Cryo-EM structure of the KLHL22 E3 ligase bound to an oligomeric metabolic enzyme

CULLIN-RING ligases constitute the largest group of E3 ubiquitin ligases. While some CULLIN family members recruit adapters before engaging further with different substrate receptors, homo-dimeric BTB-Kelch family proteins combine adapter and substrate receptor into a single polypeptide for the CULLIN3 family. However, the entire structural assembly and molecular details have not been elucidated to date. Here, we present a cryo-EM structure of the CULLIN3^RBX1 in complex with Kelch-like protein 22 (KLHL22) and a mitochondrial glutamate dehydrogenase complex I (GDH1) at 3.06 Å resolution. The structure adopts a W-shaped architecture formed by E3 ligase dimers. Three CULLIN3^KLHL22-RBX1 dimers were found to be dynamically associated with a single GDH1 hexamer. CULLIN3^KLHL22-RBX1 ligase mediated the polyubiquitination of GDH1 in vitro. Together, these results enabled the establishment of a structural model for understanding the complete assembly of BTB-Kelch proteins with CULLIN3 and how together they recognize oligomeric substrates and target them for ubiquitination.