Many
E. coli strains live harmlessly in our guts, but when they find their
way into the urinary tract, they produce pili with sticky ends that
allow them to attach to bladder cells and cause infection. Finding ways
to interfere with pili formation could help thwart urinary tract
infections, which affect millions of women around the world each year.
Previously,
Li’s group at Brookhaven/Stony Brook and colleagues at Washington
University School of Medicine and University College London solved
individual pieces of the puzzle. In 2008, they combined their efforts to
publish the first complete structure of the pore-like protein complex
that traverses the bacterial membrane and transports pili components
from the microbe cell’s interior to its outer surface.
But
the scientists were still not sure how the transporter protein’s
various parts worked to “recruit” and bring together the many subunits
that make up the pili — or how it assembled and moved these structures
through the membrane to the bacterial cell’s surface. The new work,
again combining efforts from the two teams, uses a range of imaging
techniques and computer modeling to arrive at a more complete picture of
the assembly process and transport mechanism.
“This is the first view of a protein transporter in the act of secreting its substrate,” said Li.
At
the European Synchrotron Radiation Facility in Grenoble, France, the
Washington University/UK group determined the atomic-level crystal
structure of the entire transporter protein, known as an “usher,” bound
to the sticky adhesin subunit that forms the end of a pilus and another
helper protein, called a chaperone, that shuttles the pilus subunits to
the usher one at a time. Meanwhile, Li’s group worked at the National Synchrotron Light Source at Brookhaven to produce new images of the unbound usher protein in its closed, inactive state.
“Each
group’s work tells only part of the story, but when combined, the
results provide unique insights into how the transporter works,” said
Li. “By comparing the same transporter in the closed and open state,
we’ve determined how the gate should open, and exactly how the structure
of the channel changes in response to the gate opening so the growing
pilus can reach the exterior of the membrane,” Li said.
When
no subunits are bound to the usher, the barrel like pore remains
plugged, completely sealed off. But when the first chaperoned subunit,
the adhesin, arrives, it causes a dramatic conformational change that
unplugs the pore and changes its shape from an oval to nearly circular.
“This
large conformational rearrangement in the translocation channel upon
activation by adhesin-chaperone is unprecedented for these barrel
proteins, which until now were considered rigid structures,” Li said.
The
research also reveals that the usher protein has two binding sites for
chaperone-subunit complexes. From the imaging studies and bioassays, it
appears that the two operate in concert: While one chaperone-subunit
complex remains bound as it moves through the translocation channel, the
other site is available to recruit the next chaperone-subunit complex
and add it to the growing pilus. Computer models show that the next
incoming subunit is positioned in an ideal orientation for addition to
the growing pilus structure via a “zip-in-zip-out” binding mechanism.
Blocking
or removing either of the two binding sites may therefore be a way to
inhibit pilus formation, and this idea is already being explored in new
drug-development investigations. The other details of pilus assembly
revealed by this study may suggest additional targets for new drugs.
This
research was supported by the Medical Research Council (UK), the
National Institutes of Health (US), and Laboratory Directed Research and
Development funding at Brookhaven Lab. The National Synchrotron Light
Source is supported by the DOE Office of Science.
Crystal structure of the FimDusher bound to its cognate FimC–FimH substrate
Molecular ‘Snapshots’ Capture Infectious Pili Formation
