Surface plasmon resonance imaging reveals multiple binding modes of Agrobacterium transformation mediator VirE2 to ssDNA

• Published in: Nucleic acids research Vol. 43; no. 13; pp. 6579 - 6586
• Main Authors: Kim, Sanghyun, Zbaida, David, Elbaum, Michael, Leh, Hervé, Nogues, Claude, Buckle, Malcolm
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 27.07.2015
• Subjects: Agrobacterium tumefaciens; Bacterial Proteins - metabolism; Biochemistry, Molecular Biology; DNA, Single-Stranded - metabolism; DNA-Binding Proteins - metabolism; Ion Channels - metabolism; Kinetics; Life Sciences; Models, Biological; Osmolar Concentration; Protein Binding; Structural Biology; Surface Plasmon Resonance
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkv571

VirE2 is the major secreted protein of Agrobacterium tumefaciens in its genetic transformation of plant hosts. It is co-expressed with a small acidic chaperone VirE1, which prevents VirE2 oligomerization. After secretion into the host cell, VirE2 serves functions similar to a viral capsid in protecting the single-stranded transferred DNA en route to the nucleus. Binding of VirE2 to ssDNA is strongly cooperative and depends moreover on protein-protein interactions. In order to isolate the protein-DNA interactions, imaging surface plasmon resonance (SPRi) studies were conducted using surface-immobilized DNA substrates of length comparable to the protein-binding footprint. Binding curves revealed an important influence of substrate rigidity with a notable preference for poly-T sequences and absence of binding to both poly-A and double-stranded DNA fragments. Dissociation at high salt concentration confirmed the electrostatic nature of the interaction. VirE1-VirE2 heterodimers also bound to ssDNA, though by a different mechanism that was insensitive to high salt. Neither VirE2 nor VirE1-VirE2 followed the Langmuir isotherm expected for reversible monomeric binding. The differences reflect the cooperative self-interactions of VirE2 that are suppressed by VirE1.