Predicted Loop Regions Promote Aggregation: A Study of Amyloidogenic Domains in the Functional Amyloid FapC. Issue 7 (27th March 2020)

• Record Type: Journal Article
• Authors: Nagaraj, Madhu; Ahmed, Mumdooh; Lyngsø, Jeppe; Vad, Brian Stougaard; Bøggild, Andreas; Fillipsen, Anne; Pedersen, Jan Skov; Otzen, Daniel Erik; Akbey, Ümit
• Is Part Of: Journal of molecular biology. Volume 432:Issue 7(2020)
• Page Start: 2232
• Page End: 2252
• Publication Date: 2020-03-27
• Subjects: functional amyloid fibril FapC -- solid state NMR -- SAXS -- polymorphism -- fibrillation mechanism; CHC Central hydrophobic cluster -- DARR Dipolar assisted rotational resonance -- FL Full length -- FTIR Fourier-transform infrared spectroscopy -- GdHCl Guanidine hydrochloride -- HSQC Heteronuclear single quantum coherence spectroscopy -- INEPT Insensitive nuclei enhanced by polarization transfer -- IPTG Isopropyl β-d-1-thiogalactopyranoside -- LB Luria Bertani broth -- MAS Magic angle spinning -- PCR Polymerase chain reaction -- RF Radio frequency -- RT Room temperature -- SAXS Small-Angle X-ray Scattering -- ssNMR Solid-state nuclear magnetic resonance -- TEM Transmission electron microscopy -- ThT Thioflavin T; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2020.01.044
• Languages: English
• ISSNs: 0022-2836

Abstract

Abstract: 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. Graphical abstract: Image 1 Highlights: Predicted Loop Regions Promote FapC functional amyloid Aggregation. The minimal aggregation unit consisting of a single repeat with flanking disordered regions (R3C) aggregates via secondary nucleation, in contrast to the primary nucleation favored by full-length (FL) FapC. Small-angle X-ray scattering (SAXS) on FapC FL, R3C, and remaining truncation constructs resolve two major coexisting species in the fibrillation process. Solid-state NMR spectra identified rigid parts of the FapC fibril. We assigned Cα-Cβ chemical shifts indicating predominant β-sheet topology, with some α-helix or loop chemical shifts. The mobile forms/regions of FapC were identified by INEPT NMR experiments.