Department of Genetics, Stanford University, Stanford, United States.
Department of Biology, Stanford University, Stanford, United States.
Elife. 2019 Jul 8;8:e40499. doi: 10.7554/eLife.40499.
Transient, regulated binding of globular protein domains to Short Linear Motifs (SLiMs) in disordered regions of other proteins drives cellular signaling. Mapping the energy landscapes of these interactions is essential for deciphering and perturbing signaling networks but is challenging due to their weak affinities. We present a powerful technology (MRBLE-pep) that simultaneously quantifies protein binding to a library of peptides directly synthesized on beads containing unique spectral codes. Using MRBLE-pep, we systematically probe binding of calcineurin (CN), a conserved protein phosphatase essential for the immune response and target of immunosuppressants, to the PxIxIT SLiM. We discover that flanking residues and post-translational modifications critically contribute to PxIxIT-CN affinity and identify CN-binding peptides based on multiple scaffolds with a wide range of affinities. The quantitative biophysical data provided by this approach will improve computational modeling efforts, elucidate a broad range of weak protein-SLiM interactions, and revolutionize our understanding of signaling networks.
无定形区中球形蛋白结构域与短线性基序（SLiM）的瞬时、调节性结合驱动细胞信号转导。绘制这些相互作用的能量景观对于破译和扰乱信号网络至关重要，但由于它们的亲和力较弱，因此具有挑战性。我们提出了一种强大的技术（MRBLE-pep），可同时定量测定直接在含有独特光谱代码珠上合成的肽文库中蛋白质与肽的结合。使用 MRBLE-pep，我们系统地探测了钙调神经磷酸酶（CN）与 PxIxIT SLiM 的结合，钙调神经磷酸酶是免疫反应所必需的保守蛋白磷酸酶，也是免疫抑制剂的靶标。我们发现侧翼残基和翻译后修饰对 PxIxIT-CN 亲和力至关重要，并基于多种具有广泛亲和力的支架识别出 CN 结合肽。这种方法提供的定量生物物理数据将改善计算建模工作，阐明广泛的弱蛋白-SLiM 相互作用，并彻底改变我们对信号网络的理解。
