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

International audienceVirE2 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 Lang-muir isotherm expected for reversible monomeric binding. The differences reflect the cooperative self-interactions of VirE2 that are suppressed by VirE1

In Vitro Uptake of SCH 27899 (Evernimicin) by Rat Alveolar Macrophages

The in vitro uptake of [(14)C]evernimicin ([(14)C]SCH 27899) by primary cultures of rat alveolar macrophages and hepatocytes was determined. Both cell populations exhibited linear rates of uptake. However, the initial rate of drug uptake by alveolar macrophages was about threefold higher than that by hepatocytes. These findings demonstrate that [(14)C]evernimicin is taken up by rat alveolar macrophages, supporting the likelihood that the drug is able to reach sites of infection