Zhou Peng, Zhang Yunyi, Li Kexin, Ye Haiyang, Mei Li, Shang Shuyong
Center for Informational Biology, School of Life Science and Technology, University of Electronic Science and Technology of China (UESTC), Chengdu, Sichuan, China.
College of Culinary and Food Science Engineering, Sichuan Tourism University, Chengdu, Sichuan, China.
Biotechnol Bioeng. 2025 Sep;122(9):2511-2521. doi: 10.1002/bit.29044. Epub 2025 Jun 4.
Self-controlling peptides (SCPs) are described as a specific sub-class of our previously proposed self-binding peptides (SBPs), which work as a molecular switch to control the protein function conversion between two or more biological states. In this study, a variety of artificial protein systems containing SCP with molecular switch functionality were rationally designed and systematically engineered by fusing several (weak and strong) phosphopeptide binders to the C-terminal tail of human Src SH2 domain via a flexible linker (FL), termed {SH2-> FL-> SCP} fusion protein systems. It is revealed that the intramolecular interaction between SH2 and SCP in the systems shares a similar binding behavior with the intermolecular interaction between SH2 and free phosphopeptide in structural and energetic points of view. In addition, FL is responsible for the binding dynamics of SCP to SH2, which restricts the SCP in a local region nearby SH2 binding site, thus equivalently enhancing the micro-concentration of SCP around the site, effectively increasing the collision frequency of SCP with the site, and consequently improving the apparent affinity of SCP to SH2. We carefully selected phosphopeptide binders and systematically optimized the sequence length and amino acid composition of FL, consequently resulting in a satisfactory {SH2-> poly(G)-> SCP(SIPM2-K)} fusion protein system that was demonstrated to possess molecular switch functionality, in which the SCP binding and unbinding events were reversibly triggered by externally controlling its Tyr0-phosphorylation and pTyr0-dephosphorylation, respectively.
自控肽（SCPs）被描述为我们之前提出的自结合肽（SBPs）的一个特定子类，它作为一种分子开关来控制蛋白质在两种或更多生物状态之间的功能转换。在本研究中，通过一个柔性接头（FL）将几种（弱和强）磷酸肽结合剂融合到人Src SH2结构域的C末端尾巴上，合理设计并系统构建了多种具有分子开关功能的含SCP的人工蛋白质系统，称为{SH2->FL->SCP}融合蛋白系统。结果表明，从结构和能量角度来看，系统中SH2和SCP之间的分子内相互作用与SH2和游离磷酸肽之间的分子间相互作用具有相似的结合行为。此外，FL负责SCP与SH2的结合动力学，它将SCP限制在SH2结合位点附近的局部区域，从而等效地提高了该位点周围SCP的微浓度，有效增加了SCP与该位点的碰撞频率，进而提高了SCP对SH2的表观亲和力。我们仔细选择了磷酸肽结合剂，并系统优化了FL的序列长度和氨基酸组成，最终得到了一个令人满意的{SH2->聚（G）->SCP（SIPM2-K）}融合蛋白系统，该系统被证明具有分子开关功能，其中SCP的结合和解离事件分别通过外部控制其Tyr0磷酸化和pTyr0去磷酸化而可逆地触发。
