Department of Chemistry, University of Pennsylvania , 209 South 33rd Street, Philadelphia, Pennsylvania 19104-6396, United States.
Department of Pharmaceutical Chemistry and the Cardiovascular Research Institute, University of California at San Francisco , San Francisco, California 94158-9001, United States.
J Am Chem Soc. 2018 Jan 31;140(4):1294-1304. doi: 10.1021/jacs.7b08261. Epub 2018 Jan 22.
De novo design 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 Zn and four carboxylate oxygens situated at the vertices of a distorted cube-like structure. The tetra-Zn 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 Zn 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 Zn in an unanticipated geometry, and the fourth bound multiple Zn 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.
从头设计为测试金属蛋白如何确定其金属离子辅因子的几何形状和反应性提供了一种有吸引力的方法。虽然在设计结合过渡金属离子（包括铁硫簇）的蛋白质方面已经取得了相当大的进展，但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，该簇由四个 Zn 和四个位于变形立方体形结构顶点的羧酸盐氧组成。四 Zn 簇位于四螺旋束内的埋藏部位结合，每个螺旋提供单个羧酸盐（Glu 或 Asp）和咪唑（His）配体，以及第二和第三壳层配体。总体而言，设计的位点由四个 Zn 和 16 个极性侧链组成，形成一个完全连接的氢键网络。设计的蛋白质在束的顶部和底部具有非极性核心，这些核心驱动配体残基在束的中心附近组装。围绕结合位点的非极性残基的空间位阻发生变化，以确定螺旋-螺旋包装的微小变化如何影响结合位点。合成的四个蛋白质中的两个的晶体结构与整体设计非常吻合；两者都形成了一个由侧翼第二和第三壳层相互作用稳定的变形立方体形位点，这些相互作用稳定了主要配体。第三个结构以出乎意料的几何形状结合了单个 Zn，第四个结构以部分占据的多个 Zn 结合到多个位点。通过圆二色性光谱、分析超速离心和 NMR 探测的溶液中螺旋束的金属结合和构象特性与晶体结构一致。
