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J. Biol. Chem., Vol. 283, Issue 35, 23852-23862, August 29, 2008

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

Matthew J. Footer¹, John K. Lyo, and Julie A. Theriot^¶

From the Department of Biochemistry and ^¶Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305 and Weill Medical College of Cornell University, New York, New York 10021

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 actin-based 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 (19 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.

Received for publication, May 6, 2008 , and in revised form, June 17, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant R01-AI36929. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: 279 Campus Dr. B477, Stanford, CA 94305. Fax: 650-723-6783; E-mail: footer{at}stanford.edu.

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