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A Versatile Strategy for the Semisynthetic Production of Ser65 Phosphorylated Ubiquitin and Its Biochemical and Structural Characterisation.

Han C, Pao KC, Kazlauskaite A, Muqit MM, Virdee S - Chembiochem (2015)

Bottom Line: Unexpectedly, we observed disulfide bond formation between ubiquitin molecules, and hence a novel crystal form.The method outlined provides a direct approach to study the combinatorial effects of phosphorylation on ubiquitin function.Our analysis also suggests that disulfide engineering of ubiquitin could be a useful strategy for obtaining alternative crystal forms of ubiquitin species thereby facilitating structural validation.

View Article: PubMed Central - PubMed

Affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH (UK).

No MeSH data available.


Related in: MedlinePlus

X-ray crystal structure of UbC46-pSer65. A) Two disulfide-linked UbC46-pSer65 molecules in the asymmetric unit; pSer65 and Cys46 are depicted in ball and stick representation. B) Stick representation pSer65 residue; water molecules are depicted as red spheres. The 2 Fo−Fc electron density map (1.5 σ) is shown as a blue mesh. C) Hydrogen bonding network around pSer65. D) Disulfide bond between Cys46 of two Ub molecules in the asymmetric unit. Contiguous electron density is clearly evident between the γ-sulfur atoms of Cys46 (2 Fo−Fc electron density map at 1.5 σ).
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fig03: X-ray crystal structure of UbC46-pSer65. A) Two disulfide-linked UbC46-pSer65 molecules in the asymmetric unit; pSer65 and Cys46 are depicted in ball and stick representation. B) Stick representation pSer65 residue; water molecules are depicted as red spheres. The 2 Fo−Fc electron density map (1.5 σ) is shown as a blue mesh. C) Hydrogen bonding network around pSer65. D) Disulfide bond between Cys46 of two Ub molecules in the asymmetric unit. Contiguous electron density is clearly evident between the γ-sulfur atoms of Cys46 (2 Fo−Fc electron density map at 1.5 σ).

Mentions: In our structure there are two symmetry-related Ub molecules in the asymmetric unit (RMSD 0.081 Å) in the P31 space group (Figure 3 A); this differs from the previously solved Ub-pSer65 structure.10 The observed symmetry permitted assignment of the P3121 space group. However, the two Ub molecules form a covalently linked oxidised homodimer (apparent from the contiguous electron density), linked by the two cysteines arising from the Ala46Cys mutation (Figure 3 D). Consequently we assigned the P31 space group. Wild-type non-phosphorylated ubiquitin has previously been crystallised in the P3121 space group (PDB ID: 4HK2) but our structure is based on a different crystal form. Structural conservation between molecule 2 in our structure and the reported structure of phospho-ubiquitin was high (main chain RMSD 0.659 Å). The β-strand slippage that gives rise to a reported minor conformation species10 was not observed, thus indicating that our structure also represents the major conformation, despite having a different crystal form. The electron density for pSer65 was clearly visible, thus allowing unambiguous assignment of the pSer65 rotomer (Figure 3 B). Although the orientation of pSer65 largely mirrors that in the previous structure, the hydrogen bond between a phosphate oxygen and the backbone amide nitrogen of Gln62 was less significant (3.8 vs. 3.3 Å). In our structure, which is at a higher resolution, the γ-oxygen forms a more favourable H-bond with the amide nitrogen of Gln62 thereby forming an H-bond analogous to that in the wild-type ubiquitin structure10 (Figure 3 C). The orientation of the phosphate thus appears to be dictated by a solvent-mediated hydrogen-bond network involving Thr66. A water molecule bridges a phosphate oxygen of pSer65 and the main-chain amide nitrogen of Thr66. This water molecule also hydrogen bonds with a second water that forms a hydrogen bond with the hydroxyl group of Thr66. All the described hydrogen bonds have favourable geometries (2.5–3.2 Å; Figure 3 C). However, pSer65 does exist at a packing interface and is directed towards His68 of an adjacent Ub molecule. We cannot therefore exclude the possibility that the orientation is an artefact of crystallisation, or that this interaction is maintained in solution.


A Versatile Strategy for the Semisynthetic Production of Ser65 Phosphorylated Ubiquitin and Its Biochemical and Structural Characterisation.

Han C, Pao KC, Kazlauskaite A, Muqit MM, Virdee S - Chembiochem (2015)

X-ray crystal structure of UbC46-pSer65. A) Two disulfide-linked UbC46-pSer65 molecules in the asymmetric unit; pSer65 and Cys46 are depicted in ball and stick representation. B) Stick representation pSer65 residue; water molecules are depicted as red spheres. The 2 Fo−Fc electron density map (1.5 σ) is shown as a blue mesh. C) Hydrogen bonding network around pSer65. D) Disulfide bond between Cys46 of two Ub molecules in the asymmetric unit. Contiguous electron density is clearly evident between the γ-sulfur atoms of Cys46 (2 Fo−Fc electron density map at 1.5 σ).
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Related In: Results  -  Collection

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fig03: X-ray crystal structure of UbC46-pSer65. A) Two disulfide-linked UbC46-pSer65 molecules in the asymmetric unit; pSer65 and Cys46 are depicted in ball and stick representation. B) Stick representation pSer65 residue; water molecules are depicted as red spheres. The 2 Fo−Fc electron density map (1.5 σ) is shown as a blue mesh. C) Hydrogen bonding network around pSer65. D) Disulfide bond between Cys46 of two Ub molecules in the asymmetric unit. Contiguous electron density is clearly evident between the γ-sulfur atoms of Cys46 (2 Fo−Fc electron density map at 1.5 σ).
Mentions: In our structure there are two symmetry-related Ub molecules in the asymmetric unit (RMSD 0.081 Å) in the P31 space group (Figure 3 A); this differs from the previously solved Ub-pSer65 structure.10 The observed symmetry permitted assignment of the P3121 space group. However, the two Ub molecules form a covalently linked oxidised homodimer (apparent from the contiguous electron density), linked by the two cysteines arising from the Ala46Cys mutation (Figure 3 D). Consequently we assigned the P31 space group. Wild-type non-phosphorylated ubiquitin has previously been crystallised in the P3121 space group (PDB ID: 4HK2) but our structure is based on a different crystal form. Structural conservation between molecule 2 in our structure and the reported structure of phospho-ubiquitin was high (main chain RMSD 0.659 Å). The β-strand slippage that gives rise to a reported minor conformation species10 was not observed, thus indicating that our structure also represents the major conformation, despite having a different crystal form. The electron density for pSer65 was clearly visible, thus allowing unambiguous assignment of the pSer65 rotomer (Figure 3 B). Although the orientation of pSer65 largely mirrors that in the previous structure, the hydrogen bond between a phosphate oxygen and the backbone amide nitrogen of Gln62 was less significant (3.8 vs. 3.3 Å). In our structure, which is at a higher resolution, the γ-oxygen forms a more favourable H-bond with the amide nitrogen of Gln62 thereby forming an H-bond analogous to that in the wild-type ubiquitin structure10 (Figure 3 C). The orientation of the phosphate thus appears to be dictated by a solvent-mediated hydrogen-bond network involving Thr66. A water molecule bridges a phosphate oxygen of pSer65 and the main-chain amide nitrogen of Thr66. This water molecule also hydrogen bonds with a second water that forms a hydrogen bond with the hydroxyl group of Thr66. All the described hydrogen bonds have favourable geometries (2.5–3.2 Å; Figure 3 C). However, pSer65 does exist at a packing interface and is directed towards His68 of an adjacent Ub molecule. We cannot therefore exclude the possibility that the orientation is an artefact of crystallisation, or that this interaction is maintained in solution.

Bottom Line: Unexpectedly, we observed disulfide bond formation between ubiquitin molecules, and hence a novel crystal form.The method outlined provides a direct approach to study the combinatorial effects of phosphorylation on ubiquitin function.Our analysis also suggests that disulfide engineering of ubiquitin could be a useful strategy for obtaining alternative crystal forms of ubiquitin species thereby facilitating structural validation.

View Article: PubMed Central - PubMed

Affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH (UK).

No MeSH data available.


Related in: MedlinePlus