Three-dimensional structure of the single-stranded DNA-binding protein encoded by gene V of the filamentous bacteriophage M13 and a model of its complex with single-stranded DNA

Abstract

Abstract Gene V protein (GVP) of the filamentous bacteriophage M13 is a single-stranded DNA (ssDNA) binding protein that both regulates virus DNA replication and gene expression and that, upon cooperative binding to the viral genome, forms a regular left-handed superhelical polymer in which the GVP dimers are arrayed at the outside and the ssDNA strands on the inside. It is 87 amino acids long and occurs in solution as a homodimer. The solution structure of the homodimer of the GVP mutant Tyr41-His has recently been elucidated by nuclear magnetic resonance and X-ray crystallographic techniques (Folkers et al., (1994) J. Mol. Biol. 236, 229–246; Skinner, M.M. et al. (1994) Proc. Natl. Acad. Sci. USA 91, 2071–2075). The monomer consists of a distorted five-stranded β-barrel from which three major loops protrude; one of these is involved in dimerization, another in DNA binding and a third in cooperative protein—protein interactions. To derive a model for the complex between viral DNA and GVP, a contact analysis and a series of restrained molecular dynamics simulations were employed. Contact analysis served to determine the helix parameters that permit the energetically most favourable packing of the protein molecules. Subsequently, the superhelix was built into which two extended DNA strands were modelled using restrained molecular dynamics. Specific constraints, based on nuclear magnetic resonance spin label experiments, were included to ensure that the DNA would position itself into the binding groove of the protein. The left-handed model presented is highly consistent with existing biophysical and biochemical data. A description of the protein—protein interface is given and the interaction between the protein and DNA is discussed in view of the derived model. In addition, it is described that, on the basis of the available experimental data and not imposing the left-handedness of the nucleoprotein complex, it is feasible also to build a plausible model for the complex which exhibits the opposite, i.e. right-handed, helical sense. This right-handed structure features characteristics highly similar to those of the left-handed complex. The meaning of the helical models regarding the biological role GVP fulfils in the phage replication process is discussed.