Structure-function analysis of invasion plasmid antigen C (IpaC) from Shigella flexneri.
Shigella flexneri causes a self-limiting gastroenteritis in humans, characterized by severe localized inflammation and ulceration of the colonic mucosa. Shigellosis most often targets young children in underdeveloped countries. Invasion plasmid antigen C (IpaC) has been identified as the primary effector protein for Shigella invasion of epithelial cells. Although an initial model of IpaC function has been developed, no detailed structural information is available that could assist in a better understanding of the molecular basis for its interactions with the host cytoskeleton and phospholipid membrane. We have therefore initiated structural studies of IpaC, IpaC I', (residues 101-363 deleted), and IpaC Delta H (residues 63-170 deleted). The secondary and tertiary structure of the protein was examined as a function of temperature, employing circular dichroism and high resolution derivative absorbance techniques. ANS (8-anilino-1-napthalene sulfonic acid) was used to probe the exposure of the hydrophobic surfaces under different conditions. The interaction of IpaC and these mutants with a liposome model (liposomes with entrapped fluorescein) was also examined. Domain III (residues 261-363) was studied using linker-scanning mutagenesis. It was shown that domain III contains periodic, sequence-dependent activity, suggesting helical structure in this section of the protein. In addition to these structural studies, investigation into the actin nucleation properties of IpaC was conducted, and actin nucleation by IpaC and some of the mutants was exhibited. Structure-function relationships of IpaC are discussed.
Kueltzo
Lisa A
LA
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66045, USA.
Osiecki
John
J
Barker
Jeff
J
Picking
Wendy L
WL
Ersoy
Baran
B
Picking
William D
WD
Middaugh
C Russell
CR
D000199:Actins / Q000378:metabolism; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D002942:Circular Dichroism; D008081:Liposomes / Q000737:chemistry; D010957:Plasmids; D012763:Shigella flexneri / Q000235:genetics*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013816:Thermodynamics
D000199:Actins; D000942:Antigens, Bacterial; D008081:Liposomes; C092342:IpaC protein, Shigella