Identification of a subunit interface in transthyretin amyloid fibrils: evidence for self-assembly from oligomeric building blocks.
Amyloid and prion diseases appear to stem from the conversion of normally folded proteins into insoluble, fiber-like assemblies. Despite numerous structural studies, a detailed molecular characterization of amyloid fibrils remains elusive. In particular, models of amyloid fibrils proposed thus far have not adequately defined the constituent protein subunit interactions. To further our understanding of amyloid structure, we employed thiol-specific cross-linking and site-directed spin labeling to identify specific protein-protein associations in transthyretin (TTR) amyloid fibrils. We find that certain cysteine mutants of TTR, when dimerized by chemical cross-linkers, still form fibers under typical in vitro fibrillogenic conditions. In addition, site-directed spin labeling of many residues at the natural dimer interface reveals that their spatial proximity is preserved in the fibrillar state even in the absence of cross-linking constraints. Here, we present the first view of a subunit interface in TTR fibers and show that it is very similar to one of the natural dimeric interchain associations evident in the structure of soluble TTR. The results clarify varied models of amyloidogenesis by demonstrating that transthyretin amyloid fibrils may assemble from oligomeric protein building blocks rather than structurally rearranged monomers.
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Molecular Biology Institute, School of Medicine, University of California, Los Angeles, California 90095, USA.
Altenbach
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Gingery
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Hubbell
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Yeates
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D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D003432:Cross-Linking Reagents / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011228:Prealbumin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D013113:Spin Labels; D013438:Sulfhydryl Compounds / Q000378:metabolism
D000682:Amyloid; D003432:Cross-Linking Reagents; D011228:Prealbumin; D013113:Spin Labels; D013438:Sulfhydryl Compounds; D003545:Cysteine