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 motif 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 conserved 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 investigate the structure-function relationship of Z alpha, its domain structure was studied by limited proteolysis. Proteolytic profiles indicated that Z alpha is part of a domain, Zab, of 229 amino acids (residues 133-361 in human 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 minimal core domain of 77 amino acids (positions 133-209), containing only Z alpha, which is sufficient to bind left-handed Z-DNA; however, the substrate binding is strikingly different from that of Zab. The second motif, Z beta, retains its structural integrity only in the context of Zab and does not 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, Zab becomes more resistant to proteases, suggesting that it adopts a more rigid structure when bound to its substrate, possibly with conformational changes in parts of the protein.