Department of Chemistry, Virginia Tech, Blacksburg, Virginia 24061, United States.
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States.
Biochemistry. 2024 Nov 5;63(21):2803-2815. doi: 10.1021/acs.biochem.4c00231. Epub 2024 Oct 8.
Cell cycle regulatory enzyme Pin1 both catalyzes pSer/Thr--Pro isomerization and binds the same motif separately in its WW domain. To better understand the function of Pin1, a way to separate these activities is needed. An unnatural peptide library, RCO-pSer-Pro-NHR, was designed to identify ligands specific for the Pin1 WW domain. A new solid-phase phosphorylating reagent (SPPR) containing a phosphoramidite functional group was synthesized in one step from Wang resin. The SPPR was used in the preparation of the library by parallel synthesis. The final 315-member library was screened with our WW-domain-specific, enzyme-linked enzyme-binding assay (ELEBA). Four of the best hits were resynthesized, and the competitive dissociation constants were measured by ELEBA. NMR chemical-shift perturbations (CSP) of ligands with N-labeled Pin1 were used to measure for the best four ligands directly, demonstrating that they were specific Pin1 WW domain ligands. Models of the ligands bound to the Pin1 WW domain were used to visualize the mode of binding in the WW domain.
细胞周期调控酶 Pin1 既能催化 pSer/Thr--Pro 异构化，又能分别在其 WW 结构域中结合相同的基序。为了更好地理解 Pin1 的功能，需要有一种分离这些活性的方法。设计了一种非天然肽文库 RCO-pSer-Pro-NHR，用于鉴定针对 Pin1 WW 结构域的特异性配体。一种新的含有磷酰胺基的固相磷酸化试剂（SPPR）可以一步从 Wang 树脂合成。该 SPPR 用于通过平行合成制备文库。最终的 315 成员文库通过我们的 WW 结构域特异性酶联酶结合测定法（ELEBA）进行筛选。重新合成了四个最佳命中物，并通过 ELEBA 测量了它们的竞争解离常数。与 N 标记的 Pin1 配体的 NMR 化学位移扰动（CSP）用于直接测量四个最佳配体，表明它们是特异性 Pin1 WW 结构域配体。用于可视化 WW 结构域中结合模式的配体结合到 Pin1 WW 结构域的模型。
