Spectroscopic and calorimetric analyses of invasion plasmid antigen D (IpaD) from Shigella flexneri reveal the presence of two structural domains.
Shigella flexneri is a facultative intracellular pathogen that causes severe gastroenteritis in humans. Invasion plasmid antigen D (IpaD) is an essential participant in Shigella invasion of intestinal cells, but no detailed structural information is available to help understand the proposed role of IpaD in invasion or its interaction with other invasion proteins. Therefore, the secondary and tertiary structure and thermal stability of IpaD as well as selected IpaD deletion mutants were investigated using Fourier transform infrared (FTIR), circular dichroism (CD), and both intrinsic and extrinsic fluorescence spectroscopies. The energetics of thermal unfolding were also evaluated by differential scanning calorimetry (DSC). Secondary-structure analysis by CD and FTIR suggests that that IpaD is primarily alpha-helical with characteristics of a intramolecular coiled coil. Thermal studies revealed that the unfolding of IpaD is a complex process consisting of two transitions centered near 59 and 80 degrees C. A comparison of the data obtained with the intact protein and selected deletion mutants indicated that the lower temperature transition is a reversible event attributable to the unfolding of a small domain located at the N terminus of IpaD. In contrast, the thermal unfolding of the proposed major and highly stable C-terminal domain was irreversible and led to protein aggregation. When the results are taken together, they strongly support the idea that IpaD has two independent folding domains.
Espina
Marianela
M
Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, USA.
Ausar
S Fernando
SF
Middaugh
C Russell
CR
Picking
William D
WD
Picking
Wendy L
WL
D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D002152:Calorimetry, Differential Scanning / Q000379:methods; D002942:Circular Dichroism; D010957:Plasmids; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D012763:Shigella flexneri / Q000737:chemistry* / Q000472:pathogenicity; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D014774:Virulence
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C095770:IpaD protein, Shigella flexneri; C087447:ipaB protein, Shigella; C092342:IpaC protein, Shigella