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employed cryo-EM and dynamic light scattering to reveal a twin-pore complex
stable in solution and membrane-like lipid environments (
Li et al., 2004
). Each
PapC molecule within the dimer adopts an ellipsoidal shape and has a pore of
2 nm in width. A PapC C-terminal deletion mutant still retained the ability to
form dimers but by a different manner, as the mutant packed in length-wise
fashion as opposed to the side-wise interaction observed in the wild-type crys-
tals. However, the PapC truncate did not display in vivo activity consistent with
the requirement of CTD to recruit chaperone-subunit complexes and/or alloste-
rically relocate the chaperone-adhesin complex. The ability of FimD to interact
with the PapC C-terminal truncate to rescue P pilus assembly (
So and Thanassi,
2006
), may relate more to donation of a related CTD than to a role for a dimer in
the function and/or regulation of usher activity in vivo, but further experimenta-
tion is needed to resolve these complexities.
The protomers in the side-to-side dimer were only weakly associated (
Rem-
aut et al., 2008
). This dimer only utilizes one channel within the dimer for
secretion of the growing fiber, as suggested by electron density from cryo-EM.
Further, the recent crystal structure of the FimD usher in complex with FimC-
FimH, suggests that the usher monomer and its periplasmic domains should be
sufficient for pilus assembly (
Phan et al., 2011
), without the need for side-to-
side dimerization or head-to-head dimerization mediated by an interaction of
exposed β4 strand edges from the β4-β5 hairpin that protrudes into the extracel-
lular side of the β-barrel core (
Huang et al., 2009
).
Conformational dynamics
Molecular snapshots in the mechanism of pilus assembly provide signposts for
the dynamic processes underlying the sequence of binding events at the usher
that give rise to its catalytic activity. The first step in pilus biogenesis requires
activation of the usher by displacement of plug from the channel. Early evi-
dence from trypsin susceptibility assays (
Saulino et al., 1998
) and previously
mentioned crystal structures do indeed indicate that the usher adopts at least
two distinct conformations. It has been suggested that binding of the chaper-
one-adhesin complex to the periplasmic NTD primes the usher by inducing
a conformational change in the usher that allows plug to swing away from
the pore (
Nishiyama et al., 2003
;
Phan et al., 2011
). NMR studies on isolated
FimD NTD and NTD bound to FimC-FimH give credence to this hypothesis.
The substantial chemical shift changes observed in the linker region of FimD
NTD upon binding FimC-FimH suggest a large movement in the linker (
Nishi-
yama et al., 2005
). This linker movement may tug at the β1 strand of the usher
transmembrane domain, which may in turn induce changes in conformation of
the β-barrel that facilitate plug exit from or plug re-entry into the pore. Simul-
taneously, the hinge movement may allow NTD to swing closer and unload
its chaperone-adhesin cargo to CTDs. Because the usher functions indepen-
dently of an external energy source (
Jacob-Dubuisson et al., 1994
), this large
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