Activation of the Arp2/3 complex by the Listeria acta protein. Acta binds two actin monomers and three subunits of the Arp2/3 complex.
ActA is a bacterially encoded protein that enables Listeria monocytogenes to hijack the host cell actin cytoskeleton. It promotes Arp2/3-dependent actin nucleation, but its interactions with cellular components of the nucleation machinery are not well understood. Here we show that two domains of ActA (residues 85-104 and 121-138) with sequence similarity to WASP homology 2 domains bind two actin monomers with submicromolar affinity. ActA binds Arp2/3 with a K(d) of 0.6 microm and competes for binding with the WASP family proteins N-WASP and Scar1. By chemical cross-linking, ActA, N-WASP, and Scar1 contact the same three subunits of the Arp2/3 complex, p40, Arp2, and Arp3. Interestingly, profilin competes with ActA for binding of Arp2/3, but actophorin (cofilin) does not. The minimal Arp2/3-binding site of ActA (residues 144-170) is C-terminal to both actin-binding sites and shares sequence homology with Arp2/3-binding regions of WASP family proteins. The maximal activity at saturating concentrations of ActA is identical to the most active domains of the WASP family proteins. We propose that ActA and endogenous WASP family proteins promote Arp2/3-dependent nucleation by similar mechanisms and require simultaneous binding of Arp2 and Arp3.
Zalevsky
J
J
Department of Cellular and Molecular Pharmacology, University of California School of Medicine, San Francisco, California 94129, USA.
Grigorova
I
I
Mullins
R D
RD
D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000494:pharmacology*; D001665:Binding Sites; D003598:Cytoskeletal Proteins; D006801:Humans; D007700:Kinetics; D008089:Listeria monocytogenes / Q000737:chemistry*; D008565:Membrane Proteins / Q000494:pharmacology*; D008840:Microfilament Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D051381:Rats; D017386:Sequence Homology, Amino Acid; D051300:Wiskott-Aldrich Syndrome Protein Family
C495926:ACTR2 protein, human; C495928:ACTR3 protein, human; D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins; D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D051300:Wiskott-Aldrich Syndrome Protein Family; C072980:actA protein, Listeria monocytogenes