Crystal Structures of Y41H and Y41F Mutants of Gene V Protein from Ff Phage Suggest Possible Protein-Protein Interactions in the GVP-ssDNA Complex

• Published in: Biochemistry (Easton) 1994-06, Vol.33 (25), p.7768-7778
• Main Authors: Guan, Yue, Zhang, Hong, Konings, Ruud N. H, Hilbers, Cornelis W, Terwilliger, Thomas C, Wang, Andrew H.-J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacteriophage M13; Crystallography, X-Ray; DNA, Single-Stranded - metabolism; DNA-Binding Proteins - chemistry; Fourier Analysis; Macromolecular Substances; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Binding; Protein Structure, Tertiary; Structure-Activity Relationship; Viral Proteins - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00191a004

Gene V protein (GVP) encoded by the filamentous phage Ff (M13, fl, fd) is a homodimeric protein of 87 amino acids that binds to single-stranded DNA (ssDNA) nonspecifically and cooperatively. The structure (monoclinic C2 form) of the wild-type protein has been determined and refined at 1.8-A resolution [Skinner et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 2071-2075]. The monomer structure consists of a somewhat distorted five-stranded beta-barrel core with three prominent loops: a DNA-binding loop, a dyad loop, and a dimer contact loop. The amino acid residue at position 41 plays an important role in the dimer-dimer interactions of the protein-ssDNA complex. Two Y41 mutant structures have been studied by X-ray crystallography. The Y41F GVP structure has been refined to an R-factor of 0.180 at 2.2-A resolution and is very similar to the wild-type (wt) structure (rmsd of all C alpha atoms = 0.30 A). In contrast, Y41H GVP forms a new crystal lattice in the space group P2(1)2(1)2(1) with a = 77.18 A, b = 84.17 A, and c = 28.62 A. Its structure has been solved by the molecular replacement method and refined to an R-factor of 0.170 at 2.5-A resolution. The two monomers of Y41H are crystallographically independent, and their structures remain similar to wt-GVP but with significant differences, particularly in the DNA-binding hairpin region. In both crystals, the loop (residues 36-43) that contains the Y41 residue is involved in the crystal dimer packings but in a different manner. The dimer-dimer contacts found in the wt-GVP crystal may be important for GVP aggregation in the absence of DNA. In the presence of DNA, the dimer-dimer contacts may switch to the type found in the Y41H crystal, allowing the GVP-ssDNA complex to form cooperatively. A model of the complex, consistent with existing biochemical and biophysical data, has been constructed from those crystal packing data.