Wang Xiaoyang, Xie Huabin, Zhu Zhongliang, Zhang Jiahai, Xu Chao
MOE Key Laboratory for Cellular Dynamics, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China.
FEBS J. 2023 Feb;290(3):712-723. doi: 10.1111/febs.16611. Epub 2022 Sep 11.
Enhancer of rudimentary homologue (ERH), a small protein conserved in eukaryotes, is involved in a wide spectrum of cellular events, including cell cycle progression, piRNA biogenesis, miRNA maturation and gene expression. Human ERH is recruited to replication foci by CDKN1A-interacting zinc finger protein 1 (CIZ1), and plays an important role in cell growth control. However, the molecular basis for CIZ1 recognition by ERH remains unknown. By using GST pull-down experiment, we found that a fragment within CIZ1, upstream of its first zinc finger, is sufficient for binding to ERH. We solved the structure of CIZ1-bound ERH, in which the ERH dimer binds to two CIZ1 fragments to form a 2 : 2 heterotetramer. CIZ1 forms intermolecular antiparallel β-strands with ERH, and its binding surface on ERH is distinct from those of other known ERH-binding ligands. The ERH-CIZ1 interface was further validated by mutagenesis and binding experiments. Our structural study complemented by biochemistry experiments not only provides insights into a previously unidentified ligand-binding mode for ERH but also sheds light on the understanding of evolutionarily conserved roles for ERH orthologs.
原始同源物增强子（ERH）是一种在真核生物中保守的小蛋白，参与广泛的细胞活动，包括细胞周期进程、piRNA生物合成、miRNA成熟和基因表达。人ERH通过与CDKN1A相互作用的锌指蛋白1（CIZ1）被招募到复制焦点，并在细胞生长控制中发挥重要作用。然而，ERH识别CIZ1的分子基础仍然未知。通过谷胱甘肽S-转移酶下拉实验，我们发现CIZ1中第一个锌指上游的一个片段足以与ERH结合。我们解析了与CIZ1结合的ERH的结构，其中ERH二聚体与两个CIZ1片段结合形成2:2异源四聚体。CIZ1与ERH形成分子间反平行β链，其在ERH上的结合表面与其他已知的ERH结合配体不同。通过诱变和结合实验进一步验证了ERH-CIZ1界面。我们的结构研究辅以生物化学实验，不仅为ERH以前未被识别的配体结合模式提供了见解，也为理解ERH直系同源物的进化保守作用提供了线索。
