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

Saki Fujiyama, Yoshito Abe, Taichi Takenawa, Takahiko Aramaki, Seijiro Shioi, Tsutomu Katayama, Tadashi Ueda

研究成果: ジャーナルへの寄稿学術誌査読

7 被引用数 (Scopus)


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 8 nt 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.

ジャーナルBiochimica et Biophysica Acta - Proteins and Proteomics
出版ステータス出版済み - 2月 2014

!!!All Science Journal Classification (ASJC) codes

  • 分析化学
  • 生物理学
  • 生化学
  • 分子生物学


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