Amyloid Accelerator Polyphosphate Implicated as the Mystery Density in α-Synuclein Fibrils
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Abstract
Aberrant aggregation of α-Synuclein is the pathological hallmark of a set of neurodegenerative diseases termed synucleinopathies. Recent advances in cryo-electron microscopy have led to the structural determination of the first synucleinopathy-derived α-Synuclein fibrils, which contain a non-proteinaceous, “mystery density” at the core of the protofilaments, hypothesized to be highly negatively charged. Guided by previous studies that demonstrated that polyphosphate (polyP), a universally conserved polyanion, significantly accelerates α-Synuclein fibril formation, we conducted blind docking and molecular dynamics simulation experiments to model the polyP binding site in α-Synuclein fibrils. Here we demonstrate that our models uniformly place polyP into the lysine-rich pocket, which coordinates the mystery density in patient-derived fibrils. Subsequent in vitro studies and experiments in cells revealed that substitution of the two critical lysine residues K43 and K45 leads to a loss of all previously reported effects of polyP binding on α-Synuclein, including stimulation of fibril formation, change in filament conformation and stability as well as alleviation of cytotoxicity. In summary, our study demonstrates that polyP fits the unknown electron density present in in vivo α-Synuclein fibrils and suggests that polyP exerts its functions by neutralizing charge repulsion between neighboring lysine residues.
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Figure 2.
Part of the confusion of this figure for me might be that the x axis for time in hours is different in Figure A, and you have to look closely at the x axis between them to see differences when adding polyphosphate.
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Mutational studies allowed us to validate K43 and K45 as the major polyP interaction sites in α-Syn as replacing them with alanine residues was sufficient to abolish polyP binding, and, as a direct consequence, prevented polyP to i) accelerate fibril formation, ii) stabilize α-Syn fibrils, iii) alter fibril morphology, and iv) mitigate α- Syn cytotoxicity.
I think this a great summary of results, however I found myself having to look back through the results several times to understand the model in my head. So maybe expanding on this a little more into multiple sentences to explain the effects of the mutations along with the presence/absence of polyphosphate so the model is easier to understand.
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It is noteworthy to point out that the two critical lysine residues K43 and K45 as well as H50, whose side-chains constitute the positive cluster associated with the mystery density binding in α-Syn, are part of a local hotspot known to harbor several mutations that elicit early onset familial Parkinson’s Disease.
Ah, fascinating! This gets at my earlier question
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While the mutation of H50 showed little effect on the t1/2 (∼ 40 h), substitution of either K43 or K45 with alanine reduced the t1/2 by about 2-fold (t1/2 ∼ 20h). When present in combination (i.e., α-SynK43A,K45A), we observed an even more drastic acceleration in fibril formation as reflected in a t1/2 of 8.3 hours. These results provided first evidence that this cluster of positively charged amino acids in wild-type α-Syn contributes to the slow rate of in vitro fibril formation.
This is a fascinating result! I am wondering if you could look in datasets like UK Biobank and connect if there are potential variant differences connected to these diseases. Such as if there are populations with or without these lysine residues and connect to risk for neurodegenerative diseases.
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lysine residues, suggesting that these residues are crucial for polyP binding.
I might have missed this or don't understand the conformational change between the monomer and the polymorph, but how are these residues critical for binding to polyphosphate in the polymorph but this doesn't occur in the monomer form?
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hese results were consistent with our experimental data, which failed to show any significant interaction between polyP and purified α-Syn monomers
this is fascinating
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Introduction
This is a really well written introduction, and I was able to understand most of it without having a deep molecular background myself.
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