The folding-limited nucleation of curli hints at an evolved safety mechanism for functional amyloid production

Abstract

It is nearly two decades ago that the ‘thin aggregative fimbriae’ which had been shown to enhance the biofilm formation of Salmonella enteriditis and Escherichia coli were identified as amyloid fibers. The realization that natural proteins can develop amyloidogenic traits as part of their functional repertoire instigated a search for similar proteins across all kingdoms of life. That pursuit has since unearthed dozens of candidates which now constitute the family of proteins referred to as functional amyloids (FA). FAs are promising candidates for future synthetic biology applications in that they marry the structural benefits of the amyloid fold (self-assembly and stability) while steering clear of the cytotoxicity issues that are typically linked to amyloid associated human pathologies. Unfortunately, the extreme aggregation propensity of FAs and the associated operational difficulties are restricting their adoption in real-world applications, underscoring the need for additional processes to control the amyloid reaction. Here we untangle the molecular mechanism of amyloid formation of the canonical functional amyloid curli using NMR, native mass spectrometry and cryo-electron microscopy. Our results are consistent with folding-limited one-step amyloid nucleation that has emerged as an evolutionary balance between efficient extracellular polymerization, while steering clear of pre-emptive nucleation in the periplasm. Sequence analysis of the amyloid curlin kernel suggests a finetuning of the rate of monomer folding via modulation of the secondary structure propensity of the pre-amyloid species, opening new potential avenues towards control of the amyloid reaction.

Consolidated peer review report (6 October 2023)

GENERAL ASESSMENT

This article will be of broad interest to readers in the field of protein folding, amyloid biogenesis and biotechnology. It provides new structural information on the pre-amyloid forms of a functional amyloid, curli, enabling new insight into nucleation mechanism of this protein. The authors used sequence analysis and construct design to generate a slow folding variant of the CsgA kernel, designated “slowgo”, which enabled detailed analysis of the folding kinetics using NMR. In addition they used native Mass Spectrometry and cryo-EM to identify the smallest folded CsgA species, and discovered it was a β-solenoid dimer. The authors propose a new model for curli formation, which proceeds via an initial slow folding step that places constraints on the rate of fibril formation in the cytoplasm, before efficient extracellular polymerization occurs outside the cell. Together, these results explain how curli is able to transit the cytoplasm without forming harmful aggregates and provide new insights into designer functional amyloids that will be useful in biotechnology and synthetic biology.

All three reviewers consider the work to be well-written, original, of high quality in terms of experimental data, and the experiments well-designed and controlled. The conclusions drawn from the work rely heavily on the development of the “slowgo” variant of the CsgA protein. The main suggestions to strengthen the conclusions are based on further validation of the assumptions concerning the behaviour of this species. Our recommendations for additional experiments are detailed below and aim to strengthen the conclusions made concerning this variant.

RECOMMENDATIONS

Essential revisions:

The authors mention that CsgA and its “slowgo” variant remain monomeric in a molten globule state until they transition into a dimeric folded state. The authors use NMR, DLS, and radius of gyration analysis to support this claim. Given that the monomeric state seems to be stable enough, especially for the “slowgo” variant, it would be very helpful to confirm this via a native gel. If these claims are true, CsgA should be clearly observed in the monomeric state during the first minutes of the reaction for WT, and up to 40 hours for the slowgo mutant, as indicated by the authors. In addition, the authors could run aliquots from the aggregation reaction taken at different times in denaturing gels to probe the SDS-resistance and stability of the oligomeric and amyloid species. We think this is a direct and simple way of testing the monomeric state of CsgA, which is central in the model proposed by the authors.
The authors reference the well-established sigmoidal ThT-kinetics to underpin their hypothesis that an initial slow folding step places constraints on the rate of fibril formation. However, could the initial part of the sigmoidal curve (known as lag phase before elongation) be due to other processes such as low dimerization or binding affinity of the monomers? We think it would be helpful to discuss these other possibilities or provide additional experimental evidence that these are not involved in the rate of nucleation and rate of fibril formation.
Additional analysis of the NMR data in the context of the “slowgo” construct substitutions would provide additional insight into the impact these mutations have on the folding kinetics. For example, all four substitutions detailed in the study introduce an alanine into the protein, which has a high helical propensity. Two are made in regions that promote helicity, whereas the other two variants do not promote helicity. However, it is not indicated whether these are β-solenoid strand positions or whether they are inward-facing or outward-facing. Additional clarification would help the reader understand the context of these mutations within the structure.
Similarly, additional analyses of where the regions with helical propensity lie within the context of the extended/strand portions of the folded β-solenoid secondary structure would help put the NMR data into clearer context. The regions predicted to have helical propensity are short, almost all less than a turn, and none have significant propensities above 50%; at the same time, non-extended conformations are needed for the solenoid to fold back on itself and short stretches with helical backbone angles are not necessarily disruptive. One suggestion is that Figure 5 could indicate whether each position in the repeat is extended or not in the β-solenoid structure and, correspondingly, whether the Pβ is calculated over just the extended region.
Could the authors undertake a continuous acquisition nMS experiment where they can observe the oligomerization progressing as a function of time? This would enable them to quantitively show the changes in oligomerization dynamics. If this gets too difficult to perform, the authors can expand their Supporting Figure 6 and have more granular time point data and plot the same for each oligomeric species observed for each protein-forms. This would provide strong evidence for the folding mechanism proposed.

Optional suggestions:

From Supporting Figure 6 data on time-resolved nMS it is concluded that the dimer must be on-pathway. However, depending on the rate equations, is it possible that the dimer pool is reversibly formed off-pathway but then depleted once most of the protein is incorporated into amyloids? Based on all the data together, the conclusion adopted by the authors seems likely, but perhaps the wording could be softened to indicate other possibilities do exist.
Figure 6 should be improved for clarity. Some states are unclear due to the choice of color and the design of cartoons.
Indicate temperature for the NMR studies in Fig. 1a. The rate of signal loss is faster than that seen by ThT fluorescence in Supporting Figure 1, which would suggest the possibility of a longer-lived intermediate. Is the NMR done at 25C?
The flow of the introduction would benefit from separation into more paragraphs. It would also benefit from a clearer description of the differences between the NCC mechanism and the one CsgA is thought to follow; this is an essential piece of background and strongly motivates the work here, but confusion arises from the use of the terms nucleation and oligomerisation within the two contexts.
Considering the proportion of residues exhibiting helicity and the magnitude it would be better to show much less helix in the schematic of Figure 6.
Considering the importance of Figure 2, it should be indicated how the chemical shifts were referenced, which can have significant effects on secondary shift analysis. It is indicated that TALOS was used for secondary structure predictions, but the data looks more like what one obtains from delta2d? If it was TALOS, the version should be indicated.

REVIEWING TEAM

Reviewed by:

Piere Rodriguez Aliaga, Postdoctoral Scholar, Stanford University, USA: protein biophysics, IDPs, protein misfolding and aggregation, chaperones, single molecule biophysics

Kallol Gupta, Assistant Professor, Yale University, USA: native mass spectrometry, protein folding

Jason Schnell, Associate Professor, University of Oxford, UK: protein solution NMR; protein folding

Curated by:

Simon Newstead, Professor, University of Oxford, UK

(This consolidated report is a result of peer review conducted by Biophysics Colab on version 1 of this preprint. Comments concerning minor and presentational issues have been omitted for brevity.)

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The folding-limited nucleation of curli hints at an evolved safety mechanism for functional amyloid production

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