Klemba M, Regan L
Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
Biochemistry. 1995 Aug 8;34(31):10094-100. doi: 10.1021/bi00031a034.
The tetrahedral Cys2His2 Zn(II)-binding site in the de novo designed protein Z alpha 4 [Regan, L., & Clarke, N. D. (1990) Biochemistry 29, 10878] has been studied by independently mutating each of the metal-binding ligands to alanine. The contribution of each ligand to the geometry and affinity of metal binding has been characterized using Co(II), Zn(II), and Cd(II). The results indicate that all four ligands contribute to high-affinity metal binding in Z alpha 4. Two of the four metal-site mutants retain the tetrahedral Zn(II)-binding geometry of Z alpha 4, with one water molecule presumed to bind in the vacant ligand position. These mutants provide the first examples of a demonstrated de novo tetrahedral three-coordinate site designed into a protein and as such are a first step toward the design of catalytic rather than structural Zn(II) sites. One of the metal-site mutants binds Zn(II) with either tetrahedral four-coordinate or five-coordinate geometry, while the last ligand-to-alanine substitution abolishes tetrahedral binding. The importance of ligand type for metal-binding in Z alpha 4 was investigated by characterizing two ligand-swap mutants in which a cysteine residue was replaced with a histidine. In both cases, tetrahedral metal binding was lost. Collectively, these results affirm the strategy used to design Z alpha 4 by showing that all designed liganding residues are participating in metal binding, and by suggesting that the tetrahedral geometry of the binding site is perturbed when the designed side chain ligands are replaced with alternate ligands.
通过将从头设计的蛋白质Zα4[里根，L.，&克拉克，N.D.（1990年）《生物化学》29，10878]中的每个金属结合配体独立突变为丙氨酸，对其四面体Cys2His2 Zn（II）结合位点进行了研究。使用Co（II）、Zn（II）和Cd（II）对每个配体对金属结合的几何形状和亲和力的贡献进行了表征。结果表明，所有四个配体都对Zα4中的高亲和力金属结合有贡献。四个金属位点突变体中的两个保留了Zα4的四面体Zn（II）结合几何形状，推测有一个水分子结合在空的配体位置。这些突变体提供了首个被证明设计到蛋白质中的从头四面体三配位位点的实例，因此是朝着设计催化而非结构Zn（II）位点迈出的第一步。其中一个金属位点突变体以四面体四配位或五配位几何形状结合Zn（II），而最后一个配体到丙氨酸的取代消除了四面体结合。通过表征两个半胱氨酸残基被组氨酸取代的配体交换突变体，研究了配体类型对Zα4中金属结合的重要性。在这两种情况下，四面体金属结合都丧失了。总体而言，这些结果通过表明所有设计的配体残基都参与金属结合，并通过表明当设计的侧链配体被替代配体取代时结合位点的四面体几何形状受到干扰，肯定了用于设计Zα4的策略。
