This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We performed Small Angle X-ray Scattering (SAXS) structural studies using the BioCAT beamline on a novel actin filament nucleator from Vibrio parahaemolyticus named VopL. We have found that the strong actin filament nucleation activity of VopL depends on the presence of three WH2 domains and dimerization through a C-terminal domain (DD) of unknown structure. Since this DD had no detectable similarity to any known structure in the Protein Data Bank, we used SAXS to study its structure in solution to gain further insights into the nucleation mechanism of VopL. The SAXS structure showed the expected two-fold symmetry of the dimer, consisting of a central dimerization 'knot domain', which probably comprises the predicted C-terminal coiled coil, and two diametrically opposed smaller domains. We also studied the complex of actin with VopL fragment 1W-DD (containing one of the three WH2 domains and the DD).. While the study of this complex did not yield much new information, a few inferences may be drawn: a) the structure of the DD reemerged as a stable unit in the structure of the complex with actin, b) actin subunits are bound loosely in the polymerization nucleus and c) the actin subunits bind at the interface between the central knob and small distal domains. In summary, our biochemical and structural analyses by SAXS of VopL constructs ?actin suggest that the dimerization domain binds at the pointed end of the actin filament and contains a coiled-coil region at the C-terminus.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR008630-16
Application #
8361288
Study Section
Special Emphasis Panel (ZRG1-BCMB-E (40))
Project Start
2011-01-01
Project End
2011-12-31
Budget Start
2011-01-01
Budget End
2011-12-31
Support Year
16
Fiscal Year
2011
Total Cost
$5,914
Indirect Cost
Name
Illinois Institute of Technology
Department
Other Basic Sciences
Type
Schools of Arts and Sciences
DUNS #
042084434
City
Chicago
State
IL
Country
United States
Zip Code
60616
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