RING domains act as both substrate and enzyme in a catalytic arrangement to drive self-anchored ubiquitination.

Attachment of ubiquitin (Ub) to proteins is one of the most abundant and versatile of all posttranslational modifications and affects outcomes in essentially all physiological processes. RING E3 ligases target E2 Ub-conjugating enzymes to the substrate, resulting in its ubiquitination. However, the mechanism by which a ubiquitin chain is formed on the substrate remains elusive. Here we demonstrate how substrate binding can induce a specific RING topology that enables self-ubiquitination. By analyzing a catalytically trapped structure showing the initiation of TRIM21 RING-anchored ubiquitin chain elongation, and in combination with a kinetic study, we illuminate the chemical mechanism of ubiquitin conjugation. Moreover, biochemical and cellular experiments show that the topology found in the structure can be induced by substrate binding. Our results provide insights into ubiquitin chain formation on a structural, biochemical and cellular level with broad implications for targeted protein degradation. The mechanism by which RING E3-anchored ubiquitin chains are formed is not well understood. Here, the authors solve a crystal structure of the RING E3 enzyme TRIM21 trapped in the process of self-anchored chain elongation and provide biochemical and cellular insights into the mechanism of ubiquitin conjugation.