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Review
. 2012 Apr 2;10(5):336-51.
doi: 10.1038/nrmicro2762.

The type II secretion system: biogenesis, molecular architecture and mechanism

Konstantin V Korotkov  1 , Maria Sandkvist, Wim G J Hol
Affiliations
Review

The type II secretion system: biogenesis, molecular architecture and mechanism

Konstantin V Korotkov et al. Nat Rev Microbiol. .

Abstract

Many gram-negative bacteria use the sophisticated type II secretion system (T2SS) to translocate a wide range of proteins from the periplasm across the outer membrane. The inner-membrane platform of the T2SS is the nexus of the system and orchestrates the secretion process through its interactions with the periplasmic filamentous pseudopilus, the dodecameric outer-membrane complex and a cytoplasmic secretion ATPase. Here, recent structural and biochemical information is reviewed to describe our current knowledge of the biogenesis and architecture of the T2SS and its mechanism of action.

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Figures

Figure 1
Figure 1. Type II secretion system subassemblies and biogenesis
Schematic indications of the T2SS subassemblies and their biogenesis with hypothesis for several unknown steps. For T2SS proteins only the generic capital letter (see Table 1) is shown. Ribosome, dark green; signal recognition particle (SRP), light green; Sec translocon, pink; outer membrane complex proteins, blue; pseudopilins, shades of purple; cleaved-off prepilin peptide, blue; inner membrane platform proteins, shades of green; secretion ATPase, orange. Colors of additional proteins specific for a particular biogenesis step are indicated below. The order of assembling the T2SS components is still largely unknown. For instance, although the figure suggests that pseudopilins are added to the inner membrane after the inner membrane and ATPase are assembled, it is possible that one or more pseudopilins are co-assembled with the inner-membrane complex. a) The outer membrane complex and its biogenesis. The pilotin S, light blue, is a lipoprotein, the secretin D is a dodecamer according to electron microscopy studies. The pilotin is transported to the outer membrane by the Lol sorting pathway, which consists of an ABC transporter LolCDE, a periplasmic shuttle chaperone LolA and an outer membrane chaperone LolB. b)The inner membrane platform and its biogenesis. The inner membrane platform in complex with the secretion ATPase, , , , . Although not directly demonstrated, the Sec translocon or YidC insertase are likely involved in the biogenesis of the inner membrane platform. GspD has been suggested to be involved in assembling the inner membrane platform but this still needs to be established. c) Pseudopilin processing and the almost assembled T2SS. Shown is an assembly with the tip of the pseudopilus added to the inner and outer membrane complex depicted in (a). The prepilin peptidase (PPP) is shown as a light orange box in the inner membrane. It is not known at which stage pseudopilins are added to the system. Here it is assumed that the presumed tip of the pseudopilus, , consisting of GspK•GspI•GspJ and possibly also GspH, is present in a state of the complex which is ready to add additional GspG subunits during exoprotein secretion (see also Fig. 5).Note: In some species an additional T2SS protein is present, GspN, with a predicted N-terminal TM helix, whose function and location has not yet been determined, .
Figure 2
Figure 2. Structures of the T2SS pseudopilins
a) Ribbon diagrams of pseudopilin monomers: V. cholerae GspGEpsG (PDB 3FU1) ; V. cholerae GspHEpsH (PDB 2QV8) ; ETEC GspJ, GspI and GspK (PDB 3CI0) . The N-terminal α-helix is red, the conserved β-sheet is blue and the variable region is cyan; the Ca2+ ions in GspG and GspK are shown as orange spheres. Not shown in the crystal structures are the approximately 20 N-terminal residues that form a hydrophobic α-helix. All structures are shown in the same orientation based on superposition of the N-terminal α-helix. The structures of K. oxytoca GspGPulG , V. vulnificus GspIEpsI•GspJEpsJ , , P. aeruginosa GspGXcpT and GspJXcpW are also available. b) The quasihelical GspK•GspI•GspJ heterotrimer structure (PDB 3CI0) . Cα atoms of equivalent residues Ala40GspK, Ala40GspI and Lys40GspJ are shown as red spheres. The vertical distance between those atoms is ∼10 Å. The top view along pseudohelical axis shows the right-handed arrangement of GspK, GspI and GspJ subunits, with a helical rotation angle of ∼ 100°.
Figure 3
Figure 3. Structures of the T2SS secretin and pilotin
a)Left: The crystal structure of the first three N-terminal domains of ETEC GspD (PDB 3EZJ) . The N2 domain connects to the N0–N1 domains lobe via a potentially flexible linker; the linker between the N0 and N1 domains is also flexible. Right: The structure of ETEC N0-N1-GspD (shades of blue) in complex with HR-GspC (PDB 3OSS) (yellow). b)Cryo-electron microscopy structure of V. cholerae GspD (EMDB 1763) . A cut-out side view reveals the periplasmic vestibule, a constriction, the periplasmic gate (highlighted in yellow), an extracellular chamber and an extracellular gate. The fitting of ring models of N-terminal domains of GspD is shown in the upper right. c)The crystal structure of the EHEC T2SS pilotin GspS (PDB 3SOL). The location of a potential secretin binding site, as suggested by extra electron density in the crystal structure, and a disulfide bond are indicated. The structure of K. oxytoca GspSPulS is also available .
Figure 4
Figure 4. Structures and topologies of the secretion ATPase and the inner membrane components of the T2SS
a)Left: the N1 domain of the V. cholerae secretion ATPase GspEEpsE (PDB 2BH1) ; the N0-N1 domains of the X. campestris GspEXpsE displaying open and closed conformations of the N0 domain (PDB 2D27 and 2D28) ; and the N2 and C domains of the V. cholerae secretion ATPase GspEEpsE monomer (PDB 1P9W) . Middle: a hexameric model of the V. cholerae secretion ATPase GspEEpsE based on the crystal structure of the P. aeruginosa T4aPS retraction ATPase PilT (PDB 3JVV) . Right: the different conformations of the N2 domain in a hexamer model of GspEEpsE . b) Known structures are shown in ribbon representation; domains without known structure are shown as ovals. The structures shown are: the HR domain of ETEC GspC (PDB 3OSS) and the PDZ domain of V. cholerae GspCEpsC (PDB 2I4S) ; the periplasmic domain of V. parahaemolyticus GspLEpsL (PDB 2W7V) ; the complex of the cytoplasmic domain of GspLEpsL and the N1 domain of GspEEpsE from V. cholerae (PDB 2BH1) ; the periplasmic domain of V. cholerae GspMEpsM (PDB 1UV7) ; and the first cytoplasmic domain of V. cholerae GspFEpsF (PDB 3C1Q) .
Figure 5
Figure 5. Possible mode of action of the T2SS
The hypothetical mechanism is based on numerous biochemical and structural studies (see text, Supplementary Table 1). The color code is similar to that of Fig. 1: secretion ATPase GspE, orange; inner membrane platform proteins GspC, GspF, GspL and GspM, shades of green; pseudopilus GspK•GspI•GspJ trimeric tip, silver; major pseudopilin GspG, pink; outer membrane secretin channel, shades of blue and purple. (i):Possible architecture of the T2SS prior to binding an exoprotein (see also Fig. 1). The stoichiometry of many protein components of the system is still uncertain or unknown (see text). (ii):T2SS with an exoprotein (in this case the AB5 cholera toxin; B-pentamer in gold; A subunit yellow) in the periplasmic vestibule of GspD. On recognizing an exoprotein by GspD and/or GspC, a signal is possibly transmitted to the ATPase GspE to start hydrolyzing ATP. One might speculate that a dynamic nature of the periplasmic part of the T2SS secretin, as e.g. deduced from proteolytic and electron microscopy studies, may assist in, or is essential for, allowing exoproteins to reach the periplasmic vestibule of the secretin. Exoproteins might subsequently become sequestered in this vestibule by the secretin interacting with GspC of the inner membrane platform. (iii):ATP hydrolysis leads to conformational changes in the GspE hexamer, of which we have only a glimpse from homologs , , -. These motions are transferred by the ATPase-to-pseudopilin coupling protein GspL which may be involved in adding pseudopilins, such as GspH and multiple copies of the major pseudopilins GspG, to the still short pseudopilus on the periplasmic side of the inner membrane. At some stage the exoprotein and/or the tip of the pseudopilus contact the constriction in the periplasmic vestibule of the secretin. The size of the GspKIJ tip is such that it can enter the periplasmic vestibule but not pass the constriction site without alterations in the structure of the secretin. (iv):Further addition of subunits of the major pseudopilin GspG leads to additional contacts of the exoproteins and/or the pseudopilus with the secretin GspD, resulting in conformational changes (arrows) and expulsion of the exoprotein via the open periplasmic and extracellular gates.
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