Exploring the DNA binding domain of gene V protein encoded by bacteriophage M13 with the aid of spin-labeled oligonucleotides in combination with proton NMR

Abstract

The DNA binding domain of the single-stranded DNA binding protein gene V protein encoded by the bacteriophage M13 was studied by means of 1H nuclear magnetic resonance, through use of a spin-labeled deoxytrinucleotide. The paramagnetic relaxation effects observed in the 1H-NMR spectrum of M13 GVP upon binding of the spin-labeled ligand were made manifest by means of 2D difference spectroscopy. In this way, a vast data reduction was accomplished which enabled us to check and extend the analysis of the 2D spectra carried out previously as well as to probe the DNA binding domain and its surroundings. The DNA binding domain is principally situated on two beta-loops. The major loop of the two is the so-called DNA binding loop (residues 16-28) of the protein where the residues which constitute one side of the beta-ladder (in particular, residues Ser20, Tyr26, and Leu28) are closest to the DNA spin-label. The other loop is part of the so-called dyad domain of the protein (residues 68-78), and mainly its residues at the tip are affected by the spin-label (in particular, Phe73). In addition, a part of the so-called complex domain of the protein (residues 44-51) which runs contiguous to the DNA binding loop is in close vicinity to the DNA. The NMR data imply that the DNA binding domain is divided over two monomeric units of the GVP dimer in which the DNA binding loop and the tip of the dyad loop are part of opposite monomers. The view of the GVP-ssDNA binding interaction which emerges from our data differs from previous molecular modeling proposals which were based on the GVP crystal structure (Brayer & McPherson, 1984; Hutchinson et al., 1990). These models implicate the involvement of one or two tyrosines (Tyr34, Tyr41) of the complex loop of the protein to participate in complex formation with ssDNA. In the NMR studies with the spin-labeled oligonucleotides, no indication of such interactions has been found. Other differences between the models and our NMR data are related to the structural differences found when solution and crystal structures are compared.

Secondary Structure of the Single‐stranded DNA Binding Protein Encoded by Filamentous Phage Pf3 as Determined by NMR

“…The sequences of these turns are : Glu4S-(Xaa),-Pro49-AlaSO-Gly51 in Pf3 ssDBP, and AspSO-(Xaa),-AlaS7-ProS8-Gly59 in Ff GVP. Contrary to AspSO in Ff GVP, residues 57-59 appear not to be involved in DNA binding nor protein-protein interactions (Folkers et al, 1993;Folmer et al, 1994), so their sequence similarity is probably of structural rather than functional relevance. In Fig.…”

“…Most of these residues are situated at the same side of the hairpin (Argl6, Gly18, Ser20, and Tyr26 of the Ff protein, and Argl2, Gly14, Serl6, Tyr22 of Pf3); the residues protruding to the other side are less well conserved (except for Pro2.5). DNA binding studies performed on Ff GVP have indicated that the surface formed by residues Argl6, Gly18, Ser20, Tyr26 and Leu28 is involved in the interaction with the DNA (Folkers et al, 1993). Therefore, it is likely that the corresponding residues in Pf3 ssDBP also form part of the DNA binding domain.…”

Electrostatic potential distribution of the gene V protein from Ff phage facilitates cooperative DNA binding: A model of the GVP‐ssDNA complex

“…More detailed studies regarding the interactions between GVP and DNA have been carried out (reviewed in Kansy et al, 1986). In particular, NMR and fluorescence spectroscopies have implicated several amino acids of GVP that are in contact with DNA in the GVP-ssDNA complex (King & Coleman, 1987Dick et al, 1989;Stassen et al, 1992;Folkers et al, 1993).…”

Analyses of the stability and function of three surface mutants (R82C, K69H, and L32R) of the gene V protein from Ff phage by X‐ray crystallography

“…6E) and a new salt bridge between D50 and R80 is formed. In our model of the GVP-ssDNA complex (Guan et al, 1995), R80 is involved in the interaction between ssDNA and GVP (Folkers et al, 1993). So a movement of R80 will influence the It is of interest to note that the hydrophobic L32 is found on the surface of the p-core near the dimer-dimer contact loop.…”

“…During the rolling circle replication process, GVP binds to the ssDNA cooperatively and, ultimately, a superhelical GVP-DNA complex is formed. The structure of such helical complexes has been studied by a number of biophysical methods, including electron microscopy (Gray, 1989;Olah et al, 1995), neutron scattering (Gray et al, 1982;Olah et al, 1995), NMR and fluorescence spectroscopies (King & Coleman, 1987Dick et al, 1989;Stassen et al, 1992;Folkers et al, 1993). The results of these studies have provided valuable insights regarding the interactions of GVP and ssDNA.…”