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

Abstract

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.

Role of Nse1 Subunit of SMC5/6 Complex as a Ubiquitin Ligase

“…The fact that Nse1 directly interacts with ubiquitin ( Figure 2 E) further strengthens its connection to the ubiquitination pathway and may explain the mechanism Nse1 uses to promote ubiquitin chain formation. The RING-domain E3 ligases have been shown to bind both the E2 and its covalently-bound ubiquitin to lock them in a conformation primed for catalysis and stimulate ubiquitination [ 26 , 32 , 33 , 34 ]. Therefore, based on published structures of a RING-Ubc13~Ub/Mms2 complex [ 26 ] and Nse1/3/4 [ 13 ], we modeled the Nse1–Ubc13~Ub complex ( Figure 5 ).…”

“…The RING-domain E3 ligases have been shown to bind both the E2 and its covalently-bound ubiquitin to lock them in a conformation primed for catalysis and stimulate ubiquitination [ 26 , 32 , 33 , 34 ]. Therefore, based on published structures of a RING-Ubc13~Ub/Mms2 complex [ 26 ] and Nse1/3/4 [ 13 ], we modeled the Nse1–Ubc13~Ub complex ( Figure 5 ). Accordingly, the ubiquitin that is bound to Ubc13 by a thioester bond is localized in the vicinity of the WHB domain of Nse1, which we found to be the domain mediating the Nse1-ubiquitin interaction ( Figure 2 E).…”

“…The S. pombe Nse1 and Ubc13 structures were downloaded from AlphaFold protein structure database [ 25 ]. The Nse1(vRING)-Ubc13 and Nse1-Ubc13-Ubiquitin models, most similar to the 7BBD crystal structure of the TRIM21-Ubc13-Mms2-Ubiquitin complex [ 26 ], were created manually with the help of the COZOID tool [ 27 ].…”

“…Red arrows on top indicate amino acids mutated in ( D ) and their corresponding numbers in S. pombe . ( C ) The Nse1 vRING domain (dark violet)—Ubc13 (grey) interaction model based on the crystal structure of the human TRIM21-Ubc13 [ 26 ]. The Nse1-R188 residue is red labeled.…”

TRIM21/Ro52 - Roles in Innate Immunity and Autoimmune Disease

“…Such K63-linked ubiquitination is required for targeting the TRIM21-protein (e.g. antibody) complex for degradation ( 32 ).…”