PROTEIN STRUCTURE DETERMINATION OF HUMAN Z

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The RNA editing enzyme ADAR1 contains two homologous left-handed DNA (Z-DNA) binding domains Z and Zp of which Za binds specifically and with high affinity (KD = 4 nM) to poly(br-dCdG) Z-DNA. To gain a molecular understanding of how the regulation of ADARI-mediated RNA editing relates to the recognition of Z-DNA, we are determining the high resolution structure of the Z-DNA binding protein domain Za (9.4 kD). Spectra were collected at 500, 600 and 750 MHz. Backbone assignments were obtained from 3D 15 N-edited spectra. TOCSY-HSQC spectra (35, 70 and 100 ms mixing times) provided the major part of spin system information, such as nearly all H, and many other aliphatic protons. The H protons were assigned via a 3D '5 N-HNHA spectrum. Individual spin-systems were sequentially assigned by 3D 15 N NOESY-HSQC spectra (70, 150 and 250 ms). Analysis of the NOE data has revealed that Z possesses three P-strands, called 0-strand I, II and III. Two gaps in the sequential NOE pattern arise at the positions of prolines residues, which cannot be detected by 15 N-edited experiments. A medium NH(i)-NH(i+2)-NOE in combination with the lack of a NH(i)-NH(i+l)-NOE at glycine 183 between helix III and P-strand II suggests a turn of the protein backbone. Analysis of long range NOEs in the protein backbone suggests an antiparallel P-sheet of five residues. The antiparallel P-sheet starts two residues C-terminal to helix III and is terminated by the Cterminus. Moreover, a NH(i)-NHO)- and a H,,(i-1)-NHO)-NOE were detected between P-strand I and Pstrand III, connecting the C-terminal antiparallel P-sheet with P-sheet I. Furthermore, well separated long range NOEs between the aromatic ring protons of trytophan 195 in P-sheet III to the protons of leucine 176 in the center of helix III suggests that the antiparallel P-sheet folds back onto helix III.