Active site remodelling accompanies thioester bond formation in the SUMO E1

Abstract

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 A, 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.

Figure 1. Analogs of the Ub/Ubl-adenylate and E1~Ub/Ubl tetrahedral intermediates

Chemical structures of a, Ub/Ubl-adenylate (top) and Ub-AMSN adenylate analog (bottom), b, the E1~Ub/Ubl tetrahedral intermediate (top) during thioester bond formation, the Ub/Ubl-AVSN adduct (middle), and the E1~Ub/Ubl-AVSN tetrahedral intermediate analog (bottom). Red atoms indicate modifications in AMSN and AVSN that deviate from AMP. c, DTT sensitivity of Ub/Ubl thioester and thioether adducts for human and S. cerevisiae SUMO E1s and S. pombe ubiquitin E1. d, Cross-linking assay and time course for SUMO1-AVSN, S. cerevisiae SMT3-AVSN, and S. pombe Ub-AVSN adducts to their cognate E1s. See Methods for assay conditions.

Figure 2. Structural changes in SUMO E1 accompany transitions from adenylate to tetrahedral intermediate

a, Ribbon representation for the SUMO E1/SUMO1-AMSN adenylate analog (left) and SUMO E1~SUMO1-AVSN tetrahedral intermediate analog (right). Atoms for the catalytic cysteine (Cys173), AMSN and AVSN shown as spheres with E1 domains and SUMO color-coded and labeled. N term, N-terminus. b, Elements in SUMO E1 that undergo conformational changes are color-coded and labeled. N term, N-terminus. Similar regions in E1 structures are colored gray and SUMO1 is colored yellow. c, Cartoon representation of the structures color-coded and labeled as in a and b highlighting elements that undergo remodeling.

Figure 3. Conformational changes within the Cys domain

The Cys domains of a, SUMO E1/SUMO1-AMSN, b, SUMO E1~SUMO1-AVSN, c, Ub E1/Ub complex and d, NEDD8 E1~NEDD8(t)/NEDD8(a)/Ubc12/ATP with helices labeled and depicted as tubes. Elements that undergo conformational changes colored as in Fig. 2b. Hinge points indicated by asterisks in the cross-over and re-entry loops. c, Superposition of cross-over and d, re-entry loops for E1/SUMO1-AMSN and E1~SUMO1-AVSN colored as in Fig. 2b. The catalytic cysteine (stick representation with sulfur colored green) is displaced by 34 Å during transitions between open and closed conformations. aa, amino acids

Figure 4. Active sites in E1/SUMO1-AMSN and E1~SUMO1-AVSN

a, Stereo representation (left) and schematic (right) of E1/SUMO1-AMSN depicting residues that contact the adenylate intermediate analog. b, Stereo representation (left) and schematic (right) of SUMO E1~SUMO1-AVSN depicting residues that contact the tetrahedral intermediate analog. The position analogous to SUMO1 G97 in E1~SUMO1-AVSN is denoted G97* to indicate the electrophilic center. Residues in stick representation labeled by single letter amino acid code with select waters as red spheres. Atoms colored as follows: UBA2 carbon (pink), SUMO1 carbon (yellow), oxygen (red), nitrogen (blue), and sulfur (green). Potential hydrogen bonds indicated by dashed lines.

Figure 5. Side chains required for adenylation are dispensible for formation of the tetrahedral intermediate analog

Amino acid contacts that contribute to E1 adenylation activity shown for a, E1/SUMO1/ATP·Mg, b, E1/SUMO1-AMSN adenylate analog and c, E1~SUMO1-AVSN tetrahedral intermediate analog color-coded as in Fig. 2b. Water (red) and Mg (cyan) as spheres. Dashed lines indicate potential hydrogen bonds. d, Structure–function analysis of E1 side chains depicted in a–c in assays for E1~SUMO1-AVSN cross-linking (top), E1~SUMO1 thioester formation (middle), and SUMO1-adenylate formation (bottom). Assay conditions in Methods. e, Structure–function analysis of residues in S. pombe UBA1 in assays for UBA1~Ub-AVSN cross-linking (top) and UBA1-Ub thioester formation (bottom). f, Structure-based sequence alignment of regions for human SUMO E1/SUMO1-AMSN and E1~SUMO1-AVSN, S. cerevisiae UBA1/Ub, and human NEDD8 E1/NEDD8/ATP·Mg. Gaps indicated periods. Boxes indicate conservation. Secondary structure for E1/SUMO1-AMSN and E1~SUMO1-AVSN above alignment with dashed lines indicating disorder. Conformational changes are color-coded as in Fig. 2b. Asterisks above the alignment indicate residues participating in unique interactions in the respective structures. Residues probed by mutational analysis are indicated above the alignment color-coded by activity.

Figure 6. Side chains required for formation of the thioester bond or the tetrahedral intermediate analog are dispensible for adenylation

Amino acid contacts deemed important for achieving the closed conformation are shown for a, E1/SUMO1/ATP·Mg, b, E1/SUMO1-AMSN adenylate analog and c, E1~SUMO1-AVSN tetrahedral intermediate analog colored as in Fig. 2b. Potential hydrogen bonds indicated by dashed lines. d, Structure–function analysis of E1 side chains depicted in a–c in assays for E1~SUMO1-AVSN cross-linking (top), E1-SUMO thioester formation (middle), and SUMO-adenylate formation (bottom) assays. Assays conditions described in Methods. e, Structure–function analysis of select residues in S. pombe UBA1 in assays for UBA1~Ub-AVSN cross-linking (top) and UBA1-Ub thioester formation (bottom).