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.

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