Molecular Role of Ca2+ and Hard Divalent Metal Cations on Accelerated Fibrillation and Interfibrillar Aggregation of α-Synuclein

Abstract

α-Synuclein (αSyn) is an intrinsically disordered protein, the aggregation of which is highly related to the pathology of diverse α-synucleinopathies. Various hard divalent metal cations have been shown to affect αSyn aggregation. Especially, Ca2+ is suggested to be a crucial ion due to its physiological relevance to α-synucleinopathies. However, the molecular origin of αSyn aggregation mediated by the metal ions is not fully elucidated. In this study, we revealed that hard divalent metal ions had almost identical influences on αSyn aggregation. Based on these similarities, the molecular role of Ca2+ was investigated as a representative metal ion. Herein, we demonstrated that binding of multiple Ca2+ ions induces structural transition of αSyn monomers to extended conformations, which promotes rapid αSyn fibrillation. Additionally, we observed that Ca2+ induced further interfibrillar aggregation via electrostatic and hydrophobic interactions. Our results from multiple biophysical methods, including ion mobility-mass spectrometry (IM-MS), synchrotron small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), provide detailed information on the structural change of αSyn and the aggregation process mediated by Ca2+. Overall, our study would be valuable for understanding the influence of Ca2+ on the aggregation of αSyn during the pathogenesis of α-synucleinopathies.

Effects of alpha‐synuclein post‐translational modifications on metal binding

“…The binding of multiple Ca(II) ions (up to four) to AS (Fig. ) has been demonstrated using multiple biophysical methods, including ion mobility‐mass spectrometry (IM‐MS), synchrotron small‐angle X‐ray scattering and transmission electron microscopy in combination with molecular dynamics (MD) (Han et al ). As observed with other divalent metal ions, Ca(II) was shown to induce a structural transition of AS monomers to extended conformations, promoting the exposure of the NAC region and rapid AS fibrillation.…”

“…The study concluded that Ca(II) ions also trigger non‐specific inter‐fibrillar aggregation via electrostatic and hydrophobic interactions to produce large AS aggregates (Fig. c) (Han et al ). Several studies have demonstrated that Ca(II) binds to AS through the C‐terminal region (Lowe et al ; de Laureto et al ); and it interferes with normal AS‐membrane interactions.…”

Conformational changes and the self-assembly of alpha-synuclein

“…The familial PD mutations have been shown to alter alpha-synuclein oligomerisation and subsequent aggregation (Winner et al, 2011), demonstrating a probable role for alpha-synuclein misfolding and aggregation in PD. Ligand binding has also been demonstrated to effect alpha-synuclein aggregation, with binding of some compounds such as divalent metal cations (Han, Choi and Kim, 2018) and biological polyamines (Krasnoslobodtsev et al, 2012) increasing aggregation, and others such as squalamine (Perni et al, 2017) and curcumin (Herva et al, 2014) decreasing aggregation.…”

“…The binding of many other small molecules has been investigated using IMS-MS, including investigations into spermine (Grabenauer et al, 2008), calcium (Han, Choi and Kim, 2018), dopamine (Illes-Toth, Dalton and Smith, 2013), epigallocatechin gallate (EGCG) (Konijnenberg et al, 2016) and gallic acid (Liu et al, 2014). A common finding of these studies was an increase in compact conformations upon binding of compounds known to increase alpha-synuclein aggregation, such as spermine (Grabenauer et al, 2008), and prevention of the formation of compact conformations by compounds which inhibit aggregation, such as gallic acid (Liu et al, 2014).…”

“…Certain environmental conditions have been demonstrated to be capable of inducing such collapsed conformational states, including the presence of various divalent metal ions (Santner and Uversky, 2010). While most interactions between alpha-synuclein and metals occur at the C-terminus of the protein and appear to be low-specificity (Binolfi et al, 2006), high affinity binding sites for Cu 2+ have been reported (Han, Choi and Kim, 2018). Evidence suggests alpha-synuclein is capable of binding Cu 2+ at three specific sites, shown in Figure 2.1; a high-affinity N-terminal site where copper is anchored by the freely available amino-terminal nitrogen, a site anchored by the imidazole ring of the histidine at residue 50, and a low-affinity site at the C-terminus of the protein (Binolfi et al, 2012).…”