Guffy Sharon L, Der Bryan S, Kuhlman Brian
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599-7260, USA.
Protein Eng Des Sel. 2016 Aug;29(8):327-338. doi: 10.1093/protein/gzw026. Epub 2016 Jun 29.
Structure-based protein design tests our understanding of the minimal determinants of protein structure and function. Previous studies have demonstrated that placing zinc binding amino acids (His, Glu, Asp or Cys) near each other in a folded protein in an arrangement predicted to be tetrahedral is often sufficient to achieve binding to zinc. However, few designs have been characterized with high-resolution structures. Here, we use X-ray crystallography, binding studies and mutation analysis to evaluate three alternative strategies for designing zinc binding sites with the molecular modeling program Rosetta. While several of the designs were observed to bind zinc, crystal structures of two designs reveal binding configurations that differ from the design model. In both cases, the modeling did not accurately capture the presence or absence of second-shell hydrogen bonds critical in determining binding-site structure. Efforts to more explicitly design second-shell hydrogen bonds were largely unsuccessful as evidenced by mutation analysis and low expression of proteins engineered with extensive primary and secondary networks. Our results suggest that improved methods for designing interaction networks will be needed for creating metal binding sites with high accuracy.
基于结构的蛋白质设计检验了我们对蛋白质结构和功能的最小决定因素的理解。先前的研究表明，在折叠蛋白中以预测为四面体的排列方式将锌结合氨基酸（组氨酸、谷氨酸、天冬氨酸或半胱氨酸）彼此靠近放置，通常足以实现与锌的结合。然而，很少有设计通过高分辨率结构进行表征。在这里，我们使用X射线晶体学、结合研究和突变分析，通过分子建模程序Rosetta评估三种设计锌结合位点的替代策略。虽然观察到几种设计能结合锌，但两种设计的晶体结构显示出与设计模型不同的结合构型。在这两种情况下，建模都没有准确捕捉到对确定结合位点结构至关重要的第二壳层氢键的存在或不存在。通过突变分析以及构建有广泛一级和二级网络的蛋白质的低表达证明，更明确地设计第二壳层氢键的努力在很大程度上是不成功的。我们的结果表明，为了高精度地创建金属结合位点，将需要改进设计相互作用网络的方法。
