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A covalent homodimer probing early oligomers along amyloid aggregation.

Halabelian L, Relini A, Barbiroli A, Penco A, Bolognesi M, Ricagno S - Sci Rep (2015)

Bottom Line: In this study, by mutating Ser33 to Cys, and assembling the disulphide-stabilized β2m homodimer (DimC33), such DD strand interface was locked.Three distinct crystal structures of DimC33 suggest that dimerization through the DD interface is instrumental for enhancing DimC33 aggregation propensity.Furthermore, the crystal structure of DimC33 in complex with the amyloid-specific dye Thioflavin-T pinpoints a second interface, which likely participates in the first steps of β2m aggregation.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Bioscienze, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy.

ABSTRACT
Early oligomers are crucial in amyloid aggregation; however, due to their transient nature they are among the least structurally characterized species. We focused on the amyloidogenic protein beta2-microglobulin (β2m) whose early oligomers are still a matter of debate. An intermolecular interaction between D strands of facing β2m molecules was repeatedly observed, suggesting that such interface may be relevant for β2m dimerization. In this study, by mutating Ser33 to Cys, and assembling the disulphide-stabilized β2m homodimer (DimC33), such DD strand interface was locked. Although the isolated DimC33 display a stability similar to wt β2m under native conditions, it shows enhanced amyloid aggregation propensity. Three distinct crystal structures of DimC33 suggest that dimerization through the DD interface is instrumental for enhancing DimC33 aggregation propensity. Furthermore, the crystal structure of DimC33 in complex with the amyloid-specific dye Thioflavin-T pinpoints a second interface, which likely participates in the first steps of β2m aggregation. The present data provide new insight into β2m early steps of amyloid aggregation.

No MeSH data available.


Related in: MedlinePlus

Crystal structures of DimC33_ThT and DimC33_low.(A) Cartoon representation of DimC33 assembly in the crystal structure of DimC33_ThT. The three DimC33 units are colored in cyan, green and magenta. Two ThT binding sites are visible: on the left side the 1 ThT site, on the right the 4 ThT site (the ThT molecules are colored in red). (B,C) A zoomed representation into the 1 ThT site (B) and into the 4 ThT site (C), showing ThT molecules sandwiched between the ABDE sheets of two adjacent DimC33 moieties, 2Fo-Fc omit electron density map at 1.5σ is clipped around ThT molecules. (D,E) Ribbon representation of the ABDE interface as observed (D) in the DimC33_low structure (pdb code: 4R9H); and (E) in the hexameric structure of H13F β2m (pdb code: 3CIQ).
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f4: Crystal structures of DimC33_ThT and DimC33_low.(A) Cartoon representation of DimC33 assembly in the crystal structure of DimC33_ThT. The three DimC33 units are colored in cyan, green and magenta. Two ThT binding sites are visible: on the left side the 1 ThT site, on the right the 4 ThT site (the ThT molecules are colored in red). (B,C) A zoomed representation into the 1 ThT site (B) and into the 4 ThT site (C), showing ThT molecules sandwiched between the ABDE sheets of two adjacent DimC33 moieties, 2Fo-Fc omit electron density map at 1.5σ is clipped around ThT molecules. (D,E) Ribbon representation of the ABDE interface as observed (D) in the DimC33_low structure (pdb code: 4R9H); and (E) in the hexameric structure of H13F β2m (pdb code: 3CIQ).

Mentions: DimC33 was also crystallized in the presence of 5 mM ThT. Intriguingly, the crystallographic analysis showed that five ThT molecules are hosted in the crystal asymmetric unit (AU), which contains one DimC33 moiety and two halves of a second one (i.e. two additional and independent β2m chains). As a result of crystal packing, β2m molecules belonging to independent DimC33 units interact via their ABDE β-sheets (Fig. 4a); however, two distinct ABDE interfaces can be distinguished. In one of these, four ThT molecules are wedged between the ABDE sheets of two facing β2m molecules (4 ThT site); in the other, only one ThT molecule is sandwiched between two facing ABDE sheets (1 ThT site) (Fig. 4a). In either case, binding of the ThT molecules does not induce any conformational adjustments in the β2m fold, or in the DimC33 assembly. At the 1 ThT site, the ThT molecule is sandwiched between Tyr10 residues from two facing β2m molecules; moreover, Tyr26 and Pro14 from both molecules establish van der Waals interactions with ThT (Fig. 4b). At the 4 ThT site, the four ThT molecules are stacked on each other, and in stacking contacts with Tyr10 and Tyr63 of the β2m molecules defining the binding site. Tyr26 from both β2m molecules also help accommodate the ThT hydrophobic rings (Fig. 4c).


A covalent homodimer probing early oligomers along amyloid aggregation.

Halabelian L, Relini A, Barbiroli A, Penco A, Bolognesi M, Ricagno S - Sci Rep (2015)

Crystal structures of DimC33_ThT and DimC33_low.(A) Cartoon representation of DimC33 assembly in the crystal structure of DimC33_ThT. The three DimC33 units are colored in cyan, green and magenta. Two ThT binding sites are visible: on the left side the 1 ThT site, on the right the 4 ThT site (the ThT molecules are colored in red). (B,C) A zoomed representation into the 1 ThT site (B) and into the 4 ThT site (C), showing ThT molecules sandwiched between the ABDE sheets of two adjacent DimC33 moieties, 2Fo-Fc omit electron density map at 1.5σ is clipped around ThT molecules. (D,E) Ribbon representation of the ABDE interface as observed (D) in the DimC33_low structure (pdb code: 4R9H); and (E) in the hexameric structure of H13F β2m (pdb code: 3CIQ).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588566&req=5

f4: Crystal structures of DimC33_ThT and DimC33_low.(A) Cartoon representation of DimC33 assembly in the crystal structure of DimC33_ThT. The three DimC33 units are colored in cyan, green and magenta. Two ThT binding sites are visible: on the left side the 1 ThT site, on the right the 4 ThT site (the ThT molecules are colored in red). (B,C) A zoomed representation into the 1 ThT site (B) and into the 4 ThT site (C), showing ThT molecules sandwiched between the ABDE sheets of two adjacent DimC33 moieties, 2Fo-Fc omit electron density map at 1.5σ is clipped around ThT molecules. (D,E) Ribbon representation of the ABDE interface as observed (D) in the DimC33_low structure (pdb code: 4R9H); and (E) in the hexameric structure of H13F β2m (pdb code: 3CIQ).
Mentions: DimC33 was also crystallized in the presence of 5 mM ThT. Intriguingly, the crystallographic analysis showed that five ThT molecules are hosted in the crystal asymmetric unit (AU), which contains one DimC33 moiety and two halves of a second one (i.e. two additional and independent β2m chains). As a result of crystal packing, β2m molecules belonging to independent DimC33 units interact via their ABDE β-sheets (Fig. 4a); however, two distinct ABDE interfaces can be distinguished. In one of these, four ThT molecules are wedged between the ABDE sheets of two facing β2m molecules (4 ThT site); in the other, only one ThT molecule is sandwiched between two facing ABDE sheets (1 ThT site) (Fig. 4a). In either case, binding of the ThT molecules does not induce any conformational adjustments in the β2m fold, or in the DimC33 assembly. At the 1 ThT site, the ThT molecule is sandwiched between Tyr10 residues from two facing β2m molecules; moreover, Tyr26 and Pro14 from both molecules establish van der Waals interactions with ThT (Fig. 4b). At the 4 ThT site, the four ThT molecules are stacked on each other, and in stacking contacts with Tyr10 and Tyr63 of the β2m molecules defining the binding site. Tyr26 from both β2m molecules also help accommodate the ThT hydrophobic rings (Fig. 4c).

Bottom Line: In this study, by mutating Ser33 to Cys, and assembling the disulphide-stabilized β2m homodimer (DimC33), such DD strand interface was locked.Three distinct crystal structures of DimC33 suggest that dimerization through the DD interface is instrumental for enhancing DimC33 aggregation propensity.Furthermore, the crystal structure of DimC33 in complex with the amyloid-specific dye Thioflavin-T pinpoints a second interface, which likely participates in the first steps of β2m aggregation.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Bioscienze, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy.

ABSTRACT
Early oligomers are crucial in amyloid aggregation; however, due to their transient nature they are among the least structurally characterized species. We focused on the amyloidogenic protein beta2-microglobulin (β2m) whose early oligomers are still a matter of debate. An intermolecular interaction between D strands of facing β2m molecules was repeatedly observed, suggesting that such interface may be relevant for β2m dimerization. In this study, by mutating Ser33 to Cys, and assembling the disulphide-stabilized β2m homodimer (DimC33), such DD strand interface was locked. Although the isolated DimC33 display a stability similar to wt β2m under native conditions, it shows enhanced amyloid aggregation propensity. Three distinct crystal structures of DimC33 suggest that dimerization through the DD interface is instrumental for enhancing DimC33 aggregation propensity. Furthermore, the crystal structure of DimC33 in complex with the amyloid-specific dye Thioflavin-T pinpoints a second interface, which likely participates in the first steps of β2m aggregation. The present data provide new insight into β2m early steps of amyloid aggregation.

No MeSH data available.


Related in: MedlinePlus