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

Abstract

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.

Refined Structure, DNA Binding Studies, and Dynamics of the Bacteriophage Pf3 Encoded Single-Stranded DNA Binding Protein,

“…‡ The coordinates of the energy-minimized average structure of the (uncomplexed) protein have been deposited with the Brookhaven Protein Data Bank, along with the related NMR constraints. Entry codes are 1PFS and R1PFSMR,respectively. are in accordance with all studies mentioned above (Folmer et al, 1994b;Guan et al, 1994Guan et al, , 1995.…”

Solving the molecular distance geometry problem with inaccurate distance data

“…The first one was proposed by Moré and Wu in order to validate the algorithm dgsol [22]. This set is derived from the three-dimensional structure of the fragments made up of the first 100 an 200 atoms of the chain A of protein PDB:1GPV[25,26]. For each fragment, we generated a set of constraints considering only atoms in the same residue or the neighboring residues.…”

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

“…Two 6-hairpins are labeled according to their functions. et al, 1994) as well as two mutants with changes at the tyrosine 41 position (Kinemage 2; Guan et al, 1994). The importance of Y41 is that it participates directly in the protein dimer-dimer contacts in the GVP-ssDNA complex (King & Coleman, 1988;Stassen et al, 1992).…”