Design of protein function leaps by directed domain interface evolution
Design of protein function leaps by directed domain interface evolution
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DOI：
10.1073/pnas.0801097105
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发表时间：
2008-05-06
影响因子：
11.1
通讯作者：
Koide, Shohei
中科院分区：
文献类型：
--
作者：
Huang, Jin;Koide, Akiko;Koide, Shohei
大多数执行复杂任务的天然蛋白质包含多个结构域，其中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发生的，基因重组事件连接两个或多个蛋白质结构域，产生一个新的活性位点，经常发生在新创建的结构域界面。然而，这种由不相关的结构域组合而产生的功能飞跃还没有被直接证明。在这里，我们表明，高度特异性和复杂的蛋白质功能，可以通过加入一个低亲和力的肽结合结构域与功能惰性的第二个域，随后优化的域接口。这些定向进化过程将亲和力和特异性显著增强到单个结构域无法达到的水平，分别对应于亲和力和特异性增加>500倍和> 2，000倍。X射线晶体结构显示，所得的“亲和钳”具有设计的蛤壳结构，具有由第二结构域贡献的大的额外结合表面。具有单个纳摩尔解离常数的亲和钳在免疫化学应用中优于单克隆抗体。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目标的高功能亲和试剂。自然相互作用域的普遍性和多样性表明，许多新的功能可以通过使用定向域界面进化来设计。
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.