Huang Jin, Koide Akiko, Makabe Koki, Koide Shohei
Department of Biochemistry and Molecular Biology, University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA.
Proc Natl Acad Sci U S A. 2008 May 6;105(18):6578-83. doi: 10.1073/pnas.0801097105. Epub 2008 Apr 29.
Most natural proteins performing sophisticated tasks contain multiple domains where an active site is located at the domain interface. Comparative structural analyses suggest that major leaps in protein function occur through gene recombination events that connect two or more protein domains to generate a new active site, frequently occurring at the newly created domain interface. However, such functional leaps by combination of unrelated domains have not been directly demonstrated. Here we show that highly specific and complex protein functions can be generated by joining a low-affinity peptide-binding domain with a functionally inert second domain and subsequently optimizing the domain interface. These directed evolution processes dramatically enhanced both affinity and specificity to a level unattainable with a single domain, corresponding to >500-fold and >2,000-fold increases of affinity and specificity, respectively. An x-ray crystal structure revealed that the resulting "affinity clamp" had clamshell architecture as designed, with large additional binding surface contributed by the second domain. The affinity clamps having a single-nanomolar dissociation constant outperformed a monoclonal antibody in immunochemical applications. This work establishes evolutionary paths from isolated domains with primitive function to multidomain proteins with sophisticated function and introduces a new protein-engineering concept that allows for the generation of highly functional affinity reagents to a predefined target. The prevalence and variety of natural interaction domains suggest that numerous new functions can be designed by using directed domain interface evolution.
大多数执行复杂任务的天然蛋白质都包含多个结构域，活性位点位于结构域界面处。比较结构分析表明，蛋白质功能的重大飞跃是通过基因重组事件实现的，这些事件将两个或更多蛋白质结构域连接起来，形成一个新的活性位点，该活性位点通常出现在新形成的结构域界面处。然而，这种由不相关结构域组合产生的功能飞跃尚未得到直接证明。在此，我们表明，通过将低亲和力肽结合结构域与功能惰性的第二个结构域连接起来，随后优化结构域界面，可以产生高度特异性和复杂的蛋白质功能。这些定向进化过程显著提高了亲和力和特异性，达到了单个结构域无法企及的水平，亲和力和特异性分别提高了500倍以上和2000倍以上。X射线晶体结构显示，由此产生的“亲和力钳”具有设计的蛤壳结构，第二个结构域贡献了大量额外的结合表面。在免疫化学应用中，解离常数为单纳摩尔的亲和力钳优于单克隆抗体。这项工作建立了从具有原始功能的孤立结构域到具有复杂功能的多结构域蛋白质的进化路径，并引入了一种新的蛋白质工程概念，该概念允许生成针对预定义靶标的高功能亲和力试剂。天然相互作用结构域的普遍性和多样性表明，通过定向结构域界面进化可以设计出许多新功能。
