Yang Woo Seok, Kim Seu Ha, Kim Minsang, Shin Hejeong, Lee Juyeon, Sandmann Alexander, Park Ohkmae K, Dissmeyer Nico, Song Hyun Kyu
Department of Life Sciences, Korea University, Seongbuk-gu, Seoul, South Korea.
Department of Plant Physiology and Protein Metabolism Laboratory, University of Osnabruck, Osnabruck, Germany.
Nat Commun. 2025 Aug 21;16(1):7817. doi: 10.1038/s41467-025-63282-9.
PROTEOLYSIS1 (PRT1), an N-recognin of Arabidopsis thaliana, recognizes the N-terminal aromatic hydrophobic residue (Tyr/Phe/Trp) of its substrates and ubiquitylates them for degradation by the ubiquitin-proteasome system. Herein, we report the structures of the ZZ domain of PRT1 (PRT1) in complex with bulky hydrophobic N-degron peptides. Unlike other ZZ domains, PRT1 has an unusual binding site with two hydrophobic regions. The N-terminal aromatic residues of N-degrons interact with Ile333 and Phe352 in the flexible loops, which undergo a conformational change. Notably, we identify a third residue from the N-terminus of the substrate that participates in the hydrophobic network with PRT1. Moreover, AlphaFold prediction and biochemical assays revealed that the tandem RING1 and RING2 domains of PRT1 interact intramolecularly. The dimeric RING domains in a single protein represent a unique feature among the RING-type E3 ligases. The biochemical assays using the N-terminal tyrosine-exposed substrate, BIG BROTHER, show that the intramolecular RING dimer is essential for PRT1's robust activity. Therefore, this study expands our knowledge of the structural repertoire in the N-degron pathway and provides insights into the regulation of E3 ligases containing tandem RING domains.
蛋白水解酶1（PRT1）是拟南芥的一种N-识别蛋白，可识别其底物的N端芳香族疏水残基（酪氨酸/苯丙氨酸/色氨酸），并将它们泛素化，以便通过泛素-蛋白酶体系统进行降解。在此，我们报道了与庞大的疏水性N-端降解肽结合的PRT1的ZZ结构域（PRT1-ZZ）的结构。与其他ZZ结构域不同，PRT1具有一个带有两个疏水区域的异常结合位点。N-端降解肽的N端芳香族残基与柔性环中的Ile333和Phe352相互作用，这些柔性环会发生构象变化。值得注意的是，我们从底物的N端鉴定出第三个残基，它与PRT1参与疏水网络。此外，AlphaFold预测和生化分析表明，PRT1的串联RING1和RING2结构域发生分子内相互作用。单个蛋白质中的二聚体RING结构域是RING型E3连接酶中的一个独特特征。使用N端酪氨酸暴露的底物“大兄弟”进行的生化分析表明，分子内RING二聚体对于PRT1的强大活性至关重要。因此这项研究扩展了我们对N-端降解途径中结构组成的认识，并为含有串联RING结构域的E3连接酶的调控提供了见解。
