Hayashi Akiyo, Giakoumakis Nickolaos Nikiforos, Heidebrecht Tatjana, Ishii Takashi, Panagopoulos Andreas, Caillat Christophe, Takahara Michiyo, Hibbert Richard G, Suenaga Naohiro, Stadnik-Spiewak Magda, Takahashi Tatsuro, Shiomi Yasushi, Taraviras Stavros, von Castelmur Eleonore, Lygerou Zoi, Perrakis Anastassis, Nishitani Hideo
Graduate School of Life Science, University of Hyogo, Kamigori, Japan.
Department of Biology, School of Medicine, University of Patras, Patras, Greece.
Life Sci Alliance. 2018 Dec 31;1(6):e201800238. doi: 10.26508/lsa.201800238. eCollection 2018 Dec.
The CRL4 ubiquitin ligase complex is an essential regulator of cell-cycle progression and genome stability, ubiquitinating substrates such as p21, Set8, and Cdt1, via a display of substrate degrons on proliferating cell nuclear antigens (PCNAs). Here, we examine the hierarchy of the ligase and substrate recruitment kinetics onto PCNA at sites of DNA replication. We demonstrate that the C-terminal end of Cdt2 bears a PCNA interaction protein motif (PIP box, Cdt2), which is necessary and sufficient for the binding of Cdt2 to PCNA. Cdt2 binds PCNA directly with high affinity, two orders of magnitude tighter than the PIP box of Cdt1. X-ray crystallographic structures of PCNA bound to Cdt2 and Cdt1 show that the peptides occupy all three binding sites of the trimeric PCNA ring. Mutating Cdt2 weakens the interaction with PCNA, rendering CRL4 less effective in Cdt1 ubiquitination and leading to defects in Cdt1 degradation. The molecular mechanism we present suggests a new paradigm for bringing substrates to the CRL4-type ligase, where the substrate receptor and substrates bind to a common multivalent docking platform to enable subsequent ubiquitination.
CRL4泛素连接酶复合物是细胞周期进程和基因组稳定性的重要调节因子，通过在增殖细胞核抗原（PCNA）上展示底物降解结构域，使诸如p21、Set8和Cdt1等底物发生泛素化。在此，我们研究了连接酶和底物在DNA复制位点招募到PCNA上的动力学层级。我们证明Cdt2的C末端带有一个PCNA相互作用蛋白基序（PIP框，Cdt2），这对于Cdt2与PCNA的结合是必要且充分的。Cdt2以高亲和力直接结合PCNA，比Cdt1的PIP框紧密两个数量级。与Cdt2和Cdt1结合的PCNA的X射线晶体结构表明，这些肽占据了三聚体PCNA环的所有三个结合位点。突变Cdt2会削弱与PCNA的相互作用，使CRL4在Cdt1泛素化方面效率降低，并导致Cdt1降解缺陷。我们提出的分子机制为将底物带到CRL4型连接酶提供了一种新范式，即底物受体和底物结合到一个共同的多价对接平台上，以实现后续的泛素化。
