To understand the molecular evolution of functional diversity in protein families, we comprehensively investigated the consequences of all possible mutation combinations separating two peptide-binding domains with highly divergent specificities. We analyzed the Erbin PDZ domain (Erbin-PDZ), which exhibits canonical type I specificity, and a synthetic Erbin-PDZ variant (E-14) that differs at six positions and exhibits an atypical specificity that closely resembles that of the natural Pdlim4 PDZ domain (Pdlim4-PDZ). We constructed a panel of 64 PDZ domains covering all possible transitions between Erbin-PDZ and E-14 (i.e., the panel contained variants with all possible combinations of either the Erbin-PDZ or E-14 sequence at the six differing positions). We assessed the specificity profiles of the 64 PDZ domains using a C-terminal phage-displayed peptide library containing all possible genetically encoded heptapeptides. The specificity profiles clustered into six distinct groups, showing that intermediate domains can be nodes for the evolution of divergent functions. Remarkably, three substitutions were sufficient to convert the specificity of Erbin-PDZ to that of Pdlim4-PDZ, whereas Pdlim4-PDZ contains 71 differences relative to Erbin-PDZ. X-ray crystallography revealed the structural basis for specificity transition: a single substitution in the center of the binding site, supported by contributions from auxiliary substitutions, altered the main chain conformation of the peptide ligand to resemble that of ligands bound to Pdlim4-PDZ. Our results show that a very small set of mutations can dramatically alter protein specificity, and these findings support the hypothesis whereby complex protein functions evolve by gene duplication followed by cumulative mutations.

中文翻译：

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对两个不同的PDZ域特异性之间的所有进化路径的综合分析。

为了了解蛋白质家族中功能多样性的分子进化，我们全面研究了所有可能的突变组合（其具有高度不同的特异性）分离两个肽结合结构域的后果。我们分析了具有典型I型特异性的Erbin PDZ域（Erbin-PDZ）和一个在六个位置不同并具有非常类似于天然Pdlim4 PDZ的非典型特异性的合成Erbin-PDZ变体（E-14）域（Pdlim4-PDZ）。我们构建了一个由64个PDZ域组成的面板，涵盖了Erbin-PDZ和E-14之间的所有可能过渡（即，该面板包含在六个不同位置具有Erbin-PDZ或E-14序列的所有可能组合的变体）。我们使用包含所有可能的遗传编码七肽的C末端噬菌体展示肽库评估了64个PDZ域的特异性谱。特异性概况分为六个不同的组，表明中间结构域可以是分化功能进化的节点。值得注意的是，三个取代足以将Erbin-PDZ的特异性转换为Pdlim4-PDZ的特异性，而Pdlim4-PDZ相对于Erbin-PDZ包含71个差异。X射线晶体学揭示了特异性转变的结构基础：在结合位点中心的单个取代，由辅助取代的贡献支持，改变了肽配体的主链构象，使其类似于与Pdlim4-PDZ结合的配体的主链构象。