Involvement of histidine in complex formation of PriB and single-stranded DNA

• Published in: Biochimica et biophysica acta 2014-02, Vol.1844 (2), p.299-307
• Main Authors: Fujiyama, Saki, Abe, Yoshito, Takenawa, Taichi, Aramaki, Takahiko, Shioi, Seijiro, Katayama, Tsutomu, Ueda, Tadashi
• Format: Article
• Language: English
• Subjects: Binding Sites; Cooperativity; DNA, Single-Stranded - chemistry; DNA, Single-Stranded - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Escherichia coli; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Fluorescence Resonance Energy Transfer; Histidine - genetics; Histidine - metabolism; Magnetic Resonance Spectroscopy; Models, Molecular; Mutant Proteins - chemistry; Mutant Proteins - metabolism; NMR; Oligonucleotides - metabolism; PriB; Protein Folding; Protein Interaction Domains and Motifs - physiology; Protein Structure, Quaternary; Protein Structure, Secondary; Protein–DNA interaction; Single-stranded DNA binding protein
• ISSN: 1570-9639; 0006-3002; 1878-1454
• DOI: 10.1016/j.bbapap.2013.10.015

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, 1H or 15N 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. [Display omitted] •The interaction between PriB and ssDNA showed two distinct modes.•The primary binding site was similar to the site determined by crystal structure.•PriB induced a compact state of ssDNA in the secondary binding mode.•His 64 residue of PriB contributed to the secondary binding mode.•The compact PriB-ssDNA complex was possibly involved in the cooperativity of PriB.