Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling

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114

Type III secretion systems of Gram-negative bacteria form injection devices that deliver effector proteins into eukaryotic cells during infection. They span both bacterial membranes and the extracellular space to connect with the host cell plasma membrane. Their extracellular portion is a needle-like, hollow tube that serves as a secretion conduit for effector proteins. The needle of Shigella flexneri is approximately 50-nm long and 7-nm thick and is made by the helical assembly of one protein, MxiH. We provide a 7-Å resolution 3D image reconstruction of the Shigella needle by electron cryomicroscopy, which resolves α-helices and a β-hairpin that has never been observed in the crystal and solution structures of needle proteins, including MxiH. An atomic model of the needle based on the 3D-density map, in comparison with that of the bacterial-flagellar filament, provides insights into how such a thin tubular structure is stably assembled by intricate intermolecular interactions. The map also illuminates how the needle-length control protein functions as a ruler within the central channel during export of MxiH for assembly at the distal end of the needle, and how the secretion-activation signal may be transduced through a conformational change of the needle upon host-cell contact.helical image analysis | Shigella pathogenesis | bacterial protein secretion

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Structure of a type III secretion needle at 7-Å resolution provides insights into its assembly and signaling mechanisms

Fujii

Cheung

Blanco

et al. 2012

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114

“…The C-terminal, PscF 68-85 helix is essential for polymerization of the full-length PscF, because a mutant lacking this region, PscF 1-67 , behaves as a monomer in gel filtration (SI Fig. 6) and chemical cross-linking experiments (not shown); the C-terminal region of the needle protein has also been shown to be essential for needle assembly in other T3S systems, as well as in the flagellar filament (15,22,23). In addition, PscF 68-85 is highly amphipathic.…”

Section: Resultsmentioning

confidence: 99%

“…Electron microscopy images of the bacterial flagellar filament, type III needle, and type IV pilus have revealed that the assembly of these structures involves packing of protein subunits within longitudinal helical arrays; in the case of the flagellum and type IV pilus, hydrophobic interactions have been shown to play key roles in the maintenance of the mechanical infrastructure (14,(23)(24)(25). These observations prompted us to investigate whether hydrophobic amino acids on the amphipathic C terminus of PscF could play a similar role in T3SS needle polymerization.…”

Section: Resultsmentioning

confidence: 99%

Structure of the heterotrimeric complex that regulates type III secretion needle formation

Quinaud

Plé

Job

et al. 2007

159

Type III secretion systems (T3SS), found in several Gram-negative pathogens, are nanomachines involved in the transport of virulence effectors directly into the cytoplasm of target cells. T3SS are essentially composed of basal membrane-embedded ring-like structures and a hollow needle formed by a single polymerized protein. Within the bacterial cytoplasm, the T3SS needle protein requires two distinct chaperones for stabilization before its secretion, without which the entire T3SS is nonfunctional. The 2.0-Å x-ray crystal structure of the PscE-PscF 55-85 -PscG heterotrimeric complex from Pseudomonas aeruginosa reveals that the C terminus of the needle protein PscF is engulfed within the hydrophobic groove of the tetratricopeptide-like molecule PscG, indicating that the macromolecular scaffold necessary to stabilize the T3SS needle is totally distinct from chaperoned complexes between pilus-or flagellum-forming molecules. Disruption of specific PscG-PscF interactions leads to impairment of bacterial cytotoxicity toward macrophages, indicating that this essential heterotrimer, which possesses homologs in a wide variety of pathogens, is a unique attractive target for the development of novel antibacterials.bacterial pathogenicity ͉ chaperones ͉ x-ray crystallography

“…Indeed, the outer domains of the two structures do look different both in the low-resolution maps and in their atomic structures. In flagellin, there are three outer domains: an ␣-domain near the center, a mostly ␤-(or ␤-hairpin-rich) domain at slightly higher radius, and a ␤-domain at the outside (31). The hook has two outer domains, both having ␤-folds (28) as predicted (30).…”

Section: Discussionmentioning

confidence: 99%

A partial atomic structure for the flagellar hook of Salmonella typhimurium

Shaikh

Thomas

Chen

et al. 2005

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The axial proteins of the bacterial flagellum function as a drive shaft, universal joint, and propeller driven by the flagellar rotary motor; they also form the putative protein export channel. The Nand C-terminal sequences of the eight axial proteins were predicted to form interlocking ␣-domains generating an axial tube. We report on an Ϸ1-nm resolution map of the hook from Salmonella typhimurium, which reveals such a tube made from interdigitated, 1-nm rod-like densities similar to those seen in maps of the filament. Atomic models for the two outer domains of the hook subunit were docked into the corresponding outermost features of the map. The N and C termini of the hook subunit fragment are positioned next to each other and face toward the axis of the hook. The placement of these termini would permit the residues missing in the fragment to form the rod-like features that form the core domain of the hook. We also fit the hook atomic model to an Ϸ2-nm resolution map of the hook from Caulobacter crescentus. The hook protein sequence from C. crescentus is largely homologous to that of S. typhimurium except for a large insertion (20 kDa). According to difference maps and our fitting, this insertion is found on the outer surface of the hook, consistent with our modeling of the hook.bacterial chemotaxis ͉ bacterial motility ͉ electron cryomicroscopy T he bacterial flagellum is the organ of motility for many species of bacteria. About 40 genes are needed to assemble the structure; Ϸ22 of the genes contribute structural proteins found in the completed flagellum. Of these 22 proteins, six appear to be key components of the rotary motor. An additional protein is likely to function as an adaptor connecting the motor to the axial component (1). Nine more proteins make up the axial component consisting of a rod (drive shaft), hook (universal joint), junction, filament (propeller), and cap. Two of the remaining proteins make up rings, which serve as a bushing that allows passage of the drive shaft through the cell wall and outer membrane. The rest of the proteins are associated with the flagellar-specific protein export.The rotary motor, powered by the proton-motive gradient across the cell membrane, turns the filament, which converts torque into thrust. The helical hook of the bacterial flagellum acts as a universal joint, allowing the motor to drive the filament off-axis. The hook connects the rod to the hook-filament junction, which in turn is connected to the filament. The hook, which is assembled before the more distal segments of the axial component, plays a role in the assembly of the filament. The flagellar filament elongates by subunit addition at its distal tip (2, 3). Subunits exported by the cell are thought to diffuse along a channel in the hook (and also the rod within the basal body and partially assembled filament). Three-dimensional reconstructions reveal a 3-nm channel running along the axis of the rod (D.R.T., D. G. Morgan, and D.J.D., unpublished data), hook (4), and filament (5), although higher-resolu...