The linear ubiquitin chain assembly complex (LUBAC) is the only known ubiquitin ligase that generates linear/Met1-linked ubiquitin chains. One of the LUBAC components, HOIL-1L, was recently shown to catalyse oxyester bond formation between the C-terminus of ubiquitin and some substrates. However, oxyester bond formation by LUBAC has not been directly observed. We present the first low-resolution 3D structure of LUBAC obtained by electron microscopy and report its generation of heterotypic ubiquitin chains containing linear linkages with oxyester-linked branches. We found that addition of the oxyester-bound branches depends on HOIL-1L catalytic activity. We suggest a coordinated ubiquitin relay mechanism between the two ligases supported by cross-linking mass spectrometry data, which show proximity between the catalytic RBR domains of HOIP and HOIL-1L, Mutations in the linear ubiquitin chain-binding NZF domain of HOIL-1L reduces chain branching suggesting its role in the process. In cells, these heterotypic chains were induced by TNF. In conclusion, we demonstrate that LUBAC assembles heterotypic ubiquitin chains with linear and oxyester-linked branches by the concerted action of HOIP and HOIL-1L.

中文翻译：

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线性泛素链组装复合体产生异型泛素链

线性泛素链装配复合体（LUBAC）是唯一已知的产生线性/ Met1连接的泛素链的泛素连接酶。最近显示，LUBAC的一种成分HOIL-1L可以催化泛素C末端与某些底物之间的氧酸酯键形成。但是，还没有直接观察到LUBAC形成氧酯键。我们介绍了通过电子显微镜获得的LUBAC的第一个低分辨率3D结构，并报告了其异型泛素链的生成，该泛素链包含与氧酸酯连接的分支的线性连接。我们发现氧酯结合的分支的添加取决于HOIL-1L催化活性。我们建议在交联质谱数据支持的两种连接酶之间建立协调的泛素中继机制，它们显示HOIP和HOIL-1L的催化RBR结构域之间的接近性，HOIL-1L的线性遍在蛋白链结合NZF结构域中的突变减少了链分支，表明了其在该过程中的作用。在细胞中，这些异型链是由TNF诱导的。总之，我们证明，LUBAC通过HOIP和HOIL-1L的协同作用组装具有线性和氧酸酯连接分支的异型泛素链。