TY - JOUR

T1 - Predicted Loop Regions Promote Aggregation

T2 - A Study of Amyloidogenic Domains in the Functional Amyloid FapC

AU - Nagaraj, Madhu

AU - Ahmed, Mumdooh

AU - Lyngsø, Jeppe

AU - Vad, Brian Stougaard

AU - Bøggild, Andreas

AU - Fillipsen, Anne

AU - Pedersen, Jan Skov

AU - Otzen, Daniel Erik

AU - Akbey, Ümit

N1 - Copyright © 2020 Elsevier Ltd. All rights reserved.

PY - 2020

Y1 - 2020

N2 - Protein fibrillation is traditionally associated with misfolding, loss of functional phenotype and gain of toxicity in neurodegenerative diseases. However, many organisms exploit fibrils in the form of functional amyloids (FA), as seen in bacteria such as E. coli, Salmonella, Bacillus and Pseudomonas. Here, we provide structural information and mechanistic data for fibrillation of the smallest amyloidogenic truncation unit along with the full-length version (FL) of the major amyloid protein FapC from Pseudomonas, predicted to consist of three β-hairpin-forming imperfect repeats separated by disordered regions. Using a series of truncation mutants, we establish that the putative loops (linkers) increase the rate of aggregation. The minimal aggregation unit consisting of a single repeat with flanking disordered regions (R3C) aggregates in a pathway dominated by secondary nucleation, in contrast to the primary nucleation favored by full-length (FL) FapC. SAXS on FapC FL, R3C, and remaining truncation constructs resolves two major coexisting species in the fibrillation process, namely pre-fibrillar loosely aggregated monomers and cylindrical elliptical cross section fibrils. Solid-state NMR spectra identified rigid parts of the FapC fibril. We assigned Cα-Cβ chemical shifts, indicative of a predominant β-sheet topology with some α-helix or loop chemical shifts. Our work emphasizes the complex nature of FapC fibrillation. In addition, we are able to deduce the importance of non-repeat regions (i.e. predicted loops), which enhance the amyloid protein aggregation and their influence on the polymorphism of the fibril architecture.

AB - Protein fibrillation is traditionally associated with misfolding, loss of functional phenotype and gain of toxicity in neurodegenerative diseases. However, many organisms exploit fibrils in the form of functional amyloids (FA), as seen in bacteria such as E. coli, Salmonella, Bacillus and Pseudomonas. Here, we provide structural information and mechanistic data for fibrillation of the smallest amyloidogenic truncation unit along with the full-length version (FL) of the major amyloid protein FapC from Pseudomonas, predicted to consist of three β-hairpin-forming imperfect repeats separated by disordered regions. Using a series of truncation mutants, we establish that the putative loops (linkers) increase the rate of aggregation. The minimal aggregation unit consisting of a single repeat with flanking disordered regions (R3C) aggregates in a pathway dominated by secondary nucleation, in contrast to the primary nucleation favored by full-length (FL) FapC. SAXS on FapC FL, R3C, and remaining truncation constructs resolves two major coexisting species in the fibrillation process, namely pre-fibrillar loosely aggregated monomers and cylindrical elliptical cross section fibrils. Solid-state NMR spectra identified rigid parts of the FapC fibril. We assigned Cα-Cβ chemical shifts, indicative of a predominant β-sheet topology with some α-helix or loop chemical shifts. Our work emphasizes the complex nature of FapC fibrillation. In addition, we are able to deduce the importance of non-repeat regions (i.e. predicted loops), which enhance the amyloid protein aggregation and their influence on the polymorphism of the fibril architecture.

KW - SAXS

KW - fibrillation mechanism

KW - functional amyloid fibril FapC

KW - polymorphism

KW - solid state NMR

UR - http://www.scopus.com/inward/record.url?scp=85080940818&partnerID=8YFLogxK

U2 - 10.1016/j.jmb.2020.01.044

DO - 10.1016/j.jmb.2020.01.044

M3 - Journal article

C2 - 32084414

VL - 432

SP - 2232

EP - 2252

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 7

ER -