title: ActA from Listeria monocytogenes can interact with up to four Ena/VASP homology 1 domains simultaneously.
abstract: 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 (FP(4)). 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 FP(4) sites have roughly equivalent affinities with dissociation constants of about 4 microm. Mutational analysis of the FP(4) 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 Listeria to utilize efficiently resources of the host cell microfilament for its own intracellular motility.
authors.affiliation: Department of Structural Biology, German Research Center for Biotechnology (GBF), Mascheroder Weg 1, D-38124 Braunschweig, Germany.
authors.forename: M P
authors.initials: MP
authors.lastname: Machner
authors.forename: C
authors.initials: C
authors.lastname: Urbanke
authors.forename: M
authors.initials: M
authors.lastname: Barzik
authors.forename: S
authors.initials: S
authors.lastname: Otten
authors.forename: A S
authors.initials: AS
authors.lastname: Sechi
authors.forename: J
authors.initials: J
authors.lastname: Wehland
authors.forename: D W
authors.initials: DW
authors.lastname: Heinz
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000235:genetics; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins / Q000235:genetics; D008089:Listeria monocytogenes / Q000472:pathogenicity*; D008565:Membrane Proteins / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism*; D008956:Models, Chemical; D008958:Models, Molecular; D009842:Oligopeptides / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D000098662:Vasodilator-Stimulated Phosphoprotein
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; C092744:ENA-VASP proteins; C102176:Enah protein, mouse; D008565:Membrane Proteins; D008840:Microfilament Proteins; D009842:Oligopeptides; D010446:Peptide Fragments; D010750:Phosphoproteins; D000098662:Vasodilator-Stimulated Phosphoprotein; C072980:actA protein, Listeria monocytogenes