Agrobacterial, Single-Stranded DNA-Binding Protein VirE2 and Its Complexes.

VirE2 from Agrobacterium tumefaciens is a single-stranded (ss) DNA-binding protein involved in delivery of ssT-DNA (single-stranded transfer DNA) from the agrobacterial Ti plasmid into the eukaryotic cell nucleus. The crystallized part of VirE2 was studied by X-ray diffraction, and the noncrystallized parts of the C- (40 amino acid residues [aars]) and N- (111 aars) termini of the protein, which are presumably disordered, were evaluated by computational methods. We did a molecular dynamics simulation of VirE2 without VirE1 and observed no large changes in domain orientation. The interaction of VirE2 with ssDNA and formation of ssDNA-VirE2 complexes in silico were studied. We also used computer-aided methods to design model complexes consisting from two- and four-subunit VirE2 proteins. We examined the implication of disordered sites in formation of two- and four-subunit VirE2 complexes. Formation of VirE2 dimers and tetramers within ssDNA-VirE2 complexes was demonstrated by computational methods. Using the Platinum program, we found that hydrophilic amino acids were predominant on the surface of the four-subunit VirE2 complex.

Computer evaluation of VirE2 protein complexes for ssDNA transfer ability.

The single-stranded transfer DNA from the Ti plasmid of the soil bacteria Agrobacterium nonspecifically integrates into the plant chromosome and is inherited at subsequent cell divisions. How it is transferred across host membranes is unknown, but it is believed that VirE2 proteins form a membrane-spanning pore or channel in a lipid bilayer and possibly mediate the delivery of the single-stranded transfer DNA-VirD2-VirE2 complex to the plant cell chromosomes. The aim of this work was to perform a computer simulation of VirE2's pore-forming capacity and an evaluation of constructed VirE2 complexes. The oscillating motions of complexes consisting of two and four VirE2 subunits were estimated by the molecular dynamics and normal modes methods. We did not predict any large changes in domain orientation for two and four-subunit VirE2 complexes within simulation times of 1ns. A possible gating mechanism similar to that seen in the ion channels of the complex formed from two VirE2 proteins was proposed, whereas no conformational changes were predicted inside the pore in the complex formed from four VirE2 proteins.

VirE2-dependent pores for ssDNA transfer across artificial and cell membranes.

The transfer of single-stranded (ss) T-DNA from soil bacteria of the genus Agrobacterium with the help of the VirE2 protein, which possibly mediates the delivery of ss-T-DNA across the cell membrane, was demonstrated earlier, but how VirE2 participates in ssDNA transfer across artificial and natural membranes is not known. Using computational methods, we reconstructed model structures composed of two and four VirE2 proteins and showed by the MOLE program the formation of pores with channel diameters of 1.2-1.6 and 1.4-4.6 nm in a model structure formed from two and four VirE2 molecules, respectively. Using light scattering, we recorded the size distribution for recombinant VirE2-dependent complexes in aqueous solutions and found that VirE2 in a buffer solution is present as a complex made up of two or more proteins. We revealed single, long-lived jumps in voltage-dependent membrane conductance during coincubation of planar black membranes with the VirE2 protein. On the addition of VirE2 and FAM-labeled oligonucleotides to HeLa cells, the fluorescence intensity for the cells increased by 56% as compared to that for cells incubated only with oligonucleotides.