De Novo Design of Tetranuclear Transition Metal Clusters Stabilized by Hydrogen-Bonded Networks in Helical Bundles.
De Novo Design of Tetranuclear Transition Metal Clusters Stabilized by Hydrogen-Bonded Networks in Helical Bundles.
复制标题
螺旋束中氢键网络稳定的四核过渡金属簇的从头设计。
DOI：
10.1021/jacs.7b08261
复制
发表时间：
2018
影响因子：
15
通讯作者：
Lombardi,Angela
中科院分区：
文献类型：
--
作者：
Zhang,Shao-Qing;Chino,Marco;Liu,Lijun;Tang,Youzhi;Hu,Xiaozhen;DeGrado,WilliamF;Lombardi,Angela
De novodesign提供了一种有吸引力的方法来测试金属蛋白质定义其金属离子辅因子的几何形状和反应性的机制。虽然在设计结合包括铁硫簇的过渡金属离子的蛋白质方面已经取得了相当大的进展，但是具有富氧环境的四核簇的设计尚未完成。在这里，我们描述了四核簇的设计，由四个Zn 2+和四个羧酸氧位于扭曲的立方体状结构的顶点。四-Zn 2+簇结合在四螺旋束内的掩埋位点处，每个螺旋提供单个羧酸盐（Glu或Asp）和咪唑（His）配体，以及第二和第三壳配体。总体而言，所设计的网站由四个锌+和16个极性侧链在一个完全连接的氢键网络。所设计的蛋白质在束的顶部和底部具有非极性核心，其驱动在束的中心附近的配体残基的组装。改变结合位点周围的非极性残基的空间体积，以确定螺旋-螺旋包装的细微变化如何影响结合位点。合成的四种蛋白质中的两种的晶体结构与整体设计非常一致;两者都形成了一个扭曲的立方体位点，该位点通过侧翼的第二和第三壳层相互作用来稳定主要配体。第三个结构绑定一个单一的Zn 2+在一个意想不到的几何形状，和第四个绑定多个Zn 2+在多个网站在部分占用。螺旋束在溶液中的金属结合和构象性质，探测圆二色性光谱，分析ultracenthesis，和NMR，与晶体结构是一致的。
De novodesign provides an attractive approach to test the mechanism by which metalloproteins define the geometry and reactivity of their metal ion cofactors. While there has been considerable progress in designing proteins that bind transition metal ions including iron–sulfur clusters, the design of tetranuclear clusters with oxygen-rich environments has not been accomplished. Here, we describe the design of tetranuclear clusters, consisting of four Zn2+and four carboxylate oxygens situated at the vertices of a distorted cube-like structure. The tetra-Zn2+clusters are bound at a buried site within a four-helix bundle, with each helix donating a single carboxylate (Glu or Asp) and imidazole (His) ligand, as well as second- and third-shell ligands. Overall, the designed site consists of four Zn2+and 16 polar side chains in a fully connected hydrogen-bonded network. The designed proteins have apolar cores at the top and bottom of the bundle, which drive the assembly of the liganding residues near the center of the bundle. The steric bulk of the apolar residues surrounding the binding site was varied to determine how subtle changes in helix–helix packing affect the binding site. The crystal structures of two of four proteins synthesized were in good agreement with the overall design; both formed a distorted cuboidal site stabilized by flanking second- and third-shell interactions that stabilize the primary ligands. A third structure bound a single Zn2+in an unanticipated geometry, and the fourth bound multiple Zn2+at multiple sites at partial occupancy. The metal-binding and conformational properties of the helical bundles in solution, probed by circular dichroism spectroscopy, analytical ultracentrifugation, and NMR, were consistent with the crystal structures.