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Structural insights into the pre-amyloid tetramer of ?-2-microglobulin from covalent labeling and mass spectrometry.

ABSTRACT: The main pathogenic process underlying dialysis-related amyloidosis is the accumulation of ?-2-microglobulin (?2m) as amyloid fibrils in the musculoskeletal system, and some evidence suggests that Cu(II) may play a role in ?2m amyloid formation. Cu(II)-induced ?2m fibril formation is preceded by the formation of discrete, oligomeric intermediates, including dimers, tetramers, and hexamers. In this work, we use selective covalent labeling reactions combined with mass spectrometry to investigate the amino acids responsible for mediating tetramer formation in wild-type ?2m. By comparing the labeling patterns of the monomer, dimer, and tetramer, we find evidence that the tetramer interface is formed by the interaction of D strands from one dimer unit and G strands from another dimer unit. These covalent labeling data along with molecular dynamics calculations allow the construction of a tetramer model that indicates how the protein might proceed to form even higher-order oligomers.

SUBMITTER: Mendoza VL

PROVIDER: S-EPMC3149750 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Structural insights into the pre-amyloid tetramer of β-2-microglobulin from covalent labeling and mass spectrometry.

Mendoza Vanessa Leah VL Barón-Rodríguez Mario A MA Blanco Cristian C Vachet Richard W RW

Biochemistry 20110708 31

The main pathogenic process underlying dialysis-related amyloidosis is the accumulation of β-2-microglobulin (β2m) as amyloid fibrils in the musculoskeletal system, and some evidence suggests that Cu(II) may play a role in β2m amyloid formation. Cu(II)-induced β2m fibril formation is preceded by the formation of discrete, oligomeric intermediates, including dimers, tetramers, and hexamers. In this work, we use selective covalent labeling reactions combined with mass spectrometry to investigate t ...[more]

PMID: 21718071

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Project description:Beta-2-microglobulin (beta2m) deposits as amyloid fibrils in the musculoskeletal system of patients undergoing long-term dialysis treatment as a result of kidney failure. Previous work has shown that Cu(II) binding causes beta2m to organize into nativelike dimers and tetramers that precede amyloid formation. Cu(II) is then released from higher-order oligomers before mature Cu(II)-free amyloid fibrils are formed. While some of the Cu(II)-induced structural changes that enable beta2m self-assembly are starting to be revealed, the details of how the Cu(II) binding site evolves from the monomer to the dimers and tetramers are not known. Here, we report results from three mass spectrometry (MS)-based methods that provide insight into the changing Cu-beta2m interactions. We find that monomeric beta2m binds Cu(II) via the N-terminal amine, the amide of Gln2, His31, and Asp59. In the dimer and tetramer, Asp59 is no longer bound to Cu(II), but the other residues still comprise a well-defined albeit weaker binding site that is better able to release Cu(II). Consistent with this is the observation that a fraction of the tetrameric species no longer binds Cu(II) at this weakened binding site, which agrees with a previous report that suggested the tetramer as the first Cu(II)-free oligomer. Our results also provide some insight into structural changes caused by Cu(II) binding that facilitate oligomer formation. Specifically, binding by Asp59 in the monomer requires significant movement of this residue, and we propose that this repositioning is important for establishing a pair of dimer-stabilizing salt bridges between this residue and Lys19. We also find evidence that Cu(II) binding in the N-terminal region of the monomer repels Arg3, which likely allows this residue to form a pair of dimer-stabilizing salt bridges with Glu16. Overall, our measurements suggest that the previously proposed conformational switch caused by Cu(II) binding includes not only a cis-trans isomerization at Pro32 but also the repositioning of residues that are critical for the formation of new electrostatic interactions.

Dataset Information

Structural insights into the pre-amyloid tetramer of ?-2-microglobulin from covalent labeling and mass spectrometry.

Publications

Structural insights into the pre-amyloid tetramer of β-2-microglobulin from covalent labeling and mass spectrometry.

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