Solution structure determination of the two DNA-binding domains in the Schizosaccharomyces pombe Abp1 protein by a combination of dipolar coupling and diffusion anisotropy restraints.
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 Abpl protein were defined well by a conventional NOE-based NMR methodology, the overall structure of the Abpl 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 Abpl 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 Abp 1) 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.
Kikuchi
Jun
J
RIKEN Genomic Sciences Center, Tsurumi, Yokohama, Japan.
Iwahara
Junji
J
Kigawa
Takanori
T
Murakami
Yota
Y
Okazaki
Tsuneko
T
Yokoyama
Shigeyuki
S
D016880:Anisotropy; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009904:Optical Rotation; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000737:chemistry*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry*; D012996:Solutions / Q000737:chemistry; D014157:Transcription Factors
C061886:ABF1 protein, S cerevisiae; D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D012996:Solutions; D014157:Transcription Factors; C097691:cbp1 protein, S pombe; D004247:DNA