Structural basis for antagonism of the ubiquitin ligase BIRC6 by SMAC
Structural basis for antagonism of the ubiquitin ligase BIRC6 by SMAC
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DOI：
10.1101/2022.08.30.505748
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发表时间：
2022-08
期刊：
影响因子：
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通讯作者：
Larissa Dietz;Cara J. Ellison;Carlos Riechmann;C. K. Cassidy;F. D. Felfoldi;Adán Pinto-Fernández;B. Kessler;P. R. Elliott
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文献类型：
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作者：
Larissa Dietz;Cara J. Ellison;Carlos Riechmann;C. K. Cassidy;F. D. Felfoldi;Adán Pinto-Fernández;B. Kessler;P. R. Elliott
细胞凋亡是一种遗传程序性细胞死亡，可以由内部或外部信号触发，最终激活半胱氨酸天冬氨酸酶(Caspase)这一蛋白家族1。凋亡抑制因子(IAP)家族的某些成员是前哨蛋白，通过抑制caspase来防止细胞过早死亡。IAPS又受包括第二线粒体衍生的caspase激活剂(SMAC)在内的拮抗剂的调节。含IAP重复序列的杆状病毒蛋白6(BIRC6)是一种巨型IAP，具有E2/E3两种泛素连接酶活性，并通过抑制caspase 2-7参与细胞凋亡，其机制尚不清楚。在这里，我们展示了BIRC6直接限制激活的caspase-3，并泛素化激活的caspase-3，−7和−9专门与非规范的E1，UBA6一起工作。重要的是，我们表明SMAC抑制了这两种机制。BIRC6单独的和与Smac形成的复合体的冷冻电子显微镜(Cryo-EM)结构表明，BIRC6以反平行二聚体的形式存在，每一端都有一个底物结合模块并列在催化结构域上，我们发现了多个高度保守的、对结构和功能重要的未注释结构域。通过我们的结构、生化和生物物理发现，我们发现Smac在多个位置与BIRC6结合，导致亚纳摩尔亲和力，使Smac竞争性地取代caspase，从而拮抗BIRC6介导的caspase抑制。
Apoptosis, a form of genetically programmed cell death, can be triggered by either internal or external signals ultimately activating caspases, a family of proteases1. Certain members of the inhibitors of apoptosis (IAP) family are sentinel proteins preventing untimely cell death by inhibiting caspases. IAPs are in turn regulated by antagonists including second mitochondria-derived activator of caspase (SMAC). Baculoviral IAP repeat-containing protein 6 (BIRC6), a giant IAP, possesses dual E2/E3 ubiquitin ligase activity and is implicated in apoptosis via caspase inhibition2–7. How this is achieved remains unknown. Here we show BIRC6 directly restricts activated caspase-3, and ubiquitinates activated caspases-3, −7 and −9 working exclusively with the non-canonical E1, UBA6. Importantly, we show SMAC supresses both mechanisms. Cryo-electron microscopy (cryo-EM) structures of BIRC6 alone and in complex with SMAC reveal BIRC6 exists as an anti-parallel dimer with a substrate-binding module juxtaposed to the catalytic domain at each end, and we identify multiple highly conserved unannotated domains important for architecture and function. Through our structural, biochemical and biophysical findings, we discover SMAC engages BIRC6 at multiple sites resulting in a sub-nanomolar affinity enabling SMAC to competitively displace caspases, thus antagonising BIRC6-mediated caspase inhibition.