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Salmonella Invasion Protein A (SipA)

Mutants of Salmonella typhimurium with a disruption of the Salmonella invasion protein A, or sipA, gene show an attenuated virulence in bovine intestinal models, impaired ability to invade cells, and less robust and localized membrane ruffling as compared with the wild type strain. A carboxy-terminal domain of SipA is involved in binding to F-actin, polymerization of G-actin in low salt, and reduction of the critical G-actin concentration required for polymerization. The activity of SipA enhances F-actin bundling by host fimbrin (plastin) and SipC -induced actin nucleation and bundling both in vitro and in cultured cells. Moreover, SipA prevents F-actin filament depolymerization in vitro, and it locally inhibits ADF/cofilin- and gelsolin- directed actin disassembly.

The crystal structure of the carboxy-terminal domain of S. typhimurium SipA (residues 497 to 669) has been determined, revealing this molecule to be a compact, heart shaped fold dominated by helical secondary structure measuring roughly 30x40x40 A. SipA 497-669 is active in actin binding and polymerization, and has a novel three-dimensional structure. In the crystal, the protein is ordered only between residues 513 and 657, and the N- and C-termini are positioned at opposite sides of the molecule.

Electron microscopic studies of actin-SipA 497-669 complexes show that SipA interacts with actin as a globular structure with two non-globular extensions connecting actin protomers on opposite strands. These arms are located at opposite ends of the globular density and link subdomain four of the nth actin subunit in the filament to subdomain one of subunit n+3. Previous studies with a longer construct (SipA 446-684) revealed that SipA is an extended and tubular molecule on the order of nearly 100 A long. Electron density maps obtained from electron micrographs of filaments with SipA 497-669 and SipA 446-684 were compared, and it was found that in both cases there is a central density into which the globular crystal structure can be positioned. What differs significantly is the length of the arms. Thus, the previously observed extended and tubular appearance of longer SipA constructs is due to the presence of additional polypeptide in non-globular extensions, or "arms."

The combination of the SipA crystal structure and EM data has led to a model for SipA function termed the "molecular staple." In this model, SipA possesses a globular domain for actin binding and positioning the arms, and non-globular extensions to reach out and tether actin molecules on opposing strands of the filament. Numerous deletion mutants in which the arms were truncated have supported this model, although difficulties in controlling actin polymerization in the presence of SipA have prevented high resolution crystallographic confirmation of this model.

 

Lilic M, Galkin VE, Orlova A, VanLoock MS, Egelman EH, Stebbins CE. "Salmonella SipA polymerizes actin by stapling filaments with nonglobular protein arms." Science. 2003 Sep 26;301(5641):1918-21. [Abstract]  [pdf] [pdb]

M. Lilic, V. E. Galkin, A. Orlova, M. S. Van-Loock, E. H. Egelman, and C. E. Stebbins, “A salmonella invasion protein that acts as a "molecular staple" [pdf]

 

Dr. Mirjana Lilic was the lead scientist on the SipA project. Our collaborators were the group of Dr. Edward Egelman at the University of Virginia Health Sciences Cente.