STRUCTURES OF DNA-BINDING MUTANT ZINC-FINGER DOMAINS - IMPLICATIONS FOR DNA-BINDING

Studies of Cys2-His2 zinc finger domains have revealed that the struct ures of individual finger domains in solution determined by NMR spectr oscopy are strikingly similar to the structure of fingers bound to DNA determined by X-ray diffraction. Therefore, detailed structural analy ses of single finger domains that contain amino acid substitutions kno wn to affect DNA binding in the whole protein can yield information co ncerning the structural ramifications of such mutations. We have used this approach to study two mutants in the N-terminal finger domain of ADR1, a yeast transcription factor that contains two Cys2-His2 zinc fi nger sequences spanning residues 102-159. Two point mutants at positio n 118 in the N-terminal zinc finger (ADR1b: 102-130) that adversely af fect the DNA-binding activity of ADR1 have previously been identified: H118A and H118Y. The structures of wild-type ADR1b and the two mutant zinc finger domains were determined using two-dimensional nuclear mag netic resonance spectroscopy and distance geometry and were refined us ing a complete relaxation matrix method approach (REPENT) to improve a greement between the models and the nuclear Overhauser effect spectros copy data from which they were generated, The molecular architecture o f the refined wild-type ADR1b domain is presented in detail. Compariso ns of wild-type ADR1b and the two mutants revealed that neither mutati on causes a significant structural perturbation. The structures indica te that the DNA binding properties of the His 118 mutants are dependen t on the identity of the side chain at position 118, which has been po stulated to make a direct DNA contact in the wild-type ADR1 protein. T he results suggest that the identity of the side chain at the middle D NA contact position in CYS2-HiS2 Zinc fingers may be changed with impu nity regarding the domain structure and can affect the affinity of the protein-DNA interaction.