LC3B Binds to the Autophagy Protease ATG4b with High Affinity Using a Bipartite Interface.
LC3B Binds to the Autophagy Protease ATG4b with High Affinity Using a Bipartite Interface.
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DOI：
10.1021/acs.biochem.2c00482
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发表时间：
2022-11-01
期刊：
影响因子：
2.9
通讯作者：
Arkin, Michelle R.
中科院分区：
文献类型：
--
作者：
Tang, Yinyan;Kay, Amber;Jiang, Ziwen;Arkin, Michelle R.
自噬是一种分解代谢的细胞过程，其中不需要的蛋白质和细胞器被溶酶体降解。其特征在于形成双膜自噬体，其上修饰有介导自噬体与溶酶体融合的蛋白质LC 3B。半胱氨酸蛋白酶ATG 4 b在LC 3B生命周期的两个阶段起作用。我们着手表征LC 3B和ATG 4 b之间的蛋白质-蛋白质相互作用。通过生物化学和生物物理研究，我们表明LC 3B的泛蛋白样核心（残基1-115;“LC 3B-115”），其缺乏C-末端切割位点（在残基120和121之间），在低纳摩尔范围内以令人惊讶的紧密解离常数（KD）结合全长ATG 4 b;比底物pro-LC 3B（残基1-125）或产物LC 3B-I（残基1-120）紧密10-30倍。因此，LC 3B-115是ATG 4 b介导的pro-LC 3B切割的有效抑制剂（IC 50 = 15 nM）。LC 3B-115的结合对ATG 4 b的活性位点的构象没有影响，如通过衍生自LC 3B-1残基116-120的肽底物（“底物-33”）的转换所判断的。相反，截短的ATG 4 b表明，LC 3B的结合和蛋白水解关键取决于ATG 4 b的C-末端尾，而肽底物-33的蛋白水解不需要ATG 4 b的C-末端尾。这些结果支持LC 3B-ATG 4 b结合的二分模型，其中LC 3B的核心与ATG 4 b结合，而pro-LC 3B的C末端尾部组织ATG 4 b活性位点;此外，ATG 4 b的C末端尾部对LC 3B-ATG 4 b相互作用的结合亲和力至少高1000倍，并可能包裹在LC 3B-泛素核心周围。PPI通常被描述为含有用于结合的能量“热点”;然而，在LC 3B-ATG 4 b的情况下，底物-酶复合物含有多个能量相关的结构域，其差异性地影响结合亲和力和催化效率。
Autophagy is a catabolic cellular process in which unwanted proteins and organelles are degraded by lysosomes. It is characterized by the formation of the double-membrane autophagosome decorated with LC3B, a protein that mediates autophagosomal fusion with lysosomes. The cysteine protease ATG4b acts at two stages in the life cycle of LC3B. We set out to characterize the protein–protein interaction between LC3B and ATG4b. Through biochemical and biophysical studies, we show that the ubiquitin-like core of LC3B (residues 1–115; “LC3B-115”), which lacks the C-terminal cleavage site (between residue 120 and 121), binds to full-length ATG4b with a surprisingly tight dissociation constant (KD) in the low nanomolar range; 10–30-fold tighter than that of the substrate pro-LC3B (residues 1–125) or the product LC3B-I (residues 1–120). Consequently, LC3B-115 is a potent inhibitor of the ATG4b-mediated cleavage of pro-LC3B (IC50 = 15 nM). Binding of the LC3B-115 has no effect on the conformation of the active site of ATG4b, as judged by the turnover of a peptide substrate (“substrate-33”), derived from LC3B-I residues 116–120. Conversely, truncations of ATG4b show that binding and proteolysis of LC3B critically depend on the C-terminal tail of ATG4b, whereas proteolysis of the peptide substrate-33 does not require the C-terminal tail of ATG4b. These results support a bipartite model for LC3B-ATG4b binding in which the core of LC3B binds to ATG4b and the C-terminal tail of pro-LC3B organizes the ATG4b active site; additionally, the C-terminal tail of ATG4b contributes at least 1000-fold higher binding affinity to the LC3B-ATG4b interaction and likely wraps around the LC3B-ubiquitin core. PPIs are often described as containing an energetic “hot spot” for binding; in the case of LC3B-ATG4b, however, the substrate–enzyme complex contains multiple, energetically relevant domains that differentially affect binding affinity and catalytic efficiency.
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