title: Involvement of histidine in complex formation of PriB and single-stranded DNA.
abstract: PriB is a basic 10-kDa protein that acts as a facilitator in PriA-dependent replication restart in Escherichia coli. PriB has an OB-fold dimer structure and exhibits single-stranded DNA (ssDNA)-binding activities similar to single-stranded binding protein (SSB). In this study, we examined PriB's interaction with ssDNA (oligo-dT35, -dT15, and -dT7) using heteronuclear NMR analysis. Interestingly, (1)H or (15)N chemical shift changes of the PriB main-chain showed two distinct modes using oligo-dT35. The chemical shift perturbation sites in the primary mode were consistent with the main contact site in PriB-ssDNA, which was previously determined by crystal structure analysis. The results also suggested that approximately 8nt in ssDNA was the main contact site to PriB. In the secondary mode, residues in the α-helix region (His57-Ser65) and in β4-loop3-β5 were mainly perturbed. On the other hand, we examined the state of ssDNA by FRET using 5'-Cy3- and 3'-Cy5-modified oligo-dT35. As the PriB concentration increased, two-step saturation curves were observed in the FRET assay, suggesting a compact structure of ssDNA. Moreover, we confirmed two-step PriB binding to oligo-dT35 using EMSA. The pH dependence of FRET suggested contribution of the His residues. Therefore, we prepared His mutants of PriB and found that His64 in the α-helix region contributed to the second interaction between PriB and ssDNA using FRET and EMSA. Thus, from a structural standpoint, we suggested the role of His64 on the compactness of the PriB-ssDNA complex and on the positive cooperativity of PriB.
authors.affiliation: Laboratory of Protein Structure, Function and Design, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Saki
authors.initials: S
authors.lastname: Fujiyama
authors.affiliation: Laboratory of Protein Structure, Function and Design, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Yoshito
authors.initials: Y
authors.lastname: Abe
authors.affiliation: Laboratory of Protein Structure, Function and Design, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Taichi
authors.initials: T
authors.lastname: Takenawa
authors.affiliation: Laboratory of Protein Structure, Function and Design, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Takahiko
authors.initials: T
authors.lastname: Aramaki
authors.affiliation: Radioisotope Center, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan.
authors.forename: Seijiro
authors.initials: S
authors.lastname: Shioi
authors.affiliation: Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Katayama
authors.affiliation: Laboratory of Protein Structure, Function and Design, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Electronic address: ueda@phar.kyushu-u.ac.jp.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Ueda
mesh_terms: D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs / Q000502:physiology*; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D009841:Oligonucleotides; C086004:PriB protein, E coli; D006639:Histidine
keywords: Cooperativity; EMSA; Electrophoresis Mobility Shift Assay; FRET; Fluorescence Resonance Energy Transfer; NMR; PriB; Protein–DNA interaction; SSB; Single-Stranded DNA Binding protein; Single-stranded DNA binding protein; single-stranded DNA; ssDNA