Jucovic M, Hartley R W
Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2343-7. doi: 10.1073/pnas.93.6.2343.
Barnase and barstar are trivial names of the extracellular RNase and its intracellular inhibitor produced by Bacillus amyloliquefaciens. Inhibition involves the formation of a very tight one-to-one complex of the two proteins. With the crystallographic solution of the structure of the barnase-barstar complex and the development of methods for measuring the free energy of binding, the pair can be used to study protein-protein recognition in detail. In this report, we describe the isolation of suppressor mutations in barstar that compensate for the loss in interaction energy caused by a mutation in barnase. Our suppressor search is based on in vivo selection for barstar variants that are able to protect host cells against the RNAse activity of those barnase mutants not properly inhibited by wild-type barstar. This approach utilizes a plasmid system in which barnase expression is tightly controlled to keep the mutant barnase gene silent. When expression of barnase is turned on, failure to form a complex between the mutant barnase and barstar has a lethal effect on host cells unless overcome by substitution of the wild-type barstar by a functional suppressor derivative. A set of barstar suppressors has been identified for barnase mutants with substitutions in two amino acid positions (residues 102 and 59), which are critically involved in both RNase activity and barstar binding. The mutations selected as suppressors could not have been predicted on the basis of the known protein structures. The single barstar mutation with the highest information content for inhibition of barnase (H102K) has the substitution Y30W. The reduction in binding caused by the R59E mutation in barnase can be partly reversed by changing Glu-76 of barstar, which forms a salt bridge with the Arg-59 in the wild-type complex, to arginine, thus completing an interchange of the two charges.
芽孢杆菌核酸酶（Barnase）和芽孢杆菌抗酶（Barstar）分别是解淀粉芽孢杆菌产生的细胞外核糖核酸酶及其细胞内抑制剂的通俗名称。抑制作用涉及这两种蛋白质形成非常紧密的一对一复合物。随着Barnase-Barstar复合物结构的晶体学解析以及测量结合自由能方法的发展，这一对蛋白可用于详细研究蛋白质-蛋白质识别。在本报告中，我们描述了Barstar中抑制突变的分离，这些突变可补偿Barnase突变导致的相互作用能损失。我们的抑制子搜索基于体内筛选能够保护宿主细胞免受那些未被野生型Barstar有效抑制的Barnase突变体核糖核酸酶活性影响的Barstar变体。该方法利用了一种质粒系统，其中Barnase的表达受到严格控制以使突变的Barnase基因保持沉默。当Barnase的表达开启时，除非被功能性抑制衍生物取代野生型Barstar，否则突变的Barnase与Barstar之间未能形成复合物会对宿主细胞产生致死效应。已鉴定出一组针对Barnase突变体的Barstar抑制子，这些突变体在两个氨基酸位置（第102位和第59位残基）发生了取代，这两个位置在核糖核酸酶活性和Barstar结合中都起着关键作用。基于已知的蛋白质结构无法预测所选作抑制子的突变。抑制Barnase活性信息含量最高的单个Barstar突变（H102K）发生了Y30W取代。Barnase中R59E突变导致的结合减少可通过将Barstar中与野生型复合物中的Arg-59形成盐桥的Glu-76替换为精氨酸而部分逆转，从而实现两个电荷的互换。
