Proteolytic dissection of Zab, the Z-DNA-binding domain of human ADAR1

Z alpha is a peptide motif that binds to Z-DNA with high affinity. This mot if binds to alternating dC-dG sequences stabilized in the Z-conformation by means of bromination or supercoiling, but not to B-DNA. Z alpha is part of the N-terminal region of double-stranded RNA adenosine deaminase (ADAR1) , a candidate enzyme for nuclear pre-mRNA editing in mammals. Z alpha is con served in ADAR1 from many species; in each case, there is a second similar motif, Z beta, separated from Z alpha by a more divergent linker. To invest igate the structure-function relationship of Z alpha, its domain structure was studied by limited proteolysis. Proteolytic profiles indicated that Z a lpha is part of a domain, Zab, of 229 amino acids (residues 133-361 in huma n ADAR1), This domain contains both Z alpha and Z beta as well as a tandem repeat of a 49-amino acid linker module. Prolonged proteolysis revealed a m inimal core domain of 77 amino acids (positions 133-209), containing only Z alpha, which is sufficient to bind left-handed Z-DNA; however, the substra te binding is strikingly different from that of Zab. The second motif, Z be ta, retains its structural integrity only in the context of Zab and does no t bind Z-DNA as a separate entity. These results suggest that Z alpha and Z beta act as a single bipartite domain. In the presence of substrate DNA, Z ab becomes more resistant to proteases, suggesting that it adopts a more ri gid structure when bound to its substrate, possibly with conformational cha nges in parts of the protein.