Hefei National Research Center for Cross Disciplinary Science, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China; Ministry of Education Key Laboratory for Membraneless Organelles and Cellular Dynamics, University of Science & Technology of China, Hefei, Anhui, China; Division of Life Sciences and Medicine, Center for Advanced Interdisciplinary Science and Biomedicine of IHM, University of Science and Technology of China, Hefei, Anhui, China.
Department of Physics, University of Science and Technology of China, Hefei, Anhui, China.
J Biol Chem. 2024 Aug;300(8):107605. doi: 10.1016/j.jbc.2024.107605. Epub 2024 Jul 25.
TNIP1 has been increasingly recognized as a security check to finely adjust the rate of mitophagy by disrupting the recycling of the Unc-51-like kinase complex during autophagosome formation. Through tank-binding kinase 1-mediated phosphorylation of the TNIP1 FIP200 interacting region (FIR) motif, the binding affinity of TNIP1 for FIP200, a component of the Unc-51-like kinase complex, is enhanced, allowing TNIP1 to outcompete autophagy receptors. Consequently, FIP200 is released from the autophagosome, facilitating further autophagosome expansion. However, the molecular basis by which FIP200 utilizes its claw domain to distinguish the phosphorylation status of residues in the TNIP1 FIR motif for recognition is not well understood. Here, we elucidated multiple crystal structures of the complex formed by the FIP200 claw domain and various phosphorylated TNIP1 FIR peptides. Structural and isothermal titration calorimetry analyses identified the crucial residues in the FIP200 claw domain responsible for the specific recognition of phosphorylated TNIP1 FIR peptides. Additionally, utilizing structural comparison and molecular dynamics simulation data, we demonstrated that the C-terminal tail of TNIP1 peptide affected its binding to the FIP200 claw domain. Moreover, the phosphorylation of TNIP1 Ser123 enabled the peptide to effectively compete with the peptide p-CCPG1 (the FIR motif of the autophagy receptor CCPG1) for binding with the FIP200 claw domain. Overall, our work provides a comprehensive understanding of the specific recognition of phosphorylated TNIP1 by the FIP200 claw domain, marking an initial step toward fully understanding the molecular mechanism underlying the TNIP1-dependent inhibition of mitophagy.
TNIP1 被越来越多地认为是一种安全检查，通过在自噬体形成过程中破坏 Unc-51 样激酶复合物的循环来精细调节自噬的速率。通过结合激酶 1 介导的 TNIP1 的 FIP200 相互作用区域 (FIR) 基序的磷酸化，TNIP1 与 FIP200 的结合亲和力增强，FIP200 是 Unc-51 样激酶复合物的一个组成部分，允许 TNIP1 与自噬受体竞争。因此，FIP200 从自噬体中释放出来，促进进一步的自噬体扩展。然而，FIP200 利用其爪域来区分 TNIP1 FIR 基序中残基的磷酸化状态以进行识别的分子基础尚不清楚。在这里，我们阐明了由 FIP200 爪域和各种磷酸化 TNIP1 FIR 肽形成的复合物的多个晶体结构。结构和等温滴定量热法分析确定了 FIP200 爪域中负责特异性识别磷酸化 TNIP1 FIR 肽的关键残基。此外，利用结构比较和分子动力学模拟数据，我们证明了 TNIP1 肽的 C 端尾巴影响其与 FIP200 爪域的结合。此外，TNIP1 Ser123 的磷酸化使该肽能够有效地与肽 p-CCPG1（自噬受体 CCPG1 的 FIR 基序）竞争与 FIP200 爪域的结合。总的来说，我们的工作提供了对 FIP200 爪域对磷酸化 TNIP1 特异性识别的全面理解，标志着全面理解 TNIP1 依赖性抑制自噬的分子机制迈出了第一步。
