H-1, N-15 RESONANCE ASSIGNMENT AND 3-DIMENSIONAL STRUCTURE OF CYP1 (HAP1) DNA-BINDING DOMAIN

CYP1(HAP1) is a transcriptional activator involved in the aerobic meta bolism of the yeast Saccharomyces cerevisiae. The amino acid sequence of its DNA-binding domain suggests that it belongs to the ''zinc clust er'' class. This region is indeed characterized by a pattern known to form a bimetal thiolate cluster where two zinc ions are coordinated by six cysteine residues;: Structures of two such domains, those from GA L4 and PPR1, have been solved as complexes with DNA. These domains con sist of the zinc cluster connected to a dimerization helix by a linker peptide. They recognize, as a dimer, an inverted repeat of a CGG moti f that is separated by a specific number of bases. Interestingly, the specificity of the interaction seems not to be due to the interaction between the cluster region and the DNA but rather to a fine tune betwe en the structure of the linker peptide and the number of base-pairs se parating the two CGGs. However, the CYP1 target sites fail to display such a consensus sequence. One of the two CGG sites is poorly conserve d and some experiments suggest a direct rather than an inverted repeat . Using H-1, N-15 and Cd-113 NMR spectroscopy, we have undertaken the analysis of the structural properties of the CYP1(56-126) fragment tha t consists of the zinc-cluster region, the linker peptide and a part o f the dimerization helix. We have demonstrated that the six cysteine r esidues of the peptide chelate two cadmium ions as in GAL4 and PPR1. F ifteen structures of the zinc-cluster region (residues 60 to 100) were calculated, the Linker peptide and the dimerization helix being unstr uctured under the conditions of our study. This region possesses the s ame overall fold as in GAL4 and PPR1, and most of the side-chains invo lved in the interaction with DNA are structurally conserved. This sugg ests that the CYP1 zinc-cluster region recognizes a CGG tripler in the same way as GAL4 and PPR1. In this case, the particular properties of CYP1 seem to be due to the structure of the linker peptide and/or of the dimerization helix. (C) 1996 Academic Press Limited