Structure and function of secretin in the bacterial type II protein secretion system – SECRETIN

The outer membrane barrier to protein secretion in Gram-negative bacteria is overcome by several specific secretion systems that are classified into (at least) seven major types according to sequence similarities and mechanistic features. Type II secretion systems (T2SS), which allow the secretion of hydrolytic enzymes (lipases, amylases) or virulence factors (exoproteins) into the external medium, are widespread among Gram-negative bacteria (1,2). Exoproteins are first translocated by the Sec (3) or Tat (4) translocons into the periplasm. The folded proteins are then specifically transported through the outer membrane by an ATP and proton-motive force-dependent machinery (the secreton) (5,6) composed of 12 to 15 proteins (1,2). The secreton components include several integral inner membrane proteins, pseudopilins (proteins with structural features similar to those of type IV pilins (7)), and an integral outer membrane protein called secretin, which is the focus of this project. Besides their role in protein secretion by the Type II (e.g., Klebsiella oxytoca protein PulD (8) and Pseudomonas aeruginosa protein XcpQ (9,10)) and Type III secretion systems (e.g., Yersinia enterocolitica protein YscC; (10)), secretins are also required for filamentous bacteriophage (e.g., bacteriophage f1 protein pIV; (11)) and type IV pilus (e.g., Neisseria meningitidis and P. aeruginosa PilQ; (9,12)) assembly and secretion. According to electron microscopy, 12 to 14 identical secretins form ring-like complexes with an internal channel (estimated diameters range from 5 nm (PilQ, YscC) to 10 nm (XcpQ) (9,13,14)) large enough to accommodate their substrate (9,10,15,16). The secretin channel is probably occluded, since incorporation of secretins into the Escherichia coli outer membrane causes neither leakage of periplasmic proteins nor increased sensitivity to small toxic compounds that are unable to breach the outer membrane (17). PulD, pIV, XcpQ and YscC all form very small conductance channels in lipid bilayers (13,14,16,18,19), in line with the idea that they are occluded. Specific amino acid substitutions in pIV (18,20) and N. gonorrhoeae PilQ (21) secretins increase outer membrane permeability to small compounds. As discussed in detail below, the central plug was not identified in the first 3D image of PulD obtained by cryoelectron microscopy, although examination of negatively-stained samples indicated its presence (22). A central plug was observed in the cryo-electron microscopy-based 3D model of pIV (23). By analogy with the plug or cork domain of TonB-dependent outer membrane transporters (24), the secretin plug was proposed to correspond to the N-terminal part of the protein (22). Comparison of secretin sequences led to the definition of two major domains of approximately equal length (17). The predicted domain organization was confirmed by trypsin or proteinase K proteolysis of PulD and XcpQ, producing a protease-resistant C domain (14,22) that, in the case of XcpQ, formed channels whose conductance was similar to that of the intact protein (14). These data prompted the speculation that the secretin C domain, which is well conserved, is anchored in the outer membrane by 10 to 14 potentially transmembrane amphipathic beta strands (17) characteristic of other outer membrane proteins (25). The N domain, corresponding approximately to the first half of the protein, is much less conserved than the C domain. The Erwinia chrysanthemi exoprotein pectate lyase binds to this region of its cognate secretin, OutD, indicating that it might carry a specific exoprotein recognition determinant (26). A third secretin domain that is sometimes present downstream from the C domain mediates the interaction with a protein (pilotin) that facilitates secretin insertion into the outer membrane (27-30). In the case of PulD, this domain, called S, comprises about 60 residues that bind to the pilotin PulS, a small lipoprotein that targets the PulD-PulS complex t.