Molecular Structure &Function, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 0A4.
Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5.
Nat Commun. 2017 Jan 3;8:13943. doi: 10.1038/ncomms13943.
The ubiquitin ligase SCF mediates phosphorylation-dependent elimination of numerous substrates by binding one or more Cdc4 phosphodegrons (CPDs). Methyl-based NMR analysis of the Cdc4 WD40 domain demonstrates that Cyclin E, Sic1 and Ash1 degrons have variable effects on the primary Cdc4 binding pocket. Unexpectedly, a Sic1-derived multi-CPD substrate (pSic1) perturbs methyls around a previously documented allosteric binding site for the chemical inhibitor SCF-I2. NMR cross-saturation experiments confirm direct contact between pSic1 and the allosteric pocket. Phosphopeptide affinity measurements reveal negative allosteric communication between the primary CPD and allosteric pockets. Mathematical modelling indicates that the allosteric pocket may enhance ultrasensitivity by tethering pSic1 to Cdc4. These results suggest negative allosteric interaction between two distinct binding pockets on the Cdc4 domain may facilitate dynamic exchange of multiple CPD sites to confer ultrasensitive dependence on substrate phosphorylation.
泛素连接酶 SCF 通过结合一个或多个 Cdc4 磷酸降解结构域（CPD）介导许多底物的磷酸依赖性消除。对 Cdc4 WD40 结构域进行基于甲基的 NMR 分析表明，Cyclin E、Sic1 和 Ash1 降解结构域对主要 Cdc4 结合口袋具有不同的影响。出乎意料的是，Sic1 衍生的多 CPD 底物（pSic1）扰乱了先前记录的化学抑制剂 SCF-I2 的变构结合位点周围的甲基。NMR 交叉饱和实验证实了 pSic1 与变构口袋之间的直接接触。磷酸肽亲和测量揭示了主要 CPD 和变构口袋之间的负变构通讯。数学建模表明，变构口袋可能通过将 pSic1 与 Cdc4 连接起来来增强超敏性。这些结果表明，Cdc4 结构域上两个不同结合口袋之间的负变构相互作用可能促进多个 CPD 位点的动态交换，从而赋予对底物磷酸化的超灵敏依赖性。
