Active site remodelling accompanies thioester bond formation in the SUMO E1

E1 enzymes activate ubiquitin (Ub) and ubiquitin-like (Ubl) proteins in two steps by carboxy-terminal adenylation and thioester bond formation to a conserved catalytic cysteine in the E1 Cys domain. The structural basis for these intermediates remains unknown. Here we report crystal structures for human SUMO E1 in complex with SUMO adenylate and tetrahedral intermediate analogues at 2.45 and 2.6 Å, respectively. These structures show that side chain contacts to ATP·Mg are released after adenylation to facilitate a 130 degree rotation of the Cys domain during thioester bond formation that is accompanied by remodelling of key structural elements including the helix that contains the E1 catalytic cysteine, the crossover and re-entry loops, and refolding of two helices that are required for adenylation. These changes displace side chains required for adenylation with side chains required for thioester bond formation. Mutational and biochemical analyses indicate these mechanisms are conserved in other E1s. Post-translational modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins such as SUMO regulate a broad array of cellular processes. Prior to conjugation, E1 enzymes must first activate Ub/Ubls in two steps. In the first step, E1s utilize ATP and magnesium to adenylate the C-terminal Ub/Ubl glycine, releasing pyrophosphate. In the second step, the Ub/Ubl adenylate is attacked by a conserved E1 cysteine, resulting in release of AMP and formation of a thioester bond between the C-terminal Ub/Ubl glycine and E1. Here, Olsen et al. use a combination of chemistry and structural biology to trap the E1 bound to mimics of the Ub/Ubl adenylate and thioester intermediates. These structures reveal that the E1 undergoes dramatic conformational changes and structural remodelling to create distinct active sites that propel the reaction forward. This study provides the most complete insight into the details of the E1 catalytic cycle yet observed. The post-translational modification of cellular proteins by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins — such as SUMO — regulates a broad array of cellular processes. E1 enzymes activate Ub and Ubl in two steps, by carboxy-terminal adenylation and thioester bond formation to a catalytic cysteine, but the structural basis for the intermediates remains unknown. Crystal structures for SUMO E1 in complex with SUMO adenylate and tetrahedral intermediate analogues are now reported and analysed.