title: Limited Proteolysis Reveals That Amyloids from the 3D Domain-Swapping Cystatin B Have a Non-Native β-Sheet Topology.
abstract: 3D domain-swapping proteins form multimers by unfolding and then sharing of secondary structure elements, often with native-like interactions. Runaway domain swapping is proposed as a mechanism for folded proteins to form amyloid fibres, with examples including serpins and cystatins. Cystatin C amyloids cause a hereditary form of cerebral amyloid angiopathy whilst cystatin B aggregates are found in cases of Unverricht-Lundborg Syndrome, a progressive form of myoclonic epilepsy. Under conditions that favour fibrillisation, cystatins populate stable 3D domain-swapped dimers both in vitro and in vivo that represent intermediates on route to the formation of fibrils. Previous work on cystatin B amyloid fibrils revealed that the α-helical region of the protein becomes disordered and identified the conservation of a continuous 20-residue elongated β-strand (residues 39-58), the latter being a salient feature of the dimeric 3D domain-swapped structure. Here we apply limited proteolysis to cystatin B amyloid fibrils and show that not only the α-helical N-terminal of the protein (residues 1-35) but also the C-terminal of the protein (residues 80-98) can be removed without disturbing the underlying fibril structure. This observation is incompatible with previous models of cystatin amyloid fibrils where the β-sheet is assumed to retain its native antiparallel arrangement. We conclude that our data favour a more generic, at least partially parallel, arrangement for cystatin β-sheet structure in mature amyloids and propose a model that remains consistent with available data for amyloids from either cystatin B or cystatin C.
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Davis
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester M13 9PT, UK.
authors.forename: David
authors.initials: D
authors.lastname: Holmes
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK; Faculty of Life Sciences, Manchester Institute of Biotechnology, Manchester M1 7DN, UK.
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Waltho
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK. Electronic address: r.a.staniforth@sheffield.ac.uk.
authors.forename: Rosemary A
authors.initials: RA
authors.lastname: Staniforth
mesh_terms: D000682:Amyloid / Q000737:chemistry*; D055313:Cystatin B / Q000737:chemistry* / Q000378:metabolism*; D055316:Cystatin C / Q000737:chemistry / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D059748:Proteolysis
chemical_list: D000682:Amyloid; C525030:CST3 protein, human; D055316:Cystatin C; D055313:Cystatin B; D019286:Endopeptidase K
keywords: 3D domain swapping; amyloid structure; cystatin; limited proteolysis; stefin