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: Peter J Davis, David Holmes, Jonathan P Waltho, Rosemary A Staniforth
• Journal: Journal of molecular biology (J Mol Biol) Vol. 427 Issue 15 Pg. 2418-2434 (Jul 31 2015) ISSN: 1089-8638 [Electronic] Netherlands
• PMID: 26004542 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2015. Published by Elsevier Ltd.

Chemical References

• Amyloid
• CST3 protein, human
• Cystatin C
• Cystatin B
• Endopeptidase K

Topics

• Amyloid (chemistry)
• Cystatin B (chemistry, metabolism)
• Cystatin C (chemistry, metabolism)
• Endopeptidase K (metabolism)
• Humans
• Models, Molecular
• Protein Folding
• Protein Structure, Secondary
• Protein Structure, Tertiary
• Proteolysis