title: The DNA binding mechanism of a SSB protein from Lactococcus lactis siphophage p2.
abstract: Virulent lactococcal phages of the Siphoviridae family are responsible for the industrial milk fermentation failures worldwide. Lactococcus lactis, a Gram-positive bacterium widely used for the manufacture of fermented dairy products, is subjected to infections by virulent phages, predominantly those of the 936 group, including phage p2. Among the proteins coded by lactococcal phage genomes, of special interest are those expressed early, which are crucial to efficiently carry out the phage lytic cycle. We previously identified and solved the 3D structure of lactococcal phage p2 ORF34, a single stranded DNA binding protein (SSBp2). Here we investigated the molecular basis of ORF34 binding mechanism to DNA. DNA docking on SSBp2 and Molecular Dynamics simulations of the resulting complex identified R15 as a crucial residue for ssDNA binding. Electrophoretic Mobility Shift Assays (EMSA) and Atomic Force Microscopy (AFM) imaging revealed the inability of the Arg15Ala mutant to bind ssDNA, as compared to the native protein. Since R15 is highly conserved among lactococcal SSBs, we propose that its role in the SSBp2/DNA complex stabilization might be extended to all the members of this protein family.
authors: Dept. of Veterinary Science, Università degli Studi di Parma, Parma, Italy.
authors: Erika
authors: E
authors: Scaltriti
authors: Eugenia
authors: E
authors: Polverini
authors: Stefano
authors: S
authors: Grolli
authors: Elisa
authors: E
authors: Eufemi
authors: Sylvain
authors: S
authors: Moineau
authors: Christian
authors: C
authors: Cambillau
authors: Roberto
authors: R
authors: Ramoni
mesh_terms: D017901:Bacteriophage P2 / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism / Q000821:virology*; D018625:Microscopy, Atomic Force / Q000379:methods; D062105:Molecular Docking Simulation / Q000379:methods; D056004:Molecular Dynamics Simulation; D009154:Mutation; D017510:Protein Folding; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins