Deep mutational scanning (DMS) facilitates data-driven models of protein structure and function. Here, we adapted Saturated Programmable Insertion Engineering (SPINE) as a programmable DMS technique. We validate SPINE with a reference single mutant dataset in the PSD95 PDZ3 domain and then characterize most pairwise double mutants to study epistasis. We observe wide-spread proximal negative epistasis, which we attribute to mutations affecting thermodynamic stability, and strong long-range positive epistasis, which is enriched in an evolutionarily conserved and function-defining network of “sector” and clade-specifying residues. Conditional neutrality of mutations in clade-specifying residues compensates for deleterious mutations in sector positions. This suggests that epistatic interactions between these position pairs facilitated the evolutionary expansion and specialization of PDZ domains. We propose that SPINE provides easy experimental access to reveal epistasis signatures in proteins that will improve our understanding of the structural basis for protein function and adaptation.

中文翻译：

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对成对上位性的大规模调查揭示了 PDZ 域进化扩展和专业化的机制

深度突变扫描 (DMS) 促进了蛋白质结构和功能的数据驱动模型。在这里，我们采用了饱和可编程插入工程 (SPINE) 作为可编程 DMS 技术。我们使用 PSD95 PDZ3 域中的参考单突变体数据集验证 SPINE，然后表征大多数成对双突变体以研究上位性。我们观察到广泛的近端负上位性，我们将其归因于影响热力学稳定性的突变，以及强大的远程正上位性，这在进化上保守的和功能定义的“扇区”和进化枝特异性残基网络中得到了丰富。进化枝特异性残基中突变的条件中性补偿了扇区位置中的有害突变。这表明这些位置对之间的上位相互作用促进了 PDZ 域的进化扩展和专业化。我们建议 SPINE 提供了简单的实验途径来揭示蛋白质中的上位性特征，这将提高我们对蛋白质功能和适应的结构基础的理解。