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Abstract：
The design and selection of peptides targeting cellular proteins is challenging and often yields candidates with undesired properties. Therefore we deployed a new selection system based on the twin-arginine translocase (TAT) pathway of Escherichia coli, named hitchhiker translocation (HiT) selection. A pool of α-helix encoding sequences was designed and selected for interference with the coiled coil domain (CC) of a melanoma-associated basic-helix-loop-helix-leucine-zipper (bHLHLZ) protein, the microphthalmia associated transcription factor (MITF). One predominant sequence (iM10) was enriched during selection and showed remarkable protease resistance, high solubility and thermal stability while maintaining its specificity. Furthermore, it exhibited nanomolar range affinity towards the target peptide. A mutation screen indicated that target-binding helices of increased homodimer stability and improved expression rates were preferred in the selection process. The crystal structure of the iM10/MITF-CC heterodimer (2.1Å) provided important structural insights and validated our design predictions. Importantly, iM10 did not only bind to the MITF coiled coil, but also to the markedly more stable HLHLZ domain of MITF. Characterizing the selected variants of the semi-rational library demonstrated the potential of the innovative bacterial selection approach.
摘要：
靶向细胞蛋白的肽的设计和选择是具有挑战性的,并且通常产生具有不期望的性质的候选物。因此，我们基于大肠杆菌的双精氨酸转位酶（TAT）途径部署了一种新的选择系统，名为搭便车者易位（HiT）选择。设计并选择了α-螺旋编码序列库，以干扰黑色素瘤相关的碱性-螺旋-环-螺旋-亮氨酸-拉链（bHLHLZ）蛋白的卷曲螺旋结构域（CC），小眼症相关转录因子（MITF）。一个优势序列（iM10）在选择过程中被富集，并显示出显著的蛋白酶抗性，高溶解度和热稳定性，同时保持其特异性。此外，它对目标肽表现出纳摩尔范围的亲和力。突变筛选表明,在选择过程中优选具有提高的同二聚体稳定性和提高的表达率的靶结合螺旋。iM10/MITF-CC异二聚体（2.1µ）的晶体结构提供了重要的结构见解，并验证了我们的设计预测。重要的是,iM10不仅与MITF卷曲螺旋结合，以及MITF的显著更稳定的HLHLZ结构域。表征半理性文库的所选变体证明了创新细菌选择方法的潜力。
