Molecular and Cellular Biology of Salmonella’s Infections: Structural and functional analysis of the SifA:SKIP complex.

DIACOVICH L., SOPRANO E., BIGNON C., LAFITTE D., BOURNE Y., MÉRESSE S.

Grenoble. Francia.

Workshop; Structural and Molecular Biology of Host-Pathogen Interactions.; 2008

European Synchrotron Radiation Facility (ESRF).

SifA is a Salmonella effector which is translocated into the host cytoplasm of infected cells. SifA is required for the formation of Salmonella-induced filaments and is necessary to the stability of the Salmonella containing vacuole. Recently a human protein SKIP was identified as a target for the protein SifA [1]. SKIP is necessary for the regulation of the kinesin activity associated with various organelles, including the Golgi apparatus and lysosomes. This protein has two distinct functional domains and the binding of SifA on the C-terminal, Pleckstrin homology (PH), domain of SKIP inhibits the binding of kinesin. The interaction between the purified SifA and PH domain of SKIP was characterized by pull-down, native gels, and size exclusion chromatography. We studied the stoichiometry of complex SifA-SKIP(PH) and was found to be 1:1 in molar ratio. The dissociation constant was determined by micro-calorimetry resulting 9.43 x 10-6 M. Finally, proteins crystals able for X-ray diffraction studies were obtained from the native complex SifA-SKIP(PH) and from the Seleno-methionine derivate proteins. The understanding of this kinesin modulation mechanism will provide evidence of how effectors activate a cellular signalization pathway and define its role during the course of the pathogenic process. References [1] Boucrot E, Henry T, Borg JP, Gorvel JP, Méresse S. The intracellular fate of Salmonella depends on the recruitment of kinesin. Science. 2005 May 20;308(5725):1174-8. (Premio al mejor poster).