1.B.11 The Outer Membrane Fimbrial Usher Porin (FUP) Family

The FUP family consists of a group of large proteins (700-900 amino acyl residues) present in the outer membranes of Gram-negative proteobacteria, members of the Deinococcus-Thermus group, and cyanobacteria (Dodson et al., 1993; Van Rosmalen et al., 1993; Nuccio and Bäumler, 2007). Each fimbrial usher protein acts in the fimbrial assembly process together with a periplasmic fimbrial chaperone protein (Waksman and Hultgren, 2009). They contain a large N-terminal-central domain that spans the membrane 24 times as β-strands, presumably forming a β-barrel structure and a transmembrane pore (Henderson et al., 2004). They also possess extreme N-terminal and C-terminal periplasmic domains which may function in protein folding and subunit assembly (Valent et al., 1995). The C-terminal domain of PapC is not inserted in the membrane, but is probably in the lumen of the N-terminal β-barrel, similar to the plug domain of the OMR family proteins (TC #1.B.14) (Henderson et al., 2004).

A single bacterial species such as E. coli may be capable of synthesizing numerous fimbriae, and the operon encoding the structural proteins of each fimbrium also encodes a fimbrium-specific process, usually together with one or more periplasmic chaperone protein and a fimbrium-specific outer membrane usher protein (Mol et al., 1996; Van Rosmalen et al., 1993). Phylogenetic analyses suggest that the chaperone protein and the usher protein in general evolved in parallel from their evolutionary precursor proteins. Nuccio & Bäumler (2007) have classified the usher proteins into 6 primary groups: α, β, γ, κ, π and σ. A detailed study of fimbrial usher protein evolution and phylogenetic classification has been presented (Nuccio and Bäumler, 2007).

One member of the FUP family, PapC, has been reported to form oligomeric channels, 2 nm in diameter, in the outer membrane of E. coli (Thanassi et al., 1998). However, in another report, PapC was shown to form a dimer, both in detergent solution and in the phospholipid bilayer. It forms a twin-pore complex with an inner diameter of 2 nm (Li et al., 2004; Nuccio and Bäumler, 2007). This pore must be large enough to accommodate fimbrial subunits, and maybe even partially assembled linear structures. Complexes formed by members of the FUP family may be similar to complexes formed by PulD and other related proteins involved in secretion across Gram-negative bacterial outer membranes.

The structural basis for pilus fiber assembly and secretion performed by the outer membrane assembly platform, the usher, has been revealed by the crystal structure of the translocation domain of the P pilus usher PapC and single particle cryo-electron microscopy imaging of the FimD usher bound to a translocating type 1 pilus assembly intermediate (Remaut et al., 2008). These structures provide molecular snapshots of a twinned-pore translocation machinery in action. Unexpectedly, only one pore is used for secretion, while both usher protomers are used for chaperone-subunit complex recruitment. The translocating pore itself comprises 24 beta strands and is occluded by a folded plug domain, likely gated by a conformationally constrained beta-hairpin. These structures capture the secretion of a virulence factor across the outer membrane of gram-negative bacteria.

Ushers constitute a family of bacterial outer membrane proteins responsible for the assembly and secretion of surface organelles such as the pilus. The structure at 3.15-A resolution of the usher pyelonephritis-associated pili C (PapC) translocation domain reveals a 24-stranded kidney-shaped beta-barrel, occluded by an internal plug domain (Huang et al., 2009). The dimensions of the pore allow tandem passage of individual folded pilus subunits in an upright pilus growth orientation, but they are insufficient for accommodating donor strand exchange. The molecular packing revealed by the crystal structure shows that 2 PapC molecules in head-to-head orientation interact via exposed beta-strand edges, which could be the preferred dimer interaction in solution. In vitro reconstitution of fiber assemblies suggest that PapC monomers may be sufficient for fiber assembly and secretion; both the plug domain and the C-terminal domain of PapC are required for filament assembly, whereas the N-terminal domain is mainly responsible for recruiting the chaperone-subunit complexes to the usher. The plug domain has a dual function: gating the beta-pore and participating in pilus assembly (Huang et al., 2009). Adaptive mutations in the signal peptide of the type 1 fimbrial adhesin of uropathogenic E. coli affects both fimbrial assembly and length (Ronald et al., 2008).

The plug, helix and N- and C- terminal domains regulate channel opening (Mapingire et al., 2009). The wildtype pore is in a leaky, low-conductive state most of the time, but displays frequent short-lived transitions to various open states. PapC mutants containing deletions of the plug domain, an alpha-helix that caps the plug domain, or the N- and C-terminal domains, form channels with higher open probability. Removal of the plug domain results in a channel with extremely large conductance. Thus, the plug gates the usher channel, and the periplasmic domains and alpha-helix function to modulate the gating activity of the PapC twin-pore (Mapingire et al., 2009).

Gram-negative pathogens commonly exhibit adhesive pili on their surfaces that mediate attachment to the host. The structural basis for pilus fiber assembly and secretion, performed by the outer membrane assembly platform--the usher--has been revealed by the crystal structure of the translocation domain of the P pilus usher, PapC, and single particle cryo-electron microscopy imaging of the FimD usher bound to a translocating type 1 pilus assembly intermediate (Remaut et al., 2008). These structures provide molecular snapshots of a twinned-pore translocation machinery in action. Unexpectedly, only one pore is used for secretion, while both usher protomers are used for chaperone-subunit complex recruitment. The translocating pore itself comprises 24 beta strands and is occluded by a folded plug domain, likely gated by a conformationally constrained beta-hairpin.

The PapC usher (1.B.11.2.1) contains five functional domains: a transmembrane β-barrel, a β-sandwich Plug, an N-terminal (periplasmic) domain (NTD), and two C-terminal (periplasmic) domains, CTD1 and CTD2. Volkan et al. (2012) delineated usher domain interactions between themselves and with chaperone-subunit complexes and showed that overexpression of individual usher domains inhibits pilus assembly. Prior work revealed that the Plug domain occludes the pore of the transmembrane domain of a solitary usher, but the chaperone-adhesin-bound usher has its Plug displaced from the pore, adjacent to the NTD. They demonstrated an interaction between the NTD and Plug domains that suggested a biophysical basis for usher gating. The NTD exhibits high-affinity binding to the chaperone-adhesin (PapDG) complex and low-affinity binding to the major tip subunit PapE (PapDE). CTD2 binds with lower affinity to all tested chaperone-subunit complexes except for the chaperone-terminator subunit (PapDH) and has a catalytic role in dissociating the NTD-PapDG complex, suggesting an interplay between recruitment to the NTD and transfer to CTD2 during pilus initiation. The Plug domain and the NTD-Plug complex bound all of the chaperone-subunit complexes tested including PapDH, suggesting that the Plug actively recruits chaperone-subunit complexes to the usher and is the sole recruiter of PapDH. The cooperative, active roles played by periplasmic domains of the usher to initiate, grow, and terminate a prototypical chaperone-usher pathway pilus was revealed (Volkan et al., 2012).

Usher pore gating occurs by a mechanism in which the plug resides stably within the transmembrane pore when the usher is inactive. Upon activation, the plug is translocated into the periplasmic space, where it functions in pilus assembly. A single salt bridge apparently functions in maintaining the plug in the channel of the usher PapC, and a loop between the 12th and 13th beta strands of the pore (the beta12-13 loop) facilitates pore opening. Mutation in the beta12-13 loop results in a closed PapC pore, unable to efficiently mediate pilus assembly (Volkan et al. 2013). Deletion of the PapH terminator/anchor resulted in increased OM permeability, suggesting a role for the proper anchoring of pili in retaining OM integrity. Cysteine residues in the plug and N-terminal periplasmic domains that resulted in a FimD usher with a greater propensity to exist in an open conformation, resulting in increased OM permeability but no loss in type 1 pilus assembly.

The generalized transport reaction catalyzed is:

Fimbrial subunits (periplasm) → fimbrial subunits (out)

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