 2:2 stoichiometric tetramer is observed where heterodimers, conjoined by a random coil linker, assemble back-to-back to form a tripartite DLP chain with extreme flexibility. In vitro, tetramerisation triggers GTPase activity and induces lipid binding. Liposomes are readily tethered and form tubes at high tetramer concentration. Our results provide a direct mechanism for the long-range binding and bridging of opposing membranes by a bacterial DLP pair. They also provide broad mechanistic and structural insights that are relevant to other heterotypic DLP complexes.

Membrane fission by dynamin: what we know and what we need to know.

The large GTPase dynamin is the first protein shown to catalyze membrane fission. Dynamin and its related proteins are essential to many cell functions, from endocytosis to organelle division and fusion, and it plays a critical role in many physiological functions such as synaptic transmission and muscle contraction. Research of the past three decades has focused on understanding how dynamin works. In this review, we present the basis for an emerging consensus on how dynamin functions. Three properties of dynamin are strongly supported by experimental data: first, dynamin oligomerizes into a helical polymer; second, dynamin oligomer constricts in the presence of GTP; and third, dynamin catalyzes membrane fission upon GTP hydrolysis. We present the two current models for fission, essentially diverging in how GTP energy is spent. We further discuss how future research might solve the remaining open questions presently under discussion.

Membrane remodelling in bacteria.

In bacteria the ability to remodel membrane underpins basic cell processes such as growth, and more sophisticated adaptations like inter-cell crosstalk, organelle specialisation, and pathogenesis. Here, selected examples of membrane remodelling in bacteria are presented and the diverse mechanisms for inducing membrane fission, fusion, and curvature discussed. Compared to eukaryotes, relatively few curvature-inducing proteins have been characterised so far. Whilst it is likely that many such proteins remain to be discovered, it also reflects the importance of alternative membrane remodelling strategies in bacteria where passive mechanisms for generating curvature are utilised.

LeoA, B and C from enterotoxigenic Escherichia coli (ETEC) are bacterial dynamins.

Escherichia coli (ETEC) strain H10407 contains a GTPase virulence factor, LeoA, which is encoded on a pathogenicity island and has been shown to enhance toxin release, potentially through vesicle secretion. By sequence comparisons and X-ray structure determination we now identify LeoA as a bacterial dynamin-like protein (DLP). Proteins of the dynamin family remodel membranes and were once thought to be restricted to eukaryotes. In ETEC H10407 LeoA localises to the periplasm where it forms a punctate localisation pattern. Bioinformatic analyses of leoA and the two upstream genes leoB and leoC suggest that LeoA works in concert with a second dynamin-like protein, made up of LeoB and LeoC. Disruption of the leoAB genes leads to a reduction in secretion of periplasmic Tat-GFP and outer membrane OmpA. Our data suggest a role for LeoABC dynamin-like proteins in potentiating virulence through membrane vesicle associated toxin secretion.

Structure of a type IV secretion system.

Bacterial type IV secretion systems translocate virulence factors into eukaryotic cells, distribute genetic material between bacteria and have shown potential as a tool for the genetic modification of human cells. Given the complex choreography of the substrate through the secretion apparatus, the molecular mechanism of the type IV secretion system has proved difficult to dissect in the absence of structural data for the entire machinery. Here we use electron microscopy to reconstruct the type IV secretion system encoded by the Escherichia coli R388 conjugative plasmid. We show that eight proteins assemble in an intricate stoichiometric relationship to form an approximately 3 megadalton nanomachine that spans the entire cell envelope. The structure comprises an outer membrane-associated core complex connected by a central stalk to a substantial inner membrane complex that is dominated by a battery of 12 VirB4 ATPase subunits organized as side-by-side hexameric barrels. Our results show a secretion system with markedly different architecture, and consequently mechanism, to other known bacterial secretion systems.

Dynamin architecture--from monomer to polymer.

Dynamins form a family of eukaryotic and prokaryotic proteins involved in membrane fission, fusion and restructuring. They have complex mechanisms of self-assembly, which are coupled to the tubulation and destabilization of lipid bilayers. Recent structural data has revolutionized our understanding and is now yielding detailed insights into dynamin structure, from monomer through to polymer. Traditional division of the dynamin subunit into GTPase domain, middle domain and GTPase effector domain based on sequence alignments and biochemistry is not supported by recent structural data. A unified model of dynamin architecture is presented here, based on observation that the basic dynamin fold is conserved across evolutionary kingdoms.

Structure of a bacterial dynamin-like protein lipid tube provides a mechanism for assembly and membrane curving.

Proteins of the dynamin superfamily mediate membrane fission, fusion, and restructuring events by polymerizing upon lipid bilayers and forcing regions of high curvature. In this work, we show the electron cryomicroscopy reconstruction of a bacterial dynamin-like protein (BDLP) helical filament decorating a lipid tube at approximately 11 A resolution. We fitted the BDLP crystal structure and produced a molecular model for the entire filament. The BDLP GTPase domain dimerizes and forms the tube surface, the GTPase effector domain (GED) mediates self-assembly, and the paddle region contacts the lipids and promotes curvature. Association of BDLP with GMPPNP and lipid induces radical, large-scale conformational changes affecting polymerization. Nucleotide hydrolysis seems therefore to be coupled to polymer disassembly and dissociation from lipid, rather than membrane restructuring. Observed structural similarities with rat dynamin 1 suggest that our results have broad implication for other dynamin family members.

A bacterial dynamin-like protein.

Dynamins form a superfamily of large mechano-chemical GTPases that includes the classical dynamins and dynamin-like proteins (DLPs). They are found throughout the Eukarya, functioning in core cellular processes such as endocytosis and organelle division. Many bacteria are predicted by sequence to possess large GTPases with the same multidomain architecture that is found in DLPs. Mechanistic dissection of dynamin family members has been impeded by a lack of high-resolution structural data currently restricted to the GTPase and pleckstrin homology domains, and the dynamin-related human guanylate-binding protein. Here we present the crystal structure of a cyanobacterial DLP in both nucleotide-free and GDP-associated conformation. The bacterial DLP shows dynamin-like qualities, such as helical self-assembly and tubulation of a lipid bilayer. In vivo, it localizes to the membrane in a manner reminiscent of FZL, a chloroplast-specific dynamin-related protein with which it shares sequence similarity. Our results provide structural and mechanistic insight that may be relevant across the dynamin superfamily. Concurrently, we show compelling similarity between a cyanobacterial and chloroplast DLP that, given the endosymbiotic ancestry of chloroplasts, questions the evolutionary origins of dynamins.

The crystal structure of ZapA and its modulation of FtsZ polymerisation.

FtsZ is part of a mid-cell cytokinetic structure termed the Z-ring that recruits a hierarchy of fission related proteins early in the bacterial cell cycle. The widely conserved ZapA has been shown to interact with FtsZ, to drive its polymerisation and to promote FtsZ filament bundling thereby contributing to the spatio-temporal tuning of the Z-ring. Here, we show the crystal structure of ZapA (11.6 kDa) from Pseudomonas aeruginosa at 2.8 A resolution. The electron density reveals two dimers associating via an extensive C-terminal coiled-coil protrusion to form an elongated anti-parallel tetramer. In solution, ZapA exists in a dimer-tetramer equilibrium that is strongly correlated with concentration. An increase in concentration promotes formation of the higher oligomeric state. The dimer is postulated to be the predominant physiological species although the tetramer could become significant if, as FtsZ is integrated into the Z-ring and is cross-linked, the local concentration of the dimer becomes sufficiently high. We also show that ZapA binds FtsZ with an approximate 1:1 molar stoichiometry and that this interaction provokes dramatic FtsZ polymerisation and inter-filament association as well as yielding filaments, single or bundled, more stable and resistant to collapse. Whilst in vitro dynamics of FtsZ are well characterised, its in vivo arrangement within the ultra-structural architecture of the Z-ring is yet to be determined despite being fundamental to cell division. The ZapA dimer has single 2-fold symmetry whilst the bipolar tetramer displays triple 2-fold symmetry. Given the symmetry of these ZapA oligomers and the polar nature of FtsZ filaments, the structure of ZapA carries novel implications for the inherent architecture of the Z-ring in vivo.