Brain metal dyshomeostasis and altered structural dynamics of the presynaptic protein α-synuclein (αS) are both implicated in the pathology of Parkinson's disease (PD), yet a mechanistic understanding of disease progression in the context of αS structure and metal interactions remains elusive. In this Communication, we detail the influence of iron, a prevalent redox-active brain biometal, on the aggregation propensity and secondary structure of N-terminally acetylated αS (αS), the physiologically relevant form in humans. We demonstrate that under aerobic conditions, Fe(II) commits αS to a PD-relevant oligomeric assembly, verified by the oligomer-selective A11 antibody, that does not have any parallel β-sheet character but contains a substantial right-twisted antiparallel β-sheet component based on CD analyses and descriptive deconvolution of the secondary structure. ThisαS-Fe oligomer does not develop into the β-sheet fibrils that have become hallmarks of PD, even after extended incubation, as verified by TEM imaging and the fibril-specific OC antibody. Thioflavin T (ThT), a fluorescent probe for β-sheet fibril formation, also lacks coordination to this antiparallel conformer. We further show that this oligomeric state is not observed when O is excluded, indicating a role for iron(II)-mediated O chemistry in locking this dynamic protein into a conformation that may have physiological or pathological implications.

Streptococcus sanguinis is an oral commensal bacterium, but it can colonize pre-existing heart valve vegetations if introduced into the bloodstream, leading to infective endocarditis. Loss of Mn-or Fe-cofactored virulence determinants are thought to result in weakening of this bacterium. Indeed, intracellular Mn accumulation mediated by the lipoprotein SsaB, a component of the SsaACB transporter complex, has been shown to promote virulence for endocarditis and O 2 tolerance. To delineate intracellular metal-ion abundance and redox speciation within S. sanguinis, we developed a protocol exploiting two spectroscopic techniques, Inductively coupled plasma-optical emission spectrometry (ICP-OES) and electron paramagnetic resonance (EPR) spectroscopy, to respectively quantify total intracellular metal concentrations and directly measure redox speciation of Fe and Mn within intact whole-cell samples. Addition of the cell-permeable siderophore deferoxamine shifts the oxidation states of accessible Fe and Mn from reduced-to-oxidized, as verified by magnetic moment calculations, aiding in the characterization of intracellular metal pools and metal sequestration levels for Mn 2+ and Fe. We have applied this methodology to S. sanguinis and an SsaACB knockout strain (ΔssaACB), indicating that SsaACB mediates both Mn and Fe uptake, directly influencing the metal-ion pools available for biological inorganic pathways. Mn supplementation of ΔssaACB returns total intracellular Mn to wild-type levels, but it does not restore wild-type redox speciation or distribution of metal cofactor availability for either Mn or Fe. Our results highlight the biochemical basis for S. sanguinis oxidative resistance, revealing a dynamic role for SsaACB in controlling redox homeostasis by managing the intracellular Fe/Mn composition and distribution.

The structurally dynamic amyloidogenic protein αsynuclein (αS) is universally recognized as a key player in Parkinson's disease (PD). Copper, which acts as a neuronal signaling agent, is also an effector of αS structure, aggregation, and localization in vivo. In humans, αS is known to carry an acetyl group on the starting methionine residue, capping the N-terminal free amine which was a known high-affinity Cu II binding site. We now report the first detailed characterization data using electron paramagnetic resonance (EPR) spectroscopy to describe the Cu II coordination modes of N-terminally acetylated αS ( NAc αS). Through use of EPR hyperfine structure analyses and the Peisach−Blumberg correlation, an N3O1 binding mode was established that involves the single histidine residue at position 50 and a lower population of a second Cu II -binding mode that may involve a C-terminal contribution. We additionally generated an N-terminally acetylated disease-relevant variant, NAc H50Q, that promotes a shift in the Cu II binding site to the Cterminus of the protein. Moreover, fibrillar NAc H50Q-Cu II exhibits enhanced parallel β-sheet character and increased hydrophobic surface area compared to NAc αS-Cu II and to both protein variants that lack a coordinated cupric ion. The results presented herein demonstrate the differential impact of distinct Cu II binding sites within NAc αS, revealing that C-terminal Cu II binding exacerbates the structural consequences of the H50Q missense mutation. Likewise, the global structural modifications that result from N-terminal capping augment the properties of Cu II coordination. Hence, consideration of the effect of Cu II on NAc αS and NAc H50Q misfolding may shed light on the extrinsic or environmental factors that influence PD pathology.

No abstract

No abstract