PDBsum entry 2eke

Ligase/protein binding

PDB id: 2eke

Contents

Protein chains

154 a.a. *

91 a.a. *

Waters ×375

* Residue conservation analysis

PDB id:

2eke

Name:

Ligase/protein binding

Title:

Structure of a sumo-binding-motif mimic bound to smt3p-ubc9p: conservation of a noncovalent ubiquitin-like protein-e2 complex as a platform for selective interactions within a sumo pathway

Structure:

Sumo-conjugating enzyme ubc9. Chain: a, b. Synonym: ubiquitin-conjugating enzyme e2-18 kda, ubiquitin-protein ligase, ubiquitin carrier protein 9. Engineered: yes. Ubiquitin-like protein smt3. Chain: c, d. Engineered: yes. Mutation: yes

Source:

Saccharomyces cerevisiae. Baker's yeast. Organism_taxid: 4932. Gene: ubc9. Expressed in: escherichia coli. Expression_system_taxid: 562. Gene: smt3.

Resolution:

1.90Å R-factor: 0.225 R-free: 0.250

Authors:

D.M.Duda,B.A.Schulman

Key ref:

D.M.Duda et al. (2007). Structure of a SUMO-binding-motif Mimic Bound to Smt3p-Ubc9p: Conservation of a Non-covalent Ubiquitin-like Protein-E2 Complex as a Platform for Selective Interactions within a SUMO Pathway. J Mol Biol, 369, 619-630. PubMed id: 17475278 DOI: 10.1016/j.jmb.2007.04.007

Date:

23-Mar-07 Release date: 29-May-07

Protein chains

P50623  (UBC9_YEAST) -  SUMO-conjugating enzyme UBC9 from Saccharomyces cerevisiae (strain ATCC 204508 / S288c)

Seq: 157 a.a.

Struc: 154 a.a.

Protein chains

Q12306  (SMT3_YEAST) -  Ubiquitin-like protein SMT3 from Saccharomyces cerevisiae (strain ATCC 204508 / S288c)

Seq: 101 a.a.

Struc: 91 a.a.*

* PDB and UniProt seqs differ at 8 residue positions (black crosses)

Enzyme reactions

• Enzyme class: Chains A, B: E.C.2.3.2.- - ?????

DOI no: 10.1016/j.jmb.2007.04.007

J Mol Biol 369:619-630 (2007)

PubMed id: 17475278

Structure of a SUMO-binding-motif Mimic Bound to Smt3p-Ubc9p: Conservation of a Non-covalent Ubiquitin-like Protein-E2 Complex as a Platform for Selective Interactions within a SUMO Pathway.

D.M.Duda, R.C.van Waardenburg, L.A.Borg, S.McGarity, A.Nourse, M.B.Waddell, M.A.Bjornsti, B.A.Schulman.

ABSTRACT

The SUMO ubiquitin-like proteins play regulatory roles in cell division, transcription, DNA repair, and protein subcellular localization. Paralleling other ubiquitin-like proteins, SUMO proteins are proteolytically processed to maturity, conjugated to targets by E1-E2-E3 cascades, and subsequently recognized by specific downstream effectors containing a SUMO-binding motif (SBM). SUMO and its E2 from the budding yeast Saccharomyces cerevisiae, Smt3p and Ubc9p, are encoded by essential genes. Here we describe the 1.9 A resolution crystal structure of a non-covalent Smt3p-Ubc9p complex. Unexpectedly, a heterologous portion of the crystallized complex derived from the expression construct mimics an SBM, and binds Smt3p in a manner resembling SBM binding to human SUMO family members. In the complex, Smt3p binds a surface distal from Ubc9's catalytic cysteine. The structure implies that a single molecule of Smt3p cannot bind concurrently to both the non-covalent binding site and the catalytic cysteine of a single Ubc9p molecule. However, formation of higher-order complexes can occur, where a single Smt3p covalently linked to one Ubc9p's catalytic cysteine also binds non-covalently to another molecule of Ubc9p. Comparison with other structures from the SUMO pathway suggests that formation of the non-covalent Smt3p-Ubc9p complex occurs mutually exclusively with many other Smt3p and Ubc9p interactions in the conjugation cascade. By contrast, high-resolution insights into how Smt3p-Ubc9p can also interact with downstream recognition machineries come from contacts with the SBM mimic. Interestingly, the overall architecture of the Smt3p-Ubc9p complex is strikingly similar to recent structures from the ubiquitin pathway. The results imply that non-covalent ubiquitin-like protein-E2 complexes are conserved platforms, which function as parts of larger assemblies involved in many protein post-translational regulatory pathways.

Selected figure(s)

Figure 1.
Figure 1. Structure of a Smt3p–Ubc9p complex. (a) Sequence alignment of Smt3p from S. cerevisiae and human SUMO-1, SUMO-2, and SUMO-3, with secondary structure elements indicated above, and residues identical to Smt3p highlighted in yellow. Residues in Smt3p that contact Ubc9p are denoted with cyan circles. (b) Sequence alignment of Ubc9p from S. cerevisiae with human, Arabidopsis thaliana, Schizosaccharomyces pombe, and Xenopus laevis Ubc9. Secondary structure elements are indicated above, and residues identical to S. cerevisiae Ubc9p are highlighted in cyan. Residues in Ubc9p that contact Smt3p are denoted with yellow circles. (c) Overall structure of the complex, with Smt3p in yellow and Ubc9p in cyan. Secondary structures are shown overlaid with a semi-transparent surface. Ubc9p's catalytic cysteine (Cys 93) is represented by a green sphere. The crystals form in C2 with a = 120.89 Å, b = 84.58 Å, c = 80.14 Å, and β = 124.31, and two complexes per asymmetric unit. Data were collected using the mail-in program at the 22-BM (SER-CAT, Southeast Regional Collaborative Access Team) beamline at the Advanced Photon Source. Reflection data were indexed, integrated and scaled using HKL2000.^50 Initial phases were obtained by molecular replacement using the coordinates of Ubc9p^37 as a search model in CNS.^51 Electron density for Smt3p was readily visible in initial maps. The model was built manually using O,^52 using the previous structures of Smt3p as guides,^27.^ and ^36. and refined using CNS alternating with cycles of rebuilding relying on simulated-annealing omit and composite omit maps.^51 The structure was refined from 50.0 Å to 1.9 Å. The final model has excellent geometry, with no Ramachandran outliers in disallowed regions. Statistics from data collection and refinement are given in Table 1. This and other Figures representing structures were generated with Pymol.^53 (d) Close-up view of the interface between Smt3p and Ubc9p, oriented as in (c). Smt3p is shown in yellow, with specific residues labeled in black. Ubc9p is shown in cyan, with specific residues labeled cyan. Oxygen atoms are colored red, and nitrogen atoms blue. Hydrogen bonds and salt-bridges are represented with dashes. (e) ubc9 bearing mutations in interface residues with Smt3p do not complement ubc9Δ cells. In a plasmid shuffle assay, ubc9Δ (PTY30 or PTY34) cells, transformed with a LEU2 vector containing an intron-less cDNA sequence for wild-type UBC9 or the indicated mutant allele expressed from the UBC9 promoter, were spotted onto selective medium supplemented with dextrose, as described.^[37]^ and ^54. Individual transformants were successively replica plated onto 5-FOA (US Biological) plates to cure cells of YCpUBC9·U and incubated at 26 °C, 30 °C, and 36 °C.^37 Similar results were obtained at all three temperatures, and representative results are shown for the experiment at 30 °C. Plasmids were isolated from viable YCpUBC9·U-cured strains and sequenced to verify the identity of the ubc9 allele. (f) ubc9 bearing mutations in interface residues with Smt3p do not restore resistance to genotoxic stress. The ubc9-10 mutant strain of S. cerevisiae, bearing a ubc9P123L conditional mutation, was shown previously to display increased sensitivity to a wide range of DNA damaging agents including hydroxyurea (HU) and methyl methane sulfonate (MMS).^54 Exponentially growing cultures of ubc9-10 cells (A[595] = 0.3) transformed with the indicated plasmids were serially tenfold diluted and 5 μl aliquots were spotted onto the appropriate selective media supplemented with dextrose and incubated at 36 °C. To assay cell sensitivity to HU or MMS, plates were supplemented with 5 mg/ml HU or 0.0125% MMS, respectively.^37

Figure 3.
Figure 3. Structure of a SUMO binding motif mimic bound to Smt3p–Ubc9p: structural conservation of non-covalent ubiquitin-like protein–E2 complexes as platforms for interactions within Ubl pathways. (a) Two complexes as in the crystal, where Smt3p (yellow)–Ubc9p (cyan) and Smt3p' (magenta)–Ubc9p' (blue) are related by crystallographic C2 symmetry, showing the uncleaved thrombin-site linker sequence from the adjacent, crystallographic symmetry-related Smt3p' packing in a groove in Smt3p. The linker from Smt3p extends five additional residues beyond the *. (b) Structure of a SUMO-binding motif mimic bound to Smt3p–Ubc9p. A portion of the linker from the adjacent, crystallographic symmetry-related Smt3p' is shown in magenta sticks, as it interacts with the Smt3p (yellow)–Ubc9p (cyan) complex. The N and C-terminal regions of the displayed portion of the linker are labeled to indicate directionality of the peptide-like interaction with Smt3p. Oxygen atoms are colored red, nitrogen atoms blue, and Ubc9p's catalytic Cys93 is marked with a green sphere. Hydrogen bonds and salt-bridges are represented with dashes. (c) Structures of human SUMO-1 (yellow) in complex with the SBM regions (magenta) from Nup358/RanBP2,^26 thymine DNA glycosylase,^33 and PIASX^21 are shown from left to right, oriented after superposition of SUMO-1 with Smt3p as in (b). The N and C-terminal regions of the displayed peptide-like regions of Nup358/RanBP2 and thymine DNA glycosylase, and the peptide from PIASX, to indicate directionality of the polypeptide interaction with SUMO-1. Oxygen atoms are colored red, and nitrogen atoms blue. Hydrogen bonds and salt-bridges are represented with dashes. (d) Close-up view of interactions between Smt3p and the uncleaved thrombin-site linker sequence from the adjacent, crystallographic symmetry-related Smt3p'. Smt3p is shown in yellow with black labels, and the SBM mimicking linker in magenta. Oxygen atoms are colored red, and nitrogen atoms blue. Hydrogen bonds are represented with dashes. (e) Structure-based sequence alignments of the SUMO/Smt3p-binding sequences from Nup358/RanBP2, thymine DNA glycosylase (TDG), PIASX, and the SUMO-binding motif mimic from the uncleaved thrombin-site linker sequence upstream of Smt3p' residues used for crystallization (linker). Residues mediating key hydrophobic interactions are boxed. (f) Structure of the human ubiquitin (yellow)–UbcH5 (cyan) non-covalent complex, showing non-covalent interactions between ubiquitin and a ubiquitin E2 involved in BRCA1-mediated polyubiquitin chain assembly.^40 This complex is oriented with UbcH5 in the same position as Ubc9p in (b), after superposition of UbcH5 from the complex with ubiquitin onto the structure of Ubc9p from Smt3p–Ubc9p. (g) Structure of the human ubiquitin (yellow)–MMS2 (cyan) complex, showing non-covalent interactions between ubiquitin and a non-catalytic E2 variant involved in Lys63-linked polyubiquitin chain assembly.^40 This complex is oriented with MMS2 in the same position as Ubc9p in (b), after superposition of MMS2 from the complex with ubiquitin onto the structure of Ubc9p from Smt3p–Ubc9p.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2007, 369, 619-630) copyright 2007.