Solution structure determination of the two DNA-binding domains in the Shizosaccharomyces pombe Abp1 protein by a combination of dipolar coupling and diffusion anisotropy restraints

Jun Kikuchi, Junji Iwahara, Takanori Kigawa, Yota Murakami, Tsuneko Okazaki, Shigeyuki Yokoyama

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

We have solved the solution structure of the N-terminal region of the fission yeast centromere protein, Abp1, bound to a 21-base pair DNA fragment bearing its recognition site (Mw = 30 kDa). Although the two DNA-binding domains in the Abp1 protein were defined well by a conventional NOE-based NMR methodology, the overall structure of the Abp1 protein was poorly defined, due to the lack of interdomain distance restraints. Therefore, we additionally used residual dipolar couplings measured in a weakly aligned state, and rotational diffusion anisotropies. Neither the NH residual dipolar couplings nor the backbone 15N T1/T2 data were sufficient to determine the overall structure of the Abp1 protein, due to spectral overlap. We used a combination of these two orientational restraints (residual dipolar coupling and rotational diffusion anisotropy), which significantly improved the convergence of the overall structures. The range of the observed T1/T2 ratios was wider (20-50 for the secondary structure regions of Abp1) than the previously reported data for several globular proteins, indicating that the overall shape of the Abp1•DNA complex is ellipsoid. This extended form would facilitate the recognition of the two separate sites in the relatively long DNA sequence by the DNA-binding domains of Apb1.

Original languageEnglish (US)
Pages (from-to)333-347
Number of pages15
JournalJournal of Biomolecular NMR
Volume22
Issue number4
DOIs
StatePublished - May 8 2002
Externally publishedYes

Keywords

  • Alignment tensor
  • Domain orientation
  • Residual dipolar coupling
  • Rotational diffusion anisotrophy

ASJC Scopus subject areas

  • Biochemistry
  • Spectroscopy

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