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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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Structural changes in SUMO E1 accompany transitions from adenylate to tetrahedral intermediatea, 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.
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Figure 2: Structural changes in SUMO E1 accompany transitions from adenylate to tetrahedral intermediatea, 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.

Mentions: A structure for E1/SUMO1-AMSN was determined by x-ray crystallography to 2.45 Å and refined to R/Rfree of 0.190/0.249 (Methods; Supplemental Table I). Electron density was evident for the covalent bond between SUMO1 and AMSN, thus, this adduct resembles the adenylate intermediate (Supplemental Fig. 1). This structure shares many overall similarities to structures of the SUMO E1 bound to SUMO1/ATP·Mg9, including the relative conformations of the UFD and Cys domains9 (Fig. 2). For discussion purposes, we term this the ‘open’ conformation. As expected, contacts between amino acid side chains that coordinate the magnesium ion and ATP β-γ phosphates, as observed in E1/SUMO1/ATP·Mg structures, were absent in the E1/SUMO1-AMSN structure. Another notable difference was that E1 amino acids 607–640 of UBA2 were observed in contacts with SUMO1 via a C-terminal SIM motif (aa 632–640; ELDDVIALD; Supplemental Fig. 2) although the functional significance of the SIM remains unclear because amino acids 550–640 are dispensible for human E1 activity in vitro and for yeast E1 function in vivo in S. cerevisiae9.


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

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

Structural changes in SUMO E1 accompany transitions from adenylate to tetrahedral intermediatea, 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.
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Related In: Results  -  Collection

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Figure 2: Structural changes in SUMO E1 accompany transitions from adenylate to tetrahedral intermediatea, 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.
Mentions: A structure for E1/SUMO1-AMSN was determined by x-ray crystallography to 2.45 Å and refined to R/Rfree of 0.190/0.249 (Methods; Supplemental Table I). Electron density was evident for the covalent bond between SUMO1 and AMSN, thus, this adduct resembles the adenylate intermediate (Supplemental Fig. 1). This structure shares many overall similarities to structures of the SUMO E1 bound to SUMO1/ATP·Mg9, including the relative conformations of the UFD and Cys domains9 (Fig. 2). For discussion purposes, we term this the ‘open’ conformation. As expected, contacts between amino acid side chains that coordinate the magnesium ion and ATP β-γ phosphates, as observed in E1/SUMO1/ATP·Mg structures, were absent in the E1/SUMO1-AMSN structure. Another notable difference was that E1 amino acids 607–640 of UBA2 were observed in contacts with SUMO1 via a C-terminal SIM motif (aa 632–640; ELDDVIALD; Supplemental Fig. 2) although the functional significance of the SIM remains unclear because amino acids 550–640 are dispensible for human E1 activity in vitro and for yeast E1 function in vivo in S. cerevisiae9.

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