Multimodal substrate recruitment enables CTLH-MKLN1 E3 ligase to target N-, C-, and internal degrons

Abstract

The GID/CTLH family of E3 ubiquitin ligases employs several substrate receptor subunits that recruit distinct degrons, but the substrate recognition mechanism of the CTLHMKLN1 assembly has largely remained elusive. Here, we reconstitute CTLHMKLN1-dependent ubiquitylation of three biochemically distinct substrates - MKLN1 itself, ZMYND19, and FAM72A-recruited UNG2, and determine cryo-EM structures of each substrate bound to MKLN1. The structures reveal how MKLN1's discoidin and Kelch {beta}-propeller domains engage substrates through multivalent contacts. MKLN1 recruits itself through discoidin - Kelch interactions between MKLN1 dimers, forming an assembly that competes with other substrates. ZMYND19 and FAM72A each bind to MKLN1 through loops engaging the discoidin trench domain while their C-terminal Arg residues engage the MKLN1 Kelch central channel, identifying MKLN1 as an Arg/C-degron receptor. The acetylated N-terminus of UNG2 is positioned within a Y-shaped tunnel of FAM72A, defining an adaptor-mediated Ac/N-degron recognition mechanism. Thus, our data reveal how combinatorial deployment of MKLN1 discoidin and Kelch domains enable a single receptor subunit to recognize a diverse substrate repertoire, adding to a complex picture of degron recognition across the GID/CTLH family.