Structural basis for exquisite specificity of affinity clamps, synthetic binding proteins generated through directed domain-interface evolution.
Structural basis for exquisite specificity of affinity clamps, synthetic binding proteins generated through directed domain-interface evolution.
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DOI：
10.1016/j.jmb.2009.07.067
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发表时间：
2009-10-09
影响因子：
5.6
通讯作者：
Koide S
中科院分区：
文献类型：
--
作者：
Huang J;Makabe K;Biancalana M;Koide A;Koide S
我们最近建立了一种新的蛋白质工程策略，称为“定向结构域界面进化”，通过连接两个蛋白质结构域，然后优化它们之间的界面，产生一个结合位点。采用这种策略，我们已经产生了合成的两个域的“亲和钳”使用PDZ和纤连蛋白III型（FN 3）域作为积木。虽然这些亲和钳对靶肽的亲和力都比潜在的PDZ结构域显著更高，但在靶特异性方面发现了两种不同类型的亲和钳。一种类型保守的父母PDZ结构域的特异性，和其他显着增加的特异性。在这里，我们使用肽噬菌体展示库和扫描突变来表征它们的特异性谱，这表明高特异性亲和钳的识别位点显着扩大。高特异性亲和钳的晶体结构显示出与不被PDZ结构域识别的肽配体的一部分的广泛接触，从而使亲和钳在特异性方面的改进合理化。与另一种亲和钳结构的比较显示，尽管两者在PDZ和FN3结构域之间具有广泛的接触，但它们在两个结构域的相对位置上表现出较大的偏移。我们的研究结果表明，连接域可以快速融合和发展为一个单一的功能模块，域接口的固有可塑性允许产生不同的活性位点的地形。定向结构域界面进化的这些属性为产生具有广泛功能的合成蛋白质提供了简便的方法。
We have recently established a new protein-engineering strategy termed “directed domain-interface evolution” that generates a binding site by linking two protein domains and then optimizing the interface between them. Employing this strategy, we have generated synthetic two-domain “affinity clamps” using PDZ and fibronectin type III (FN3) domains as the building blocks. While these affinity clamps all had significantly higher affinity toward a target peptide than the underlying PDZ domain, two distinct types of affinity clamps were found in terms of target specificity. One type conserved the specificity of the parent PDZ domain, and the other dramatically increased the specificity. Here, we characterized their specificity profiles using peptide phage-display libraries and scanning mutagenesis, which suggested a significantly enlarged recognition site of the high-specificity affinity clamps. The crystal structure of a high-specificity affinity clamp showed extensive contacts with a portion of the peptide ligand that is not recognized by the PDZ domain, thus rationalizing the affinity clamp’s improvement in specificity. A comparison with another affinity clamp structure revealed that, although both had extensive contacts between PDZ and FN3 domains, they exhibited a large offset in the relative position of the two domains. Our results indicate that linked domains could rapidly fuse and evolve as a single functional module and that the inherent plasticity of domain interfaces allows for the generation of diverse active-site topography. These attributes of directed domain-interface evolution provide facile means to generate synthetic proteins with a broad range of functions.
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影响因子：
5.6
作者：
Gilbreth, Ryan N.;Esaki, Kaori;Koide, Shohei
通讯作者：
Koide, Shohei
影响因子：
56.9
作者：
Dahiyat, BI;Mayo, SL
通讯作者：
Mayo, SL
影响因子：
64.8
作者：
Looger, LL;Dwyer, MA;Hellinga, HW
通讯作者：
Hellinga, HW
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
DOI：
10.1073/pnas.0404387101
发表时间：
2004-08-10
影响因子：
11.1
作者：
Kaplan, J;DeGrado, WF
通讯作者：
DeGrado, WF