Three-dimensional Reconstruction of Agrobacterium VirE2 Protein with Single-stranded DNA

• Published in: The Journal of biological chemistry Vol. 279; no. 24; pp. 25359 - 25363
• Main Authors: Abu-Arish, Asmahan, Frenkiel-Krispin, Daphna, Fricke, Tobin, Tzfira, Tzvi, Citovsky, Vitaly, Wolf, Sharon Grayer, Elbaum, Michael
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 11.06.2004; American Society for Biochemistry and Molecular Biology
• Subjects: Active Transport, Cell Nucleus; Agrobacterium tumefaciens; Bacterial Proteins; DNA, Single-Stranded - chemistry; DNA-Binding Proteins - chemistry; Imaging, Three-Dimensional; Ion Channels - chemistry; Microscopy, Electron; Nucleic Acid Conformation; Rhizobium - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M401804200

Agrobacterium tumefaciens infects plant cells by a unique mechanism involving an interkingdom genetic transfer. A single-stranded DNA substrate is transported across the two cell walls along with the bacterial virulence proteins VirD2 and VirE2. A single VirD2 molecule covalently binds to the 5′-end of the single-stranded DNA, while the VirE2 protein binds stoichiometrically along the length of the DNA, without sequence specificity. An earlier transmission/scanning transmission electron microscopy study indicated a solenoidal (“telephone coil”) organization of the VirE2-DNA complex. Here we report a three-dimensional reconstruction of this complex using electron microscopy and single-particle image-processing methods. We find a hollow helical structure of 15.7-nm outer diameter, with a helical rise of 51.5 nm and 4.25 VirE2 proteins/turn. The inner face of the protein units contains a continuous wall and an inward protruding shelf. These structures appear to accommodate the DNA binding. Such a quaternary arrangement naturally sequesters the DNA from cytoplasmic nucleases and suggests a mechanism for its nuclear import by decoration with host cell factors. Coexisting with the helices, we also found VirE2 tetrameric ring structures. A two-dimensional average of the latter confirms the major features of the three-dimensional reconstruction.