Close Packing of Listeria monocytogenes ActA, a Natively Unfolded Protein, Enhances F-actin Assembly without Dimerization

Abstract

Studies of the biochemistry of Listeria monocytogenes virulence protein ActA have typically focused on the behavior of bacteria in complex systems or on the characterization of the protein after expression and purification. Although prior in vivo work has proposed that ActA forms dimers on the surface of L. monocytogenes, dimerization has not been demonstrated in vitro, and little consideration has been given to the surface environment where ActA performs its pivotal role in bacterial actinbased motility. We have synthesized and characterized an ActA dimer and provide evidence that the two ActA molecules do not interact with each other even when tethered together. However, we also demonstrate that artificial dimers provide superior activation of actin nucleation by the Arp2/3 complex compared with monomers and that increased activation of the Arp2/3 complex by dimers may be a general property of Arp2/3 activators. It appears that the close packing (ϳ19 nm) of ActA molecules on the surface of L. monocytogenes is so dense that the kinetics of actin nucleation mimic that of synthetic ActA dimers. We also present observations indicating that ActA is a natively unfolded protein, largely random coil that is responsible for many of the unique physical properties of ActA including its extended structure, aberrant mobility during SDS-PAGE, and ability to resist irreversible denaturation upon heating.

Structural and biochemical characterization of two binding sites for nucleation-promoting factor WASp-VCA on Arp2/3 complex

“…Cells have multiple mechanisms to cluster VCAs for parallel interactions with Arp2/3 complex: close packing of independent VCAs on cell membranes (35); tandem activity-bearing VCA motifs (12,13); or assembly on scaffold proteins (17,(36)(37)(38) including adapters with two SH3 domains that bind proline-rich domains of two nucleation-promoting factors (NPFs) (13,39). Activation of these crowded NPFs exposes their C-terminal VCA motifs, allowing interactions with Arp2/3 complex and actin monomers.…”

Mechanism of a Concentration-dependent Switch between Activation and Inhibition of Arp2/3 Complex by Coronin

“…Trimerization of Crn1 Is Required for Activation-Oligomerization of WASp/SCAR proteins greatly enhances their potency (39,41,42), so we asked whether oligomerization of Crn1 influences its activation potential. We used analytical ultracentrifugation to demonstrate that Crn1 trimerizes (Fig.…”

Autophagy targeting ofListeria monocytogenesand the bacterial countermeasure

“…Infection of MDCK/pGFP-LC3 cells with surface. 25 Crosslinking experiments and calibrated immunofluorescence studies indicate that enough space exists around a single ActA molecule to interact with one copy of the Arp2/3 complex, one VASP tetramer, four profilactin complexes and actin filaments, thus forming a core motility machinery on the bacterial surface. 26 Our findings, together with these studies, strongly support the premise that the coverage of the bacterial surface with densely packed ActA moieties and interacting host proteins disguises the bacterium as a subcellular organelle, thus protecting it from autophagic recognition by the host cell (Fig.…”