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Ligand binding and aggregation of pathogenic SOD1.

Wright GS, Antonyuk SV, Kershaw NM, Strange RW, Samar Hasnain S - Nat Commun (2013)

Bottom Line: Superoxide dismutase-1 mutations decrease protein stability and promote aggregation.We find both compounds interact with superoxide dismutase-1 at a key region identified at the core of the superoxide dismutase-1 fibrillar aggregates, β-barrel loop II-strand 3, rather than the proposed dimer interface site.This illustrates the need for direct structural observations when developing compounds for protein-targeted therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Liverpool L69 7ZB, UK.

ABSTRACT
Mutations in the gene encoding Cu/Zn superoxide dismutase-1 cause amyotrophic lateral sclerosis. Superoxide dismutase-1 mutations decrease protein stability and promote aggregation. The mutant monomer is thought to be an intermediate in the pathway from the superoxide dismutase-1 dimer to aggregate. Here we find that the monomeric copper-apo, zinc-holo protein is structurally perturbed and the apo-protein aggregates without reattainment of the monomer-dimer equilibrium. Intervention to stabilize the superoxide dismutase-1 dimer and inhibit aggregation is regarded as a potential therapeutic strategy. We describe protein-ligand interactions for two compounds, Isoproterenol and 5-fluorouridine, highlighted as superoxide dismutase-1 stabilizers. We find both compounds interact with superoxide dismutase-1 at a key region identified at the core of the superoxide dismutase-1 fibrillar aggregates, β-barrel loop II-strand 3, rather than the proposed dimer interface site. This illustrates the need for direct structural observations when developing compounds for protein-targeted therapeutics.

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5-FUrd and Isoproterenol bound to I113T SOD1.(a) 5-FUrd with respect to the SOD1 dimer (4A7S) in grey and light grey; strands 2, 3 and 6 of the SOD1 β-barrel are coloured yellow, purple and green, respectively. (b) 2Fo-Fc electron density map of 5-FUrd contoured at 1σ at the Trp32-binding site with the SOD1 surface coloured by amino acid hydrophobicity. (c) Hydrogen bonding between I113T SOD1 and 5-FUrd shown with black dashed line. (d) Isoproterenol with respect to the SOD1 dimer (4A7T) coloured as in a. (e) 2Fo-Fc electron density map of Isoproterenol contoured at 1σ at the Trp32-binding site coloured as above. Little electron density is seen around the Isoproterenol tail; however, atoms in the catechol have an average B factor of 24.9 Å2 and compare well with those of the Glu21 and Glu100 side chain carboxylates (average 23.0 Å2), indicating similar occupancy. (f) Hydrogen bonding between I113T SOD1 and Isoproterenol shown with black dashed line. Omit maps of both 5-FUrd and Isoproterenol bound to I113T SOD1 can be found in Supplementary Fig. S10.
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f5: 5-FUrd and Isoproterenol bound to I113T SOD1.(a) 5-FUrd with respect to the SOD1 dimer (4A7S) in grey and light grey; strands 2, 3 and 6 of the SOD1 β-barrel are coloured yellow, purple and green, respectively. (b) 2Fo-Fc electron density map of 5-FUrd contoured at 1σ at the Trp32-binding site with the SOD1 surface coloured by amino acid hydrophobicity. (c) Hydrogen bonding between I113T SOD1 and 5-FUrd shown with black dashed line. (d) Isoproterenol with respect to the SOD1 dimer (4A7T) coloured as in a. (e) 2Fo-Fc electron density map of Isoproterenol contoured at 1σ at the Trp32-binding site coloured as above. Little electron density is seen around the Isoproterenol tail; however, atoms in the catechol have an average B factor of 24.9 Å2 and compare well with those of the Glu21 and Glu100 side chain carboxylates (average 23.0 Å2), indicating similar occupancy. (f) Hydrogen bonding between I113T SOD1 and Isoproterenol shown with black dashed line. Omit maps of both 5-FUrd and Isoproterenol bound to I113T SOD1 can be found in Supplementary Fig. S10.

Mentions: 5-FUrd is a fluorinated analogue of uridine monophosphate (UMP), but lacks the phosphate group at the 5′-position. On the basis of our previous investigations35, it would be expected to bind at the UMP-binding site between the zinc and electrostatic loops; however, 5-FUrd binding at Trp32 is the only observable interaction (4A7S, Fig. 5a–c, Supplementary Figs S6 and S10, and Table 1). The fluorouracil group engages in aromatic stacking with Trp32, and the side chain of Ser98 acts as a hydrogen bond donor to the fluorouracil carbonyl adjacent to fluorine. In this instance, the indole of Trp32 displays two conformations. The ribose group shows significantly lower occupancy, as it is free to rotate about the anomeric C–N bond and projects away from the binding site into solvent. Although the reason for the switch in binding site between UMP and 5-FUrd is not fully clear, it is reasonable to suggest that although Trp32 is the preferred site, the highly polar nature of the phosphate present in UMP confers a destabilizing effect that precludes its binding there.


Ligand binding and aggregation of pathogenic SOD1.

Wright GS, Antonyuk SV, Kershaw NM, Strange RW, Samar Hasnain S - Nat Commun (2013)

5-FUrd and Isoproterenol bound to I113T SOD1.(a) 5-FUrd with respect to the SOD1 dimer (4A7S) in grey and light grey; strands 2, 3 and 6 of the SOD1 β-barrel are coloured yellow, purple and green, respectively. (b) 2Fo-Fc electron density map of 5-FUrd contoured at 1σ at the Trp32-binding site with the SOD1 surface coloured by amino acid hydrophobicity. (c) Hydrogen bonding between I113T SOD1 and 5-FUrd shown with black dashed line. (d) Isoproterenol with respect to the SOD1 dimer (4A7T) coloured as in a. (e) 2Fo-Fc electron density map of Isoproterenol contoured at 1σ at the Trp32-binding site coloured as above. Little electron density is seen around the Isoproterenol tail; however, atoms in the catechol have an average B factor of 24.9 Å2 and compare well with those of the Glu21 and Glu100 side chain carboxylates (average 23.0 Å2), indicating similar occupancy. (f) Hydrogen bonding between I113T SOD1 and Isoproterenol shown with black dashed line. Omit maps of both 5-FUrd and Isoproterenol bound to I113T SOD1 can be found in Supplementary Fig. S10.
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f5: 5-FUrd and Isoproterenol bound to I113T SOD1.(a) 5-FUrd with respect to the SOD1 dimer (4A7S) in grey and light grey; strands 2, 3 and 6 of the SOD1 β-barrel are coloured yellow, purple and green, respectively. (b) 2Fo-Fc electron density map of 5-FUrd contoured at 1σ at the Trp32-binding site with the SOD1 surface coloured by amino acid hydrophobicity. (c) Hydrogen bonding between I113T SOD1 and 5-FUrd shown with black dashed line. (d) Isoproterenol with respect to the SOD1 dimer (4A7T) coloured as in a. (e) 2Fo-Fc electron density map of Isoproterenol contoured at 1σ at the Trp32-binding site coloured as above. Little electron density is seen around the Isoproterenol tail; however, atoms in the catechol have an average B factor of 24.9 Å2 and compare well with those of the Glu21 and Glu100 side chain carboxylates (average 23.0 Å2), indicating similar occupancy. (f) Hydrogen bonding between I113T SOD1 and Isoproterenol shown with black dashed line. Omit maps of both 5-FUrd and Isoproterenol bound to I113T SOD1 can be found in Supplementary Fig. S10.
Mentions: 5-FUrd is a fluorinated analogue of uridine monophosphate (UMP), but lacks the phosphate group at the 5′-position. On the basis of our previous investigations35, it would be expected to bind at the UMP-binding site between the zinc and electrostatic loops; however, 5-FUrd binding at Trp32 is the only observable interaction (4A7S, Fig. 5a–c, Supplementary Figs S6 and S10, and Table 1). The fluorouracil group engages in aromatic stacking with Trp32, and the side chain of Ser98 acts as a hydrogen bond donor to the fluorouracil carbonyl adjacent to fluorine. In this instance, the indole of Trp32 displays two conformations. The ribose group shows significantly lower occupancy, as it is free to rotate about the anomeric C–N bond and projects away from the binding site into solvent. Although the reason for the switch in binding site between UMP and 5-FUrd is not fully clear, it is reasonable to suggest that although Trp32 is the preferred site, the highly polar nature of the phosphate present in UMP confers a destabilizing effect that precludes its binding there.

Bottom Line: Superoxide dismutase-1 mutations decrease protein stability and promote aggregation.We find both compounds interact with superoxide dismutase-1 at a key region identified at the core of the superoxide dismutase-1 fibrillar aggregates, β-barrel loop II-strand 3, rather than the proposed dimer interface site.This illustrates the need for direct structural observations when developing compounds for protein-targeted therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Liverpool L69 7ZB, UK.

ABSTRACT
Mutations in the gene encoding Cu/Zn superoxide dismutase-1 cause amyotrophic lateral sclerosis. Superoxide dismutase-1 mutations decrease protein stability and promote aggregation. The mutant monomer is thought to be an intermediate in the pathway from the superoxide dismutase-1 dimer to aggregate. Here we find that the monomeric copper-apo, zinc-holo protein is structurally perturbed and the apo-protein aggregates without reattainment of the monomer-dimer equilibrium. Intervention to stabilize the superoxide dismutase-1 dimer and inhibit aggregation is regarded as a potential therapeutic strategy. We describe protein-ligand interactions for two compounds, Isoproterenol and 5-fluorouridine, highlighted as superoxide dismutase-1 stabilizers. We find both compounds interact with superoxide dismutase-1 at a key region identified at the core of the superoxide dismutase-1 fibrillar aggregates, β-barrel loop II-strand 3, rather than the proposed dimer interface site. This illustrates the need for direct structural observations when developing compounds for protein-targeted therapeutics.

Show MeSH
Related in: MedlinePlus