Jha Ramesh K, Chakraborti Subhendu, Kern Theresa L, Fox David T, Strauss Charlie E M
Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico, 87545.
Proteins. 2015 Jul;83(7):1327-40. doi: 10.1002/prot.24828. Epub 2015 May 29.
Structure-based rational mutagenesis for engineering protein functionality has been limited by the scarcity and difficulty of obtaining crystal structures of desired proteins. On the other hand, when high-throughput selection is possible, directed evolution-based approaches for gaining protein functionalities have been random and fortuitous with limited rationalization. We combine comparative modeling of dimer structures, ab initio loop reconstruction, and ligand docking to select positions for mutagenesis to create a library focused on the ligand-contacting residues. The rationally reduced library requirement enabled conservative control of the substitutions by oligonucleotide synthesis and bounding its size within practical transformation efficiencies (∼ 10(7) variants). This rational approach was successfully applied on an inducer-binding domain of an Acinetobacter transcription factor (TF), pobR, which shows high specificity for natural effector molecule, 4-hydroxy benzoate (4HB), but no native response to 3,4-dihydroxy benzoate (34DHB). Selection for mutants with high transcriptional induction by 34DHB was carried out at the single-cell level under flow cytometry (via green fluorescent protein expression under the control of pobR promoter). Critically, this selection protocol allows both selection for induction and rejection of constitutively active mutants. In addition to gain-of-function for 34DHB induction, the selected mutants also showed enhanced sensitivity and response for 4HB (native inducer) while no sensitivity was observed for a non-targeted but chemically similar molecule, 2-hydroxy benzoate (2HB). This is unique application of the Rosetta modeling protocols for library design to engineer a TF. Our approach extends applicability of the Rosetta redesign protocol into regimes without a priori precision structural information.
基于结构的合理诱变来改造蛋白质功能，一直受到所需蛋白质晶体结构获取困难且稀缺的限制。另一方面，当高通量筛选可行时，基于定向进化获得蛋白质功能的方法具有随机性和偶然性，且合理性有限。我们结合二聚体结构的比较建模、从头环重建和配体对接，来选择诱变位置以创建一个聚焦于配体接触残基的文库。合理降低的文库需求使得能够通过寡核苷酸合成保守地控制取代，并将其大小限制在实际转化效率范围内（约10^7个变体）。这种合理方法成功应用于不动杆菌转录因子（TF）pobR的诱导物结合结构域，该结构域对天然效应分子4 - 羟基苯甲酸（4HB）具有高特异性，但对3,4 - 二羟基苯甲酸（34DHB）无天然反应。在流式细胞术下单细胞水平上进行对34DHB具有高转录诱导作用的突变体筛选（通过pobR启动子控制下的绿色荧光蛋白表达）。至关重要的是，这种筛选方案既能筛选诱导作用，又能排除组成型活性突变体。除了获得对34DHB诱导的功能外，所选突变体对4HB（天然诱导物）也表现出增强的敏感性和反应，而对非靶向但化学结构相似的分子2 - 羟基苯甲酸（2HB）未观察到敏感性。这是Rosetta建模协议在文库设计中用于改造转录因子的独特应用。我们的方法将Rosetta重新设计协议的适用性扩展到没有先验精确结构信息的情况。
