James
Bann
(Wichita State University)
Structure and Mechanism of CS1 Pilus Assembly
Pili
are hair-like surface fibers that mediate attachment and colonization
of pathogenic bacteria to host tissues. The long-term goal of my research
is to understand the mechanism of assembly of pili as a prelude to the
development of therapeutics aimed at preventing pilus assembly. The CS1
pilus system from enterotoxigenic Escherichia coli is encoded on the cooBACD
operon, and is composed of multiple CooA subunits that have at the distal
tip a single subunit of CooD required for adherence to intestinal cells.
Additionally, both CooC, a 95 kDa outer membrane protein, and CooB, a
periplasmic chaperone, are required for pilus assembly. The specific hypothesis
is that the assembly of CS1 pili is initiated by a conformational change
in the outer membrane protein CooC upon binding of CooB alone or as a
complex with CooD or CooA. The hypothesis is based on limited data that
indicate that 1) CooC is an integral outer membrane protein, 2) expression
of cooC is required for export of pili to the cell surface and 3) CooB
copurifies with CooC and stabilizes CooC against degradation. Therefore
CooB, either by itself or as a complex with CooA or CooD, initiates a
conformational change in CooC that allows transport of subunits to the
surface. The specific aims are to:
1. Characterize the native structure and oligomerization state of CooC. The formation of a defined structure will be determined by (i) the stability to denaturant, (ii) stability to proteolysis, and (iii) dispersion of 19F-NMR chemical shifts. The oligomerization state of CooC will be determined by analytical gel filtration.
2. Define structural changes in CooC. Purified CooB or CooB-subunit complexes
will be added to purified CooC labeled site-specifically with p-fluoro-phenylalanine,
and structural changes in CooC determined by 19F-NMR. The 31 phenylalanines
are well dispersed throughout the sequence. Specific regions that interact
with CooB or with CooB-subunit complexes will be identified, and mutagenesis
of residues in regions of CooC will be correlated with in vivo studies
of complementation of a cooC- cooBAD operon. The regions identified as
important for binding will guide the development of peptide therapeutics
aimed at attenuating binding.
3. Determine the role of CooC in the mechanism of polymerization of CooA.
Preliminary data indicate that CooA forms a stable 1:1 complex with a
CooB, suggesting that polymerization of CooA requires other components
such as CooC. The CooB-A complex will be added to purified CooC in the
absence and presence of a CooB-CooD complex, and polymerization of CooA
will be determined by (i) gel filtration, (ii) fluorescence polarization
and (iii) native PAGE followed by western blotting with a CooA specific
antibody.
Copyright
© 2005
Center for
Biomedical Research Excellence in Protein Structure and Function
Department
of Medicinal Chemistry, School of Pharmacy
The University
of Kansas
Contact webmaster with comments about this web site.
