Limits...
Active site remodelling accompanies thioester bond formation in the SUMO E1.

Olsen SK, Capili AD, Lu X, Tan DS, Lima CD - Nature (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Structural Biology, Sloan-Kettering Institute, New York, New York 10065, USA.

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.

Show MeSH
Side chains required for adenylation are dispensible for formation of the tetrahedral intermediate analogAmino acid contacts that contribute to E1 adenylation activity shown for a, E1/SUMO1/ATP·Mg9, 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/Ub10, and human NEDD8 E1/NEDD8/ATP·Mg8. 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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2866016&req=5

Figure 5: Side chains required for adenylation are dispensible for formation of the tetrahedral intermediate analogAmino acid contacts that contribute to E1 adenylation activity shown for a, E1/SUMO1/ATP·Mg9, 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/Ub10, and human NEDD8 E1/NEDD8/ATP·Mg8. 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.

Mentions: The E1/SUMO1-AMSN structure revealed that side chains contacting the Mg ion or ATP β-γ phosphates in E1/SUMO1/ATP·Mg were no longer involved in contacts to the adenylate analog (Fig. 5a,b). Furthermore, the E1~SUMO1-AVSN structure showed that many of these residues were fully displaced from the active site and replaced with residues from the Cys domain during thioester bond formation (Fig. 5c). These data suggest that residues required for adenylation should be dispensible for the thioester formation half reaction. The reverse should also hold true. To test this hypothesis, we mutated residues in the SUMO and Ub E1 and assayed these mutant E1s for their ability to form the adenylate, thioester adduct, or tetrahedral intermediate via cross-linking to the Ub/Ubl-AVSN adduct.


Active site remodelling accompanies thioester bond formation in the SUMO E1.

Olsen SK, Capili AD, Lu X, Tan DS, Lima CD - Nature (2010)

Side chains required for adenylation are dispensible for formation of the tetrahedral intermediate analogAmino acid contacts that contribute to E1 adenylation activity shown for a, E1/SUMO1/ATP·Mg9, 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/Ub10, and human NEDD8 E1/NEDD8/ATP·Mg8. 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2866016&req=5

Figure 5: Side chains required for adenylation are dispensible for formation of the tetrahedral intermediate analogAmino acid contacts that contribute to E1 adenylation activity shown for a, E1/SUMO1/ATP·Mg9, 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/Ub10, and human NEDD8 E1/NEDD8/ATP·Mg8. 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.
Mentions: The E1/SUMO1-AMSN structure revealed that side chains contacting the Mg ion or ATP β-γ phosphates in E1/SUMO1/ATP·Mg were no longer involved in contacts to the adenylate analog (Fig. 5a,b). Furthermore, the E1~SUMO1-AVSN structure showed that many of these residues were fully displaced from the active site and replaced with residues from the Cys domain during thioester bond formation (Fig. 5c). These data suggest that residues required for adenylation should be dispensible for the thioester formation half reaction. The reverse should also hold true. To test this hypothesis, we mutated residues in the SUMO and Ub E1 and assayed these mutant E1s for their ability to form the adenylate, thioester adduct, or tetrahedral intermediate via cross-linking to the Ub/Ubl-AVSN adduct.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Structural Biology, Sloan-Kettering Institute, New York, New York 10065, USA.

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.

Show MeSH