Bacteria often produce extracellular amyloid fibres
via
a multi-component
secretion system. Aggregation-prone, unstructured subunits cross the periplasm
and are secreted through the outer membrane, after which they self-assemble.
Here, significant progress is presented towards solving the high-resolution
crystal structure of the novel amyloid transporter FapF from
Pseudomonas
,
which facilitates the secretion of the amyloid-forming polypeptide FapC across
the bacterial outer membrane. This represents the first step towards obtaining
structural insight into the products of the
Pseudomonas fap
operon. Initial
attempts at crystallizing full-length and N-terminally truncated constructs by
refolding techniques were not successful; however, after preparing FapF
106–430
from the membrane fraction, reproducible crystals were obtained using the
sitting-drop method of vapour diffusion. Diffraction data have been processed
to 2.5 A
̊
resolution. These crystals belonged to the monoclinic space group
C
121,
with unit-cell parameters
a
= 143.4,
b
= 124.6,
c
= 80.4 A
̊
,

=

= 90,

= 96.32

and three monomers in the asymmetric unit. It was found that the switch to
complete detergent exchange into C8E4 was crucial for forming well diffracting
crystals, and it is suggested that this combined with limited proteolysis is a
potentially useful protocol for membrane

-barrel protein crystallography. The
three-dimensional structure of FapF will provide invaluable information on the
mechanistic differences of biogenesis between the curli and Fap functional
amyloid systems.