title: Exclusion of the native alpha-helix from the amyloid fibrils of a mixed alpha/beta protein.
abstract: Members of the cystatin superfamily are involved in an inherited form of cerebral amyloid angiopathy and readily form amyloid fibrils in vitro. We have determined the structured core of human stefin B (cystatin B) amyloid fibrils using quenched hydrogen exchange and NMR. The core contains residues from four of the five strands of the native beta-sheet, delimited by unprotected loop regions analogous to those of the native monomeric structure. However, non-native features are also apparent, the most striking of which is the exclusion of the native alpha-helix. Before forming amyloid in vitro, cystatins dimerise via 3D domain swapping, and assemble into tetramers with trans to cis isomerism of a conserved proline. In the fibril, the hinge loop that forms an extended beta-structure in the dimer remains protected, consistent with the domain-swapping interface being maintained. However, the fibril data are not compatible with a simple 3D domain-swapping model for amyloid formation, and the displacement of the helix points to alternative packing arrangements of native-like beta-structure, in which proline isomerism is important in preventing steric clashing.
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK.
authors.forename: Gareth J
authors.initials: GJ
authors.lastname: Morgan
authors.forename: Silva
authors.initials: S
authors.lastname: Giannini
authors.forename: Andrea M
authors.initials: AM
authors.lastname: Hounslow
authors.forename: C Jeremy
authors.initials: CJ
authors.lastname: Craven
authors.forename: Eva
authors.initials: E
authors.lastname: Zerovnik
authors.forename: Vito
authors.initials: V
authors.lastname: Turk
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Waltho
authors.forename: Rosemary A
authors.initials: RA
authors.lastname: Staniforth
mesh_terms: D000682:Amyloid / Q000737:chemistry* / Q000648:ultrastructure; D016229:Amyloid beta-Peptides / Q000737:chemistry*; D015891:Cystatins / Q000737:chemistry; D003903:Deuterium / Q000737:chemistry*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006859:Hydrogen / Q000737:chemistry*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013237:Stereoisomerism; D013997:Time Factors
chemical_list: D000682:Amyloid; D016229:Amyloid beta-Peptides; D015891:Cystatins; D006859:Hydrogen; D011392:Proline; D003903:Deuterium