Limited Proteolysis Reveals That Amyloids from the 3D Domain-Swapping Cystatin B Have a Non-Native β-Sheet Topology

• Published in: Journal of molecular biology Vol. 427; no. 15; pp. 2418 - 2434
• Main Authors: Davis, Peter J., Holmes, David, Waltho, Jonathan P., Staniforth, Rosemary A.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 31.07.2015
• Subjects: 3D domain swapping; Amyloid - chemistry; amyloid structure; cystatin; Cystatin B - chemistry; Cystatin B - metabolism; Cystatin C - chemistry; Cystatin C - metabolism; Endopeptidase K - metabolism; Humans; limited proteolysis; Models, Molecular; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Proteolysis; stefin; MS; NT; AFM; EM; MPL; stefin; 3D domain swapping; STEM; HX; CT; amyloid structure; limited proteolysis; cystatin; TEM
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2015.05.014

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. [Display omitted] •The cystatins are thought to form amyloid fibrils via a 3D domain-swapping mechanism.•Both the N-terminal 35 and the C-terminal 18 residues of cystatin B can be cleaved from fibrils without disturbing their structural integrity.•Cystatin B fibrils have a non-native topology incompatible with 3D domain swapping.•Cystatins are a good model for understanding amyloids in general.