Hoffman R C, Horvath S J, Klevit R E
Department of Biochemistry, University of Washington, Seattle 98195.
Protein Sci. 1993 Jun;2(6):951-65. doi: 10.1002/pro.5560020609.
Studies of Cys2-His2 zinc finger domains have revealed that the structures of individual finger domains in solution determined by NMR spectroscopy are strikingly similar to the structure of fingers bound to DNA determined by X-ray diffraction. Therefore, detailed structural analyses of single finger domains that contain amino acid substitutions known to affect DNA binding in the whole protein can yield information concerning 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 finger sequences spanning residues 102-159. Two point mutants at position 118 in the N-terminal zinc finger (ADR1b: 102-130) that adversely affect 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 magnetic resonance spectroscopy and distance geometry and were refined using a complete relaxation matrix method approach (REPENT) to improve agreement between the models and the nuclear Overhauser effect spectroscopy data from which they were generated. The molecular architecture of the refined wild-type ADR1b domain is presented in detail. Comparisons of wild-type ADR1b and the two mutants revealed that neither mutation causes a significant structural perturbation. The structures indicate that the DNA binding properties of the His 118 mutants are dependent on the identity of the side chain at position 118, which has been postulated to make a direct DNA contact in the wild-type ADR1 protein. The results suggest that the identity of the side chain at the middle DNA contact position in Cys2-His2 zinc fingers may be changed with impunity regarding the domain structure and can affect the affinity of the protein-DNA interaction.
对Cys2-His2锌指结构域的研究表明，通过核磁共振光谱法测定的溶液中单个指状结构域的结构与通过X射线衍射测定的与DNA结合的指状结构惊人地相似。因此，对包含已知会影响整个蛋白质中DNA结合的氨基酸取代的单个指状结构域进行详细的结构分析，可以得出有关此类突变的结构后果的信息。我们已采用这种方法来研究酵母转录因子ADR1的N端指状结构域中的两个突变体，ADR1包含两个跨越残基102 - 159的Cys2-His2锌指序列。先前已鉴定出N端锌指（ADR1b：102 - 130）中第118位的两个点突变体，它们对ADR1的DNA结合活性有不利影响：H118A和H118Y。使用二维核磁共振光谱法和距离几何学确定了野生型ADR1b和两个突变锌指结构域的结构，并使用完全弛豫矩阵方法（REPENT）进行了优化，以提高模型与从中生成的核Overhauser效应光谱数据之间的一致性。详细介绍了优化后的野生型ADR1b结构域的分子结构。野生型ADR1b与两个突变体的比较表明，这两个突变均未引起明显的结构扰动。这些结构表明，His 118突变体的DNA结合特性取决于第118位侧链的性质，据推测该侧链在野生型ADR1蛋白中与DNA直接接触。结果表明，Cys2-His2锌指中与DNA中间接触位置的侧链性质可能在不影响结构域结构的情况下发生改变，并会影响蛋白质与DNA相互作用的亲和力。
