Phosphorylation of the mitochondrial autophagy receptor Nix enhances its interaction with LC3 proteins.
Phosphorylation of the mitochondrial autophagy receptor Nix enhances its interaction with LC3 proteins.
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DOI：
10.1038/s41598-017-01258-6
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发表时间：
2017-04-25
影响因子：
4.6
通讯作者：
Novak I
中科院分区：
文献类型：
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作者：
Rogov VV;Suzuki H;Marinković M;Lang V;Kato R;Kawasaki M;Buljubašić M;Šprung M;Rogova N;Wakatsuki S;Hamacher-Brady A;Dötsch V;Dikic I;Brady NR;Novak I
线粒体自噬受体Nix与LC 3/GABARAP蛋白相互作用，将线粒体靶向到自噬体中进行降解。在这里，我们提出的证据磷酸化驱动的调节的Nix：LC 3B的相互作用。等温滴定量热法和核磁共振表明，与非磷酸化序列相比，丝氨酸34/35磷酸化Nix LC 3相互作用区（LIR）与LC 3B的亲和力增强了约100倍，并形成了非常刚性的复合物。此外，与含有谷氨酸作为磷酸模拟残基的Nix LIR肽复合的LC 3B的晶体结构和NMR实验表明，LIR磷酸化通过在Nix LIR的磷酸化丝氨酸与LC 3B中的Arg 11、Lys 49和Lys 51之间形成两个额外的氢键来稳定Nix：LC 3B复合物。在LC 3B中将Lys 51取代为Ala消除了拟磷酸化Nix突变体的结合。在功能上，丝氨酸34/35磷酸化增强HeLa细胞中自噬体向线粒体的募集。总之，这项研究提供了细胞，生物化学和生物物理证据表明，磷酸化的LIR域的Nix增强线粒体自噬受体的参与。
The mitophagy receptor Nix interacts with LC3/GABARAP proteins, targeting mitochondria into autophagosomes for degradation. Here we present evidence for phosphorylation-driven regulation of the Nix:LC3B interaction. Isothermal titration calorimetry and NMR indicate a ~100 fold enhanced affinity of the serine 34/35-phosphorylated Nix LC3-interacting region (LIR) to LC3B and formation of a very rigid complex compared to the non-phosphorylated sequence. Moreover, the crystal structure of LC3B in complex with the Nix LIR peptide containing glutamic acids as phosphomimetic residues and NMR experiments revealed that LIR phosphorylation stabilizes the Nix:LC3B complex via formation of two additional hydrogen bonds between phosphorylated serines of Nix LIR and Arg11, Lys49 and Lys51 in LC3B. Substitution of Lys51 to Ala in LC3B abrogates binding of a phosphomimetic Nix mutant. Functionally, serine 34/35 phosphorylation enhances autophagosome recruitment to mitochondria in HeLa cells. Together, this study provides cellular, biochemical and biophysical evidence that phosphorylation of the LIR domain of Nix enhances mitophagy receptor engagement.