CsgA gatekeeper residues control nucleation but not stability of functional amyloid

TY - JOUR

T1 - CsgA gatekeeper residues control nucleation but not stability of functional amyloid

AU - Olsen, William P

AU - Courtade, Gaston

AU - Peña-Díaz, Samuel

AU - Nagaraj, Madhu

AU - Sønderby, Thorbjørn V

AU - Mulder, Frans A A

AU - Malle, Mette G

AU - Otzen, Daniel E

PY - 2024/10

Y1 - 2024/10

N2 - Functional amyloids, beneficial to the organism producing them, are found throughout life, from bacteria to humans. While disease-related amyloids form by uncontrolled aggregation, the fibrillation of functional amyloid is regulated by complex cellular machinery and optimized sequences, including so-called gatekeeper residues such as Asp. However, the molecular basis for this regulation remains unclear. Here we investigate how the introduction of additional gatekeeper residues affects fibril formation and stability in the functional amyloid CsgA from E. coli. Step-wise introduction of additional Asp gatekeepers gradually eliminated fibrillation unless preformed fibrils were added, illustrating that gatekeepers mainly affect nucleus formation. Once formed, the mutant CsgA fibrils were just as stable as wild-type CsgA. HSQC NMR spectra confirmed that CsgA is intrinsically disordered, and that the introduction of gatekeeper residues does not alter this ensemble. NMR-based Dark-state Exchange Saturation Transfer (DEST) experiments on the different CsgA variants, however, show a decrease in transient interactions between monomeric states and the fibrils, highlighting a critical role for these interactions in the fibrillation process. We conclude that gatekeeper residues affect fibrillation kinetics without compromising structural integrity, making them useful and selective modulators of fibril properties.

AB - Functional amyloids, beneficial to the organism producing them, are found throughout life, from bacteria to humans. While disease-related amyloids form by uncontrolled aggregation, the fibrillation of functional amyloid is regulated by complex cellular machinery and optimized sequences, including so-called gatekeeper residues such as Asp. However, the molecular basis for this regulation remains unclear. Here we investigate how the introduction of additional gatekeeper residues affects fibril formation and stability in the functional amyloid CsgA from E. coli. Step-wise introduction of additional Asp gatekeepers gradually eliminated fibrillation unless preformed fibrils were added, illustrating that gatekeepers mainly affect nucleus formation. Once formed, the mutant CsgA fibrils were just as stable as wild-type CsgA. HSQC NMR spectra confirmed that CsgA is intrinsically disordered, and that the introduction of gatekeeper residues does not alter this ensemble. NMR-based Dark-state Exchange Saturation Transfer (DEST) experiments on the different CsgA variants, however, show a decrease in transient interactions between monomeric states and the fibrils, highlighting a critical role for these interactions in the fibrillation process. We conclude that gatekeeper residues affect fibrillation kinetics without compromising structural integrity, making them useful and selective modulators of fibril properties.

KW - Escherichia coli Proteins/chemistry

KW - Amyloid/chemistry

KW - Escherichia coli/genetics

KW - Protein Stability

KW - Nuclear Magnetic Resonance, Biomolecular

KW - Mutation

U2 - 10.1002/pro.5178

DO - 10.1002/pro.5178

M3 - Journal article

C2 - 39302107

SN - 0961-8368

VL - 33

JO - Protein Science

JF - Protein Science

IS - 10

M1 - e5178

ER -