Dimerization energetics of curli fiber subunits CsgA and CsgB

Dunbar, Martha; DeBenedictis, Elizabeth P.; Keten, Sinan

doi:10.1038/s41524-019-0164-5

Cited by 12 publications

(12 citation statements)

References 48 publications

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“…A persistent feature of pre-amyloid in curli and other functional amyloid systems is the role of weak transient interactions in preventing amyloid formation and guiding the protein for export and subsequent templating 35,36,[48][49][50] , The transience of interactions between CsgC/CsgE and CsgA, as observed by fast exchange behaviour in NMR titration experiments 35,36,49 , is likely important for the progressive handling and delicate manipulation of the dynamic pre-amyloid ensemble. Such fine tuning of this early stage of amyloid formation enables the bacteria to deliver subunits in the appropriate state for efficient and homogeneous fibre format at the surface.…”

Section: Discussionmentioning

confidence: 99%

NMR insights into the pre-amyloid ensemble and secretion targeting of the curli subunit CsgA

Sewell

Stylianou

et al. 2020

Sci Rep

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The biofilms of Enterobacteriaceae are fortified by assembly of curli amyloid fibres on the cell surface. Curli not only provides structural reinforcement, but also facilitates surface adhesion. To prevent toxic intracellular accumulation of amyloid precipitate, secretion of the major curli subunit, csgA, is tightly regulated. In this work, we have employed solution state NMR spectroscopy to characterise the structural ensemble of the pre-fibrillar state of CsgA within the bacterial periplasm, and upon recruitment to the curli pore, CsgG, and the secretion chaperone, CsgE. We show that the N-terminal targeting sequence (N) of CsgA binds specifically to CsgG and that its subsequent sequestration induces a marked transition in the conformational ensemble, which is coupled to a preference for CsgE binding. these observations lead us to suggest a sequential model for binding and structural rearrangement of CsgA at the periplasmic face of the secretion machinery.

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Section: Discussionmentioning

confidence: 99%

NMR insights into the pre-amyloid ensemble and secretion targeting of the curli subunit CsgA

Sewell

Stylianou

et al. 2020

Sci Rep

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“…The terminal repeats of CsgA have previously been experimentally verified to form amyloid-like aggregates [96,97], and we therefore decided to focus primarily on APRs within the terminal repeats of CsgA. This is further supported by the predicted β-helical structure of CsgA, which implies that curli amyloid fibrils are formed through stacking of CsgA monomers by intermolecular R1-R5 contacts [45,98,99]. APRs identified in R1 and R5 were then used as the basis for the design of tandem repeat peptides consisting of two identical hepta-sequences (containing the APR identified in either R1 or R5) linked by a flexible (Gly-Ser) or rigid (Pro-Pro) linker.…”

Section: Structure-based Design Of Anti-fuba Peptidesmentioning

confidence: 93%

Functional Bacterial Amyloids: Understanding Fibrillation, Regulating Biofilm Fibril Formation and Organizing Surface Assemblies

2022

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Functional amyloid is produced by many organisms but is particularly well understood in bacteria, where proteins such as CsgA (E. coli) and FapC (Pseudomonas) are assembled as functional bacterial amyloid (FuBA) on the cell surface in a carefully optimized process. Besides a host of helper proteins, FuBA formation is aided by multiple imperfect repeats which stabilize amyloid and streamline the aggregation mechanism to a fast-track assembly dominated by primary nucleation. These repeats, which are found in variable numbers in Pseudomonas, are most likely the structural core of the fibrils, though we still lack experimental data to determine whether the repeats give rise to β-helix structures via stacked β-hairpins (highly likely for CsgA) or more complicated arrangements (possibly the case for FapC). The response of FuBA fibrillation to denaturants suggests that nucleation and elongation involve equal amounts of folding, but protein chaperones preferentially target nucleation for effective inhibition. Smart peptides can be designed based on these imperfect repeats and modified with various flanking sequences to divert aggregation to less stable structures, leading to a reduction in biofilm formation. Small molecules such as EGCG can also divert FuBA to less organized structures, such as partially-folded oligomeric species, with the same detrimental effect on biofilm. Finally, the strong tendency of FuBA to self-assemble can lead to the formation of very regular two-dimensional amyloid films on structured surfaces such as graphite, which strongly implies future use in biosensors or other nanobiomaterials. In summary, the properties of functional amyloid are a much-needed corrective to the unfortunate association of amyloid with neurodegenerative disease and a testimony to nature’s ability to get the best out of a protein fold.

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“…The mapping was completed using MODELLER and produced a monomer of 64 residues containing two β-strand repeats and a flexible 22-residue N-terminus. Next, the R1/R5 monomers were stacked into a multimer by aligning the interface between R1 of one monomer and R5 of another monomer to the same R1–R5 interface of the CsgA dimers created in ref . This procedure was repeated to build multimer structures made up of 6 stacked monomers.…”

Section: Methodsmentioning

confidence: 99%

Curli-Mediated Self-Assembly of a Fibrous Protein Scaffold for Hydroxyapatite Mineralization

et al. 2020

Self Cite

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Nanostructures formed by self-assembled peptides have been increasingly exploited as functional materials for a wide variety of applications, from biotechnology to energy. However, it is sometimes challenging to assemble free short peptides into functional supramolecular structures, since not all peptides have the ability to self-assemble. Here, we report a self-assembly mechanism for short functional peptides that we derived from a class of fiber-forming amyloid proteins called curli. CsgA, the major subunit of curli fibers, is a self-assembling β-helical subunit composed of five pseudorepeats (R1–R5). We first deleted the internal repeats (R2, R3, R4), known to be less essential for the aggregation of CsgA monomers into fibers, forming a truncated CsgA variant (R1/R5). As a proof-of-concept to introduce functionality in the fibers, we then genetically substituted the internal repeats by a hydroxyapatite (HAP)-binding peptide, resulting in a R1/HAP/R5 construct. Our method thus utilizes the R1/R5-driven self-assembly mechanism to assemble the HAP-binding peptide and form hydrogel-like materials in macroscopic quantities suitable for biomineralization. We confirmed the expression and fibrillar morphology of the truncated and HAP-containing curli-like amyloid fibers. X-ray diffraction and TEM showed the functionality of the HAP-binding peptide for mineralization and formation of nanocrystalline HAP. Overall, we show that fusion to the R1 and R5 repeats of CsgA enables the self-assembly of functional peptides into micron long fibers. Further, the mineral-templating ability that the R1/HAP/R5 fibers possesses opens up broader applications for curli proteins in the tissue engineering and biomaterials fields.

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Dimerization energetics of curli fiber subunits CsgA and CsgB

Cited by 12 publications

References 48 publications

NMR insights into the pre-amyloid ensemble and secretion targeting of the curli subunit CsgA

NMR insights into the pre-amyloid ensemble and secretion targeting of the curli subunit CsgA

Functional Bacterial Amyloids: Understanding Fibrillation, Regulating Biofilm Fibril Formation and Organizing Surface Assemblies

Curli-Mediated Self-Assembly of a Fibrous Protein Scaffold for Hydroxyapatite Mineralization

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