Abstract
Type IV pili are extracellular polymers of the major pilin subunit. These subunits are held together in the pilus filament by hydrophobic interactions among their N-terminal α-helices, which also anchor the pilin subunits in the inner membrane prior to pilus assembly. Type IV pilus assembly involves a conserved group of proteins that span the envelope of Gram-negative bacteria. Among these is a set of minor pilins, so named because they share their hydrophobic N-terminal polymerization/membrane anchor segment with the major pilins but are much less abundant. Minor pilins influence pilus assembly and retraction, but their precise functions are not well defined. The Type IV pilus systems of enterotoxigenic Escherichia coli and Vibrio cholerae are among the simplest of Type IV pilus systems and possess only a single minor pilin. Here we show that the enterotoxigenic E. coli minor pilins CofB and LngB are required for assembly of their respective Type IV pili, CFA/III and Longus. Low levels of the minor pilins are optimal for pilus assembly, and CofB can be detected in the pilus fraction. We solved the 2.0 Å crystal structure of N-terminally truncated CofB, revealing a pilin-like protein with an extended C-terminal region composed of two discrete domains connected by flexible linkers. The C-terminal region is required for CofB to initiate pilus assembly. We propose a model for CofB-initiated pilus assembly with implications for understanding filament growth in more complex Type IV pilus systems as well as the related Type II secretion system.
Highlights
The enterotoxigenic E. coli (ETEC) Type IVb pilus systems each possess a single minor pilin
Low levels of the minor pilins are optimal for pilus assembly, and CofB can be detected in the pilus fraction
We propose a model for CofB-initiated pilus assembly with implications for understanding filament growth in more complex Type IV pilus systems as well as the related Type II secretion system
Summary
The enterotoxigenic E. coli (ETEC) Type IVb pilus systems each possess a single minor pilin. Crystal Structure and Function of the ETEC Minor Pilin CofB assembly requires a core assembly machinery composed of the major pilin subunit, a prepilin peptidase that removes the signal peptide and adds a methyl group to the N-terminal amine of residue 1 [12,13,14], a cytoplasmic assembly ATPase that powers the addition of each subunit to the growing pilus [15,16,17], an inner membrane core protein (sometimes called the platform protein) of unknown function (18 –20), and an outer membrane secretin channel [21,22,23,24,25] This core assembly machinery is conserved in the bacterial Type II secretion (T2S) system, which polymerizes “pseudopilin” subunits into a periplasmic “pseudopilus” that extrudes protein substrates across the outer membrane without itself forming an extracellular filament [26, 27]. We report here a high resolution x-ray crystal structure of N-terminally truncated CofB, the sole minor pilin from the ETEC CFA/III pilus system, and show that CofB mediates CFA/III pilus assembly
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