Structural ubiquitin contributes to K48 linkage-specificity of the HECT ligase Tom1

Homologous to E6AP C-Terminus (HECT) ubiquitin ligases play key roles in essential pathways such as DNA repair, cell cycle control or protein quality control. Tom1 is one of five HECT ubiquitin E3 ligases encoded in the S. cerevisiae genome and prototypical for a ligase with pleiotropic functions such as ubiquitin chain amplification, orphan quality control and DNA damage response. Structures of full-length HECT ligases, including the Tom1 ortholog HUWE1, have been reported, but how domains beyond the conserved catalytic module contribute to catalysis remains largely elusive. Here, through cryogenic electron microscopy (cryo-EM) of Tom1 during an active ubiquitylation cycle, we demonstrate that the extended domain architecture directly contributes to activity. We identify a Tom1-ubiquitin architecture during ubiquitylation involving a non-canonical ubiquitin binding site in the solenoid shape of Tom1. We demonstrate that this ubiquitin binding site coordinates a structural ubiquitin contributing to the fidelity of K48 poly-ubiquitin chain assembly.