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J. Biol. Chem., Vol. 281, Issue 49, 37628-37635, December 8, 2006

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Topology of the VirB4 C Terminus in the Agrobacterium tumefaciens VirB/D4 Type IV Secretion System^*

Olga Draper¹, Rebecca Middleton¹², Michaeleen Doucleff^¶23, and Patricia C. Zambryski⁴

From the Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California 94720, Department of Chemistry, University of California, Berkeley, Berkeley, California 94720, and ^¶Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720

Gram-negative type IV secretion systems (T4SSs) transfer proteins and DNA to eukaryotic and/or prokaryotic recipients resulting in pathogenesis or conjugative DNA transfer. VirB4, one of the most conserved proteins in these systems, has both energetic and structural roles in substrate translocation. We previously predicted a structural model for the large C-terminal domain (residues 425-789) of VirB4 of Agrobacterium tumefaciens. Here we have defined a homology-based structural model for Agrobacterium VirB11. Both VirB4 and VirB11 models predict hexameric oligomers. Yeast two-hybrid interactions define peptides in the C terminus of VirB4 and the N terminus of VirB11 that interact with each other. These interactions were mapped onto the homology models to predict direct interactions between the hexameric interfaces of VirB4 and VirB11 such that the VirB4 C terminus stacks above VirB11 in the periplasm. In support of this, fractionation and Western blotting show that the VirB4 C terminus is localized to the membrane and periplasm rather than the cytoplasm of cells. Additional high resolution yeast two-hybrid results demonstrate interactions between the C terminus of VirB4 and the periplasmic portions of VirB1, VirB8, and VirB10. Genetic studies reveal dominant negative interactions and thus function of the VirB4 C terminus in vivo. The above data are integrated with the existing body of literature to propose a structural, periplasmic role for the C-terminal half of the Agrobacterium VirB4 protein.

Received for publication, July 5, 2006 , and in revised form, September 18, 2006.

^* 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ Supported by National Science Foundation Grant 0343566 (to P. Z.).

² These authors contributed equally to this work.

³ Supported by a University of California Fellowship.

⁴ To whom correspondence should be addressed: Dept. of Plant and Microbial Biology, University of California, Berkeley, CA 94720. Tel.: 510-643-9203; E-mail: zambrysk{at}nature.berkeley.edu.

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