ActA from Listeria monocytogenes Can Interact with Up to Four Ena/VASP Homology 1 Domains Simultaneously210

• Published in: The Journal of biological chemistry 2001-10, Vol.276 (43), p.40096-40103
• Main Authors: Machner, Matthias P., Urbanke, Claus, Barzik, Melanie, Otten, Sonja, Sechi, Antonio S., Wehland, Jürgen, Heinz, Dirk W.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M104279200

The facultative intracellular human pathogenic bacterium Listeria monocytogenes actively recruits host actin to its surface to achieve motility within infected cells. The bacterial surface protein ActA is solely responsible for this process by mimicking fundamental steps of host cell actin dynamics. ActA, a modular protein, contains an N-terminal actin nucleation site and a central proline-rich motif of the 4-fold repeated consensus sequence FPPPP (FP4). This motif is specifically recognized by members of the Ena/VASP protein family. These proteins additionally recruit the profilin-G-actin complex increasing the local concentration of G-actin close to the bacterial surface. By using analytical ultracentrifugation, we show that a single ActA molecule can simultaneously interact with four Ena/VASP homology 1 (EVH1) domains. The four FP4 sites have roughly equivalent affinities with dissociation constants of about 4 µm. Mutational analysis of the FP4 motifs indicate that the phenylalanine is mandatory for ActA-EVH1 interaction, whereas in each case exchange of the third proline was tolerated. Finally, by using sedimentation equilibrium centrifugation techniques, we demonstrate that ActA is a monomeric protein. By combining these results, we formulate a stoichiometric model to describe how ActA enables Listeriato utilize efficiently resources of the host cell microfilament for its own intracellular motility.