Basis for a ubiquitin-like protein thioester switch toggling E1–E2 affinity

Abstract

Ubiquitin-like proteins (UBLs) are conjugated by dynamic E1-E2-E3 enzyme cascades. E1 enzymes activate UBLs by catalysing UBL carboxy-terminal adenylation, forming a covalent E1 throught UBL thioester intermediate, and generating a thioester-linked E2 throught UBL product, which must be released for subsequent reactions. Here we report the structural analysis of a trapped UBL activation complex for the human NEDD8 pathway, containing NEDD8's heterodimeric E1 (APPBP1-UBA3), two NEDD8s (one thioester-linked to E1, one noncovalently associated for adenylation), a catalytically inactive E2 (Ubc12), and MgATP. The results suggest that a thioester switch toggles E1-E2 affinities. Two E2 binding sites depend on NEDD8 being thioester-linked to E1. One is unmasked by a striking E1 conformational change. The other comes directly from the thioester-bound NEDD8. After NEDD8 transfer to E2, reversion to an alternate E1 conformation would facilitate release of the E2 throught NEDD8 thioester product. Thus, transferring the UBL's thioester linkage between successive conjugation enzymes can induce conformational changes and alter interaction networks to drive consecutive steps in UBL cascades.

Mechanistic Studies on Activation of Ubiquitin and Di-ubiquitin-like Protein, FAT10, by Ubiquitin-like Modifier Activating Enzyme 6, Uba6

“…6B and data not shown). It has been found that an E1-E2 transthiolation event requires significant conformational rearrangements of the E1 enzyme (31,34,35). Crystallography studies with the NAE ternary complex and Ubc12 show that the core domain of Ubc12 associates with the UFD of NAE and a peptide-like extension on Ubc12 docks into a groove in the adenylation domain, adjacent to the ATP-binding site on NAE (35).…”

“…Crystallography studies with the NAE ternary complex and Ubc12 show that the core domain of Ubc12 associates with the UFD of NAE and a peptide-like extension on Ubc12 docks into a groove in the adenylation domain, adjacent to the ATP-binding site on NAE (35). It is hypothesized that formation of NEDD8-adenylate on the NAEϳNEDD8 thioester induces a rotation in the UFD domain of almost 120°, which enables NAE to bind Ubc12 and align it in prime position for transthiolation (35). After the NEDD8 thioester is transferred to Ubc12, the loss of the covalent bond with NEDD8 induces the UFD of NAE to rotate back (35).…”

“…It has been found that an E1-E2 transthiolation event requires significant conformational rearrangements of the E1 enzyme (31,34,35). Crystallography studies with the NAE ternary complex and Ubc12 show that the core domain of Ubc12 associates with the UFD of NAE and a peptide-like extension on Ubc12 docks into a groove in the adenylation domain, adjacent to the ATP-binding site on NAE (35). It is hypothesized that formation of NEDD8-adenylate on the NAEϳNEDD8 thioester induces a rotation in the UFD domain of almost 120°, which enables NAE to bind Ubc12 and align it in prime position for transthiolation (35).…”

“…It is hypothesized that formation of NEDD8-adenylate on the NAEϳNEDD8 thioester induces a rotation in the UFD domain of almost 120°, which enables NAE to bind Ubc12 and align it in prime position for transthiolation (35). After the NEDD8 thioester is transferred to Ubc12, the loss of the covalent bond with NEDD8 induces the UFD of NAE to rotate back (35). Considering the mechanism proposed for NAE, we hypothesize that high concentrations of ATP could trap the UFD domain of Uba6 in a confirmation that is unfavorable for UBL charging.…”

Conformational Transition Associated with E1-E2 Interaction in Small Ubiquitin-like Modifications

“…During the transfer of Ublp from E1 to E2, the E2 enzyme is recruited to the E1-SUMO thioester conjugate by binding to multiple sites on E1, including the Cys domain (the domain containing the active Cys residue) and the ubiquitin-like (Ubl) domain (10,11). Among these multiple binding sites, the Ubl domain has the highest binding affinity, and thus it is the key E2-binding site.…”