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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 (╬▓2m) whose early oligomers are still a matter of debate. An intermolecular interaction between D strands of facing ╬▓2m molecules was repeatedly observed, suggesting that such interface may be relevant for ╬▓2m dimerization. In this study, by mutating Ser33 to Cys, and assembling the disulphide-stabilized ╬▓2m homodimer (DimC33), such DD strand interface was locked. Although the isolated DimC33 display a stability similar to wt ╬▓2m 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 ╬▓2m aggregation. The present data provide new insight into ╬▓2m early steps of amyloid aggregation.

No MeSH data available.


Related in: MedlinePlus

Crystal structure of DimC33 and DD strand interface.(A) Ribbon model of one of the two chains in DimC33 (blue) superposed on the structure of wt ╬▓2m (green). ╬▓-strands are labeled according to the standard ╬▓2m nomenclature, Ser33/Cys33 are shown in sticks. (B) A zoomed view into the DD interface of four superposed crystal structures of DimC33_ThT (green, pdb code: 4RA3), hexameric structure of H13F ╬▓2m (magenta, pdb code: 3CIQ), DimC50 (cyan, pdb code: 3TM6) and DimC20 (yellow, pdb code: 3TLR), showing the main residues involved in the DD interface as sticks model. (C) Superposition of three ╬▓2m non-covalently assembled dimers built through the DD strand interface with DimC33: DimC33_ThT in green, hexameric structure of H13F ╬▓2m in magenta, DimC50 structure in cyan, and DimC20 structure in yellow. (D) Stereo view of the DD strand interface built by the facing ╬▓2m molecules as observed in the structure of DimC33_high. The main residues involved in the interface are shown as sticks.
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f1: Crystal structure of DimC33 and DD strand interface.(A) Ribbon model of one of the two chains in DimC33 (blue) superposed on the structure of wt ╬▓2m (green). ╬▓-strands are labeled according to the standard ╬▓2m nomenclature, Ser33/Cys33 are shown in sticks. (B) A zoomed view into the DD interface of four superposed crystal structures of DimC33_ThT (green, pdb code: 4RA3), hexameric structure of H13F ╬▓2m (magenta, pdb code: 3CIQ), DimC50 (cyan, pdb code: 3TM6) and DimC20 (yellow, pdb code: 3TLR), showing the main residues involved in the DD interface as sticks model. (C) Superposition of three ╬▓2m non-covalently assembled dimers built through the DD strand interface with DimC33: DimC33_ThT in green, hexameric structure of H13F ╬▓2m in magenta, DimC50 structure in cyan, and DimC20 structure in yellow. (D) Stereo view of the DD strand interface built by the facing ╬▓2m molecules as observed in the structure of DimC33_high. The main residues involved in the interface are shown as sticks.

Mentions: In order to elucidate the structural bases of oligomer formation we focused on the amyloidogenic protein beta-2 microglobulin (╬▓2m), whose native fold is structurally well characterized, and amyloid formation in vitro has been thoroughly described7. ╬▓2m is a 99-residue globular protein with a typical immunoglobulin-like fold, composed of seven ╬▓-strands arranged in two ╬▓-sheets, named ABDE and CFG, respectively, according to standard nomenclature of the composing ╬▓-strands (Fig. 1a); the two sheets are internally linked by a disulphide bond8. ╬▓2m is an aggregation-prone protein responsible for two types of amyloid-related diseases: the wild type (wt) protein is responsible for Dialysis-Related Amyloidosis (DRA)9, while a severe hereditary systemic amyloidosis is linked to the pathological ╬▓2m D76N mutant10. Physiologically, ╬▓2m is degraded in the kidneys; DRA patients typically suffer from kidney dysfunction that results in ╬▓2m accumulation in the serum following dialysis. Over the years, ╬▓2m aggregates in the skeletal joints, bones and muscles, resulting in bone fragility and movement impairment11.


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 structure of DimC33 and DD strand interface.(A) Ribbon model of one of the two chains in DimC33 (blue) superposed on the structure of wt ╬▓2m (green). ╬▓-strands are labeled according to the standard ╬▓2m nomenclature, Ser33/Cys33 are shown in sticks. (B) A zoomed view into the DD interface of four superposed crystal structures of DimC33_ThT (green, pdb code: 4RA3), hexameric structure of H13F ╬▓2m (magenta, pdb code: 3CIQ), DimC50 (cyan, pdb code: 3TM6) and DimC20 (yellow, pdb code: 3TLR), showing the main residues involved in the DD interface as sticks model. (C) Superposition of three ╬▓2m non-covalently assembled dimers built through the DD strand interface with DimC33: DimC33_ThT in green, hexameric structure of H13F ╬▓2m in magenta, DimC50 structure in cyan, and DimC20 structure in yellow. (D) Stereo view of the DD strand interface built by the facing ╬▓2m molecules as observed in the structure of DimC33_high. The main residues involved in the interface are shown as sticks.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4588566&req=5

f1: Crystal structure of DimC33 and DD strand interface.(A) Ribbon model of one of the two chains in DimC33 (blue) superposed on the structure of wt ╬▓2m (green). ╬▓-strands are labeled according to the standard ╬▓2m nomenclature, Ser33/Cys33 are shown in sticks. (B) A zoomed view into the DD interface of four superposed crystal structures of DimC33_ThT (green, pdb code: 4RA3), hexameric structure of H13F ╬▓2m (magenta, pdb code: 3CIQ), DimC50 (cyan, pdb code: 3TM6) and DimC20 (yellow, pdb code: 3TLR), showing the main residues involved in the DD interface as sticks model. (C) Superposition of three ╬▓2m non-covalently assembled dimers built through the DD strand interface with DimC33: DimC33_ThT in green, hexameric structure of H13F ╬▓2m in magenta, DimC50 structure in cyan, and DimC20 structure in yellow. (D) Stereo view of the DD strand interface built by the facing ╬▓2m molecules as observed in the structure of DimC33_high. The main residues involved in the interface are shown as sticks.
Mentions: In order to elucidate the structural bases of oligomer formation we focused on the amyloidogenic protein beta-2 microglobulin (╬▓2m), whose native fold is structurally well characterized, and amyloid formation in vitro has been thoroughly described7. ╬▓2m is a 99-residue globular protein with a typical immunoglobulin-like fold, composed of seven ╬▓-strands arranged in two ╬▓-sheets, named ABDE and CFG, respectively, according to standard nomenclature of the composing ╬▓-strands (Fig. 1a); the two sheets are internally linked by a disulphide bond8. ╬▓2m is an aggregation-prone protein responsible for two types of amyloid-related diseases: the wild type (wt) protein is responsible for Dialysis-Related Amyloidosis (DRA)9, while a severe hereditary systemic amyloidosis is linked to the pathological ╬▓2m D76N mutant10. Physiologically, ╬▓2m is degraded in the kidneys; DRA patients typically suffer from kidney dysfunction that results in ╬▓2m accumulation in the serum following dialysis. Over the years, ╬▓2m aggregates in the skeletal joints, bones and muscles, resulting in bone fragility and movement impairment11.

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 (╬▓2m) whose early oligomers are still a matter of debate. An intermolecular interaction between D strands of facing ╬▓2m molecules was repeatedly observed, suggesting that such interface may be relevant for ╬▓2m dimerization. In this study, by mutating Ser33 to Cys, and assembling the disulphide-stabilized ╬▓2m homodimer (DimC33), such DD strand interface was locked. Although the isolated DimC33 display a stability similar to wt ╬▓2m 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 ╬▓2m aggregation. The present data provide new insight into ╬▓2m early steps of amyloid aggregation.

No MeSH data available.


Related in: MedlinePlus