Luiza Deszcz

Eukaryotic cells have evolved sophisticated quality control mechanisms to eliminate aggregation-prone proteins that compromise cellular health. Central to this defense is the ubiquitin-proteasome system, where UBR4 acts as an essential E4 ubiquitin ligase, amplifying degradation marks on defective proteins. Cryo–electron microscopy analysis of UBR4 in complex with its cofactors KCMF1 and CALM1 reveals a massive 1.3-megadalton ring structure, featuring a central substrate-binding arena and flexibly attached catalytic units. Our structure shows how UBR4 binds substrate and extends lysine-48–specific ubiquitin chains. Efficient substrate targeting depends on both preubiquitination and specific N-degrons, with KCMF1 acting as a key substrate filter. The architecture of the E4 megacomplex is conserved across eukaryotes, but species-specific adaptations allow UBR4 to perform its precisely tuned quality control function in diverse cellular environments.

Inhibitor of apoptosis proteins (IAPs) bind to pro-apoptotic proteases, keeping them inactive and preventing cell death. The atypical ubiquitin ligase BIRC6 is the only essential IAP, additionally functioning as a suppressor of autophagy. We performed a structure-function analysis of BIRC6 in complex with caspase-9, HTRA2, SMAC, and LC3B, which are critical apoptosis and autophagy proteins. Cryo–electron microscopy structures showed that BIRC6 forms a megadalton crescent shape that arcs around a spacious cavity containing receptor sites for client proteins. Multivalent binding of SMAC obstructs client binding, impeding ubiquitination of both autophagy and apoptotic substrates. On the basis of these data, we discuss how the BIRC6/SMAC complex can act as a stress-induced hub to regulate apoptosis and autophagy drivers.

Summary: Inhibitor of apoptosis proteins (IAPs) bind to pro-apoptotic proteases, keeping them inactive and preventing cell death. BIRC6 is an exceptionally large, multidomain IAP that inhibits its targets by means of its atypical ubiquitin ligase activity and in addition, functions as an inhibitor of autophagy by depleting LC3B. Little is known of the mechanisms by which BIRC6 interacts with its targets and fulfills these two roles. Here, we determined the cryo-EM structure of BIRC6 alone and in complex with two mitochondrial pro-apoptotic proteins, HTRA2 and SMAC. We show BIRC6 is an antiparallel homodimer that forms a crescent shape that arcs around a spacious cavity. The cavity is surrounded by binding sites for client proteins, where they interact with the flexible UBC domain that mediates ubiquitin ligation. Functional data reveal that multivalent binding of SMAC in the central cavity obstructs substrate binding, impeding ubiquitination of both autophagy and apoptotic target proteins. Together our data reveal the molecular mechanisms of how SMAC specifically binds and inhibits BIRC6 to promote apoptosis, and how this regulatory mechanism also extends to autophagy substrates. The interaction sites are hot spots of cancer and atrophy mutations, highlighting the importance of carefully balancing the interplay between BIRC6 and SMAC.