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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.

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Conformational changes within the Cys domainThe Cys domains of a, SUMO E1/SUMO1-AMSN, b, SUMO E1~SUMO1-AVSN, c, Ub E1/Ub complex10 and d, NEDD8 E1~NEDD8(t)/NEDD8(a)/Ubc12/ATP11 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
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Figure 3: Conformational changes within the Cys domainThe Cys domains of a, SUMO E1/SUMO1-AMSN, b, SUMO E1~SUMO1-AVSN, c, Ub E1/Ub complex10 and d, NEDD8 E1~NEDD8(t)/NEDD8(a)/Ubc12/ATP11 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

Mentions: Rotation of the Cys domain is accompanied by a 125 degree change in the path of the cross-over loop and an orthogonal change in path for the re-entry loop. Analysis of the cross-over loop shows its trajectory is altered over several residues (aa 164–168) while changes in the re-entry loop are localized to Gly381 and Asn382 (Fig. 3; Supplemental Fig. 5). Furthermore, several elements that were structured in the Cys domain in the open conformation become disordered in the closed conformation, including helices g3 and g4 and the loop joining them, the loop between H10 and H11 (g7 is disordered), and the loop between H11 and H12 (Fig. 2c). Perhaps most relevant for thioester bond formation, the H6 helix that contains the active site cysteine (aa 172–178) melts in the closed conformation and the catalytic cysteine is now observed adjacent to the catalytic machinery of the adenylation pocket and the SUMO adenylate (Fig. 2 and 3).


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

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

Conformational changes within the Cys domainThe Cys domains of a, SUMO E1/SUMO1-AMSN, b, SUMO E1~SUMO1-AVSN, c, Ub E1/Ub complex10 and d, NEDD8 E1~NEDD8(t)/NEDD8(a)/Ubc12/ATP11 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
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Related In: Results  -  Collection

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Figure 3: Conformational changes within the Cys domainThe Cys domains of a, SUMO E1/SUMO1-AMSN, b, SUMO E1~SUMO1-AVSN, c, Ub E1/Ub complex10 and d, NEDD8 E1~NEDD8(t)/NEDD8(a)/Ubc12/ATP11 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
Mentions: Rotation of the Cys domain is accompanied by a 125 degree change in the path of the cross-over loop and an orthogonal change in path for the re-entry loop. Analysis of the cross-over loop shows its trajectory is altered over several residues (aa 164–168) while changes in the re-entry loop are localized to Gly381 and Asn382 (Fig. 3; Supplemental Fig. 5). Furthermore, several elements that were structured in the Cys domain in the open conformation become disordered in the closed conformation, including helices g3 and g4 and the loop joining them, the loop between H10 and H11 (g7 is disordered), and the loop between H11 and H12 (Fig. 2c). Perhaps most relevant for thioester bond formation, the H6 helix that contains the active site cysteine (aa 172–178) melts in the closed conformation and the catalytic cysteine is now observed adjacent to the catalytic machinery of the adenylation pocket and the SUMO adenylate (Fig. 2 and 3).

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