Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2010 Oct;17(10):1226-32.
doi: 10.1038/nsmb.1910. Epub 2010 Sep 19.

Structure of the cholera toxin secretion channel in its closed state

Steve L Reichow  1 , Konstantin V Korotkov, Wim G J Hol, Tamir Gonen
Affiliations
Comparative Study

Structure of the cholera toxin secretion channel in its closed state

Steve L Reichow et al. Nat Struct Mol Biol. 2010 Oct.

Abstract

The type II secretion system (T2SS) is a macromolecular complex spanning the inner and outer membranes of Gram-negative bacteria. Remarkably, the T2SS secretes folded proteins, including multimeric assemblies such as cholera toxin and heat-labile enterotoxin from Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. The major outer membrane T2SS protein is the 'secretin' GspD. Cryo-EM reconstruction of the V. cholerae secretin at 19-Å resolution reveals a dodecameric structure reminiscent of a barrel, with a large channel at its center that contains a closed periplasmic gate. The GspD periplasmic domain forms a vestibule with a conserved constriction, and it binds to a pentameric exoprotein and to the trimeric tip of the T2SS pseudopilus. By combining our results with structures of the cholera toxin and T2SS pseudopilus tip, we provide a structural basis for a possible secretion mechanism of the T2SS.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Purification and electron cryomicroscopy of cholera toxin secretion channel VcGspD
(a) VcGspD channels were purified to homogeneity by size-exclusion chromatography. Intact channels eluted as ∼0.9 MDa species (arrow). (b) Coomassie blue stained SDS-PAGE of purified VcGspD (∼74 kDa). (c) Electron micrograph of vitrified VcGspD channels. (inset, top panel) Representative class averages of VcGspD particles. In top views, looking down the channel axis, the particles appear as a ring with a punctate density located at the center of the channel and weaker densities “spokes” surround the channel. Side views (indicated with arrow heads in (c)) appear as long striated channels with a strong central density or “plug” that bisects the center of the channel (inset, bottom panel).
Figure 2
Figure 2. Three-dimensional electron cryomicroscopy reconstruction of VcGspD
(a, c and d) Side, top and bottom views, respectively, of the dodecameric VcGspD reconstruction at 19 Å resolution. In side view, three domains are identified from bottom to top as the periplasmic domain, the outer membrane domain and extracellular cap. (b) A slice view through the channel reveals the periplasmic vestibule, a constriction, the periplasmic gate, an extracellular chamber and an extracellular gate. The extracellular chamber is ∼100 Å wide whereas the periplasmic vestibule is ∼75 Å in diameter. The periplasmic constriction narrows the vestibule to ∼55 Å.
Figure 3
Figure 3. The secretin architecture is conserved in different secretion systems
(a) Domain architecture of secretins from the T2SS, the T3SS, the filamentous phage assembly system and the T4 pilus biogenesis system (T4PBS). Members of the secretin super-family contain a C-terminal secretin core homology domain (cyan),. The T2SS secretins generally contain four periplasmic subdomains, termed N0-N3. The N0 subdomain (dark blue) is located at the N-terminus and is followed by the three structurally homologous subdomains, N1-N3 (blue, green and dark green, respectively). A T2SS specific domain, termed the S-domain (grey), is located at the very C-terminus. Secretins from other systems share a similar architecture, composed of the secretin domain and at least two periplasmic subdomains that are structurally equivalent to N0 and N3 of VcGspD. (b) Structural comparison of the VcGspD density (blue, left) to single particle reconstructions of the T3SS in its close state (center) (EMDB 122426) and to the fully assembled T3SS needle complex in its open state (right) (EMDB 161724). The outer membrane T3SS secretin sits on top of a large inner membrane complex (gold, center and right). VcGspD appears to be in its closed state (left compared with center).
Figure 4
Figure 4. The periplasmic GspD domain contains a conserved N3 constriction and binds to the T2SS exoprotein and pseudopilus tip complex
(a) Fitting of twelve member ring models of the VcGspD N-terminal periplasmic domains (N0-N3) into the VcGspD density map. The N0 domain (dark blue) and N1 domain (light blue) are anchored at the bottom of the VcGspD density map. This places the N2 domain (light green) into the central periplasmic domain density and the N3 domain (dark green) into the periplasmic constriction. This placement correlates well with protease cleavage experiments that cut at N3 (asterisk),. (b) (top) Sensorgram showing binding of the B-pentamer of heat-labile enterotoxin (B5) to immobilized EcGspD. Relative units (RU, vertical) are plotted as a function of time (in seconds, horizontal). (middle) Global fit of equilibrium measurements (RE) depicted above versus B5 concentrations (M) gives a dissociation constant (Kd) of ∼15.5 μM. (bottom) Sensorgram showing the binding of the pseudopilus tip complex EcGspK-GspI-GspJ to immobilized EcGspD. (c) Fitting of the cholera toxin AB5 heterohexamer (, PDB ID 1S5E) (A subunit yellow; B subunits gold) into the VcGspD periplasmic vestibule. The ∼65 Å wide cholera toxin molecule fits well within the ∼75 Å vestibule formed by the N0-N2 domains but would not fit though the ∼55 Å wide constriction composed of the N3 domain.
Figure 5
Figure 5. Piston driven mechanism for protein secretion
Illustration of key structural steps proposed to occur during protein secretion across the outer membrane by the type 2 secretion system. (left) The T2SS secretin (blue) is in its closed state. Large exoproteins, such as the cholera toxin AB5 heterohexamer (gold), are proposed to interact with the pseudopilus tip complex (grey) positioned at the inner membrane beneath the secretin channel, or bind directly to the secretin. (middle) The pseudopilus would then extend acting like a piston to push the toxin into the periplasmic vestibule of the secretin and onto the constriction. (right) The interaction between the N3 domain (constriction) and large exoproteins like the toxin could act as the trigger to open the periplasmic gate, allowing exoproteins to enter the extracellular chamber of the secretin. The final step would involve opening of the extracellular gate, allowing exoproteins to be secreted (See also Discussion).
See this image and copyright information in PMC

References

    1. Cianciotto NP. Type II secretion: a protein secretion system for all seasons. Trends Microbiol. 2005;13:581–588. - PubMed
    1. Hirst TR, Sanchez J, Kaper JB, Hardy SJ, Holmgren J. Mechanism of toxin secretion by Vibrio cholerae investigated in strains harboring plasmids that encode heat-labile enterotoxins of Escherichia coli. Proc Natl Acad Sci U S A. 1984;81:7752–6. - PMC - PubMed
    1. Streatfield SJ, et al. Intermolecular interactions between the A and B subunits of heat-labile enterotoxin from Escherichia coli promote holotoxin assembly and stability in vivo. Proc Natl Acad Sci U S A. 1992;89:12140–4. - PMC - PubMed
    1. Sixma TK, et al. Crystal structure of a cholera toxin-related heat-labile enterotoxin from E. coli. Nature. 1991;351:371–377. - PubMed
    1. Tauschek M, Gorrell RJ, Strugnell RA, Robins-Browne RM. Identification of a protein secretory pathway for the secretion of heat-labile enterotoxin by an enterotoxigenic strain of Escherichia coli. Proc Natl Acad Sci U S A. 2002;99:7066–71. - PMC - PubMed

Publication types

MeSH terms

Substances