Intracellular Metal Speciation in Streptococcus sanguinis Establishes SsaACB as Critical for Redox Maintenance.

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 O2 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 Mn2+ 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.

Biometals as conformational modulators of α-synuclein photochemical crosslinking.

Metal dyshomeostasis has long been linked to Parkinson's disease (PD), and the amyloidogenic protein α-synuclein (αS) is universally recognized as a key player in PD pathology. Structural consequences upon coordination of copper and iron to αS have gained attention due to significant dyshomeostasis of both metals in the PD brain. Protein-metal association can navigate protein folding in distinctive pathways based on the identity of the bio-metal in question. In this work, we employed photo-chemical crosslinking of unmodified proteins (PICUP) to evaluate these potential metal ion-induced structural alterations in the folding dynamics of N-terminally acetylated αS (NAcαS) following metal coordination. Through fluorescence analysis and immunoblotting analyses following photoirradiation, we discovered that coordination of iron obstructs copper-promoted crosslinking. The absence of intra-molecular crosslinking upon iron association further supports its C-terminal coordination site and suggests a potential role for iron in mitigating nearby post-translational modification of tyrosine residues. Decreased fluorescence emission upon synergistic coordination of both copper and iron highlighted that although copper acts as a conformational promotor of NAcαS crosslinking, iron inhibits analogous conformational changes within the protein. The metal coordination preferences of NAcαS suggest that both competitive binding sites as well as dual metal coordination contribute to the changes in folding dynamics, unveiling unique structural orientations for NAcαS that have a direct and measureable influence on photoinitiated dityrosine crosslinks. Moreover, our findings have physiological implications in that iron overload, as is associated with PD-insulted brain tissue, may serve as a conformational block of copper-promoted protein oxidation.

C-Terminal CuII Coordination to α-Synuclein Enhances Aggregation.

The structurally dynamic amyloidogenic protein α-synuclein (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 CuII binding site. We now report the first detailed characterization data using electron paramagnetic resonance (EPR) spectroscopy to describe the CuII coordination modes of N-terminally acetylated αS (NAcαS). 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 CuII-binding mode that may involve a C-terminal contribution. We additionally generated an N-terminally acetylated disease-relevant variant, NAcH50Q, that promotes a shift in the CuII binding site to the C-terminus of the protein. Moreover, fibrillar NAcH50Q-CuII exhibits enhanced parallel ß-sheet character and increased hydrophobic surface area compared to NAcαS-CuII and to both protein variants that lack a coordinated cupric ion. The results presented herein demonstrate the differential impact of distinct CuII binding sites within NAcαS, revealing that C-terminal CuII 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 CuII coordination. Hence, consideration of the effect of CuII on NAcαS and NAcH50Q misfolding may shed light on the extrinsic or environmental factors that influence PD pathology.

Iron Redox Chemistry Promotes Antiparallel Oligomerization of α-Synuclein.

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 (NAcαS), the physiologically relevant form in humans. We demonstrate that under aerobic conditions, Fe(II) commits NAcαS to a PD-relevant oligomeric assembly, verified by the oligomer-selective A11 antibody, that does not have any parallel ß-sheet character but contains a substantial right-twisted antiparallel ß-sheet component based on CD analyses and descriptive deconvolution of the secondary structure. This NAcαS-FeII oligomer does not develop into the ß-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 ß-sheet fibril formation, also lacks coordination to this antiparallel conformer. We further show that this oligomeric state is not observed when O2 is excluded, indicating a role for iron(II)-mediated O2 chemistry in locking this dynamic protein into a conformation that may have physiological or pathological implications.