Abstract
Type IV pili are polymeric fibers which protrude from the cell surface and play a critical role in adhesion and invasion by pathogenic bacteria. The secretion of pili across the periplasm and outer membrane is mediated by a specialized secretin protein, PilQ, but the way in which this large channel is formed is unknown. Using NMR, we derived the structures of the periplasmic domains from N. meningitidis PilQ: the N-terminus is shown to consist of two β-domains, which are unique to the type IV pilus-dependent secretins. The structure of the second β-domain revealed an eight-stranded β-sandwich structure which is a novel variant of the HSP20-like fold. The central part of PilQ consists of two α/β fold domains: the structure of the first of these is similar to domains from other secretins, but with an additional α-helix which links it to the second α/β domain. We also determined the structure of the entire PilQ dodecamer by cryoelectron microscopy: it forms a cage-like structure, enclosing a cavity which is approximately 55 Å in internal diameter at its largest extent. Specific regions were identified in the density map which corresponded to the individual PilQ domains: this allowed us to dock them into the cryoelectron microscopy density map, and hence reconstruct the entire PilQ assembly which spans the periplasm. We also show that the C-terminal domain from the lipoprotein PilP, which is essential for pilus assembly, binds specifically to the first α/β domain in PilQ and use NMR chemical shift mapping to generate a model for the PilP:PilQ complex. We conclude that passage of the pilus fiber requires disassembly of both the membrane-spanning and the β-domain regions in PilQ, and that PilP plays an important role in stabilising the PilQ assembly during secretion, through its anchorage in the inner membrane.
Highlights
Type IV pili are long (1–5 mm), mechanically strong polymers which extend from the surfaces of many Gram-negative bacteria, including Neisseria meningitidis, Pseudomonas aeruginosa and Vibrio cholerae [1,2]
The structure of a type III secretion system (T3SS) secretin can be extracted from the 10 Aresolution cryoelectron microscopy density map of the Salmonella needle complex: this shows the secretin in an open state, with the needle passing through both ends of the chamber [15]
The process of assembly of pili is complicated: it requires the cooperative action of a group of proteins which span both the inner and outer membranes in bacteria
Summary
Type IV pili are long (1–5 mm), mechanically strong polymers which extend from the surfaces of many Gram-negative bacteria, including Neisseria meningitidis, Pseudomonas aeruginosa and Vibrio cholerae [1,2]. They are known to mediate a variety of functions, including attachment to host cell surface receptors during infection [3], natural DNA competence [4] and a phenomenon termed twitching motility, a flagellum-independent process which enables some bacteria to move rapidly (1 mm/s21) across surfaces [5]. The structure of a type III secretion system (T3SS) secretin can be extracted from the 10 Aresolution cryoelectron microscopy density map of the Salmonella needle complex: this shows the secretin in an open state, with the needle passing through both ends of the chamber [15]
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