pH- and concentration-dependent supramolecular assembly of a fungal defensin plectasin variant into helical non-amyloid fibrils
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Self-assembly and fibril formation play important roles in protein behaviour. Amyloid fibril formation is well-studied due to its role in neurodegenerative diseases and characterized by refolding of the protein into predominantly β-sheet form. However, much less is known about the assembly of proteins into other types of supramolecular structures. Using cryo-electron microscopy at a resolution of 1.97 Å, we show that a triple-mutant of the anti-microbial peptide plectasin, PPI42, assembles into helical non-amyloid fibrils. The in vitro anti-microbial activity was determined and shown to be enhanced compared to the wildtype. Plectasin contains a cysteine-stabilised α-helix-β-sheet structure, which remains intact upon fibril formation. Two protofilaments form a right-handed protein fibril. The fibril formation is reversible and follows sigmoidal kinetics with a pH- and concentration dependent equilibrium between soluble monomer and protein fibril. This high-resolution structure reveals that α/β proteins can natively assemble into fibrils.
| Original language | English |
|---|---|
| Article number | 3162 |
| Journal | Nature Communications |
| Volume | 13 |
| Issue number | 1 |
| ISSN | 2041-1723 |
| DOIs | |
| Publication status | Published - Dec 2022 |
Bibliographical note
Funding Information:
This work was funded by European Union’s Horizon 2020 research and innovation program (grant agreement no. 675074) (P.H., A.N., W.S. and G.H.P.). We thank Birgitte Andersen and Ida Ahlmann Ellingsgaard (Novozymes A/S) for their input on previous plectasin studies, supply of purified material and their help on the activity measurements of plectasin. We thank Rahmi K. Elfa for her work on the crystallization of the plectasin wildtype. We thank Ingemar André (Lund University) for useful discussions about surface accessible areas in protein fibrils. We thank Thom Leiding and Mattias Törnquist (Probationlabs) for their input on the setup of simultaneous pH and light scattering measurements. We acknowledge the provision of in-house experimental time from the CM01 facility at the ESRF. This work used the EM facilities at the Grenoble Instruct-ERIC Center (ISBG; UMS 3518 CNRS CEA-UGA-EMBL) (E.K. and C.M.-D.) with support from the French Infrastructure for Integrated Structural Biology (FRISBI; ANR-10-INSB-05-02) and GRAL (G.E. and G.S.), a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003) within the Grenoble Partnership for Structural Biology. The IBS Electron Microscope facility is supported by the Auvergne Rhône-Alpes Region, the Fonds Feder, the Fondation pour la Recherche Médicale and GIS-IBiSA. We acknowledge MAX IV Laboratory for time on BioMAX under Proposal [20190334] (P.H.). Research conducted at MAX IV, a Swedish national user facility, is supported by the Swedish Research council under contract 2018-07152, the Swedish Governmental Agency for Innovation Systems under contract 2018-04969, and Formas under contract 2019-02496. NMR spectra were recorded using the 800 MHz spectrometer at the NMR Center DTU, supported by the Villum Foundation (S.M.).
Funding Information:
This work was funded by European Union’s Horizon 2020 research and innovation program (grant agreement no. 675074) (P.H., A.N., W.S. and G.H.P.). We thank Birgitte Andersen and Ida Ahlmann Ellingsgaard (Novozymes A/S) for their input on previous plectasin studies, supply of purified material and their help on the activity measurements of plectasin. We thank Rahmi K. Elfa for her work on the crystallization of the plectasin wildtype. We thank Ingemar André (Lund University) for useful discussions about surface accessible areas in protein fibrils. We thank Thom Leiding and Mattias Törnquist (Probationlabs) for their input on the setup of simultaneous pH and light scattering measurements. We acknowledge the provision of in-house experimental time from the CM01 facility at the ESRF. This work used the EM facilities at the Grenoble Instruct-ERIC Center (ISBG; UMS 3518 CNRS CEA-UGA-EMBL) (E.K. and C.M.-D.) with support from the French Infrastructure for Integrated Structural Biology (FRISBI; ANR-10-INSB-05-02) and GRAL (G.E. and G.S.), a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003) within the Grenoble Partnership for Structural Biology. The IBS Electron Microscope facility is supported by the Auvergne Rhône-Alpes Region, the Fonds Feder, the Fondation pour la Recherche Médicale and GIS-IBiSA. We acknowledge MAX IV Laboratory for time on BioMAX under Proposal [20190334] (P.H.). Research conducted at MAX IV, a Swedish national user facility, is supported by the Swedish Research council under contract 2018-07152, the Swedish Governmental Agency for Innovation Systems under contract 2018-04969, and Formas under contract 2019-02496. NMR spectra were recorded using the 800 MHz spectrometer at the NMR Center DTU, supported by the Villum Foundation (S.M.).
Publisher Copyright:
© 2022, The Author(s).
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