PI: Knight (SLU)
Project: Structure/function studies of virulence associated
adhesion organelles from pathogenic Gram negative bacteria
Funding ends: 30 June, 2002
Type 1 pili projects
Type 1 pili are adhesive fibers, shown to be specifically
required for Echerichia coli colonisation and pathogenesis
of the urinary tract. The organelles mediate specific
adhesion to a-D-mannosides via the adhesion protein
FimH, situated at the tip of the pilus. The structure
of the adhesion protein FimH in complex with it's chaperone
FimC was solved by S. Knight and D. Choudhury to 2.5
Å resolution last year. FimH is a two domain
protein, consisting of one pilin domain and one lectin
domain. The pilin domain binds to the chaperone, and
is also making contacts to the next subunit in the
pilus. The lectin domain mediates carbohydrate-binding,
although the exact way of binding is yet uncertain.
In order to get a better understanding of FimH-carbohydrate
interactions, a truncated version of FimH has been
produced, where only the lectin domain is kept. This
truncated protein is soluble in the absence of chaperone,
and his-tagged for easier purification. A protocol
for expression and purification for the protein has
been developed, and crystallisation trials have started.
The aim is to get high-resolution structures with different
mannose-derivatives bound to FimH. To complement the
crystallographic studies, binding studies of the same
derivatives to FimH have been initiated, using the
Surface Plasmon Resonance method.
The major subunit of the type 1 pilus is FimA, and the
structure of this protein has not yet been solved.
A problem in purifying this protein is the formation
of multimers of FimA. I have therefore initiated attempts
to modify the protein by cutting of the strand which
is donated to the next subunit in the pilus. This should
prevent multimer formation, and make the construct
easier to purify and crystallise.
The flagellar chaperone FlgN
The bacterial flagellum is comprised of a long helical
propeller filament, attached to a flexible hook by
two hook-associated proteins (HAPs), FlgK and FlgL.
To prevent premature oligomerisation in the cytosol,
the HAPs are suggested to exist in complex with their
chaperone FlgN. The mechanism of building a flagellum
is quite different from constructing a pilus, so we
expect the structure of the cytosolic chaperone FlgN
to be quite different from the periplasmic chaperones
from the pili-system. In cooperation with Professor
C. Hughes, Cambridge, we have expressed and purified
FlgN, and initiated crystallographic trials.
Publication
Knight SD, Berglund J, Choudhury D: "Bacterial
adhesins: structural studies reveal chaperone function
and pilus biogenesis". Curr. Opin. Chem. Biol.
2000, 4:653-660.
Conferences and posters
* Tällberg, SBNet conference in structural biology,
poster.
* Linköping, Annual meeting of the Swedish biophysical
society, poster.
Latest update at 5 March, 2001.