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C-terminal Cu Coordination to #-Synuclein Enhances Aggregation Dinendra L Abeyawardhane, Denver R Heitger, Ricardo D Fernández, Ashley K. Forney, and Heather R. Lucas ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00448 • Publication Date (Web): 01 Nov 2018 Downloaded from http://pubs.acs.org on November 3, 2018
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ACS Chemical Neuroscience
C-terminal CuII Coordination to α-Synuclein Enhances Aggregation Dinendra L. Abeyawardhane, Denver R. Heitger, Ricardo D. Fernández, Ashley K. Forney, Heather R. Lucas* Department of Chemistry, Virginia Commonwealth University, Richmond, VA 23284
ABSTRACT 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). Using EPR hyperfine structure and the Peisach-Blumberg correlation, an N3O1 binding mode is 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 Cterminus of the protein. Moreover, fibrillar NAcH50Q-CuII exhibits enhanced parallel β-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.
Keywords: N-acetylated α-synuclein, missense mutation, metal dyshomeostasis, EPR spectroscopy, Parkinson’s disease, aggregation
INTRODUCTION Aggregation of the neuronal protein α-synuclein (αS) is implicated in the death of dopaminergic neurons in the substantia nigra pars compacta and thus the progression of Parkinson’s disease (PD).1-4 Indeed, PD is characterized on a histopathological level by the presence of Lewy bodies, insoluble proteinaceous plaques comprised largely of misfolded and aggregated αS.5 Cerebral imaging and metal staining analyses have long linked copper dysregulation to neurodegeneration.6-9 Redox active copper can promote protein misfolding, aberrant oxidative chemistry, and/or alterations in cellular localization.10, 11 On the contrary, copper is also an important brain nutrient that is crucial for neuronal signaling mechanisms.12 Teasing out the molecular determinants of NAcαS aggregation propensity and the influence of cupric ions are of the utmost importance to understanding disease progression in PD and other synucleinopathies.
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In humans, αS is constitutively acetylated at the N-terminus.13, 14 Recently, a number of reports have detailed the often dramatic impact of this co-translational modification on the helical folding propensity, membrane binding properties, and fibrillar structure of this amyloidogenic protein.15-23 The enhanced helical conformational tendency of N-terminally acetylated αS (NAcαS) also promotes the generation of a metastable tetrameric population of NAcαS among human tissue extracts and human cell lines that was recently isolated and reported by us from a recombinant expression platform.24 The dominant intrinsically disordered conformation of the protein deservedly garners the most attention since it has a high aggregation potential towards the formation of oligomeric and/or fibrillar assemblies depending on the local environmental factors.25 N-terminal acetylation has been demonstrated to have a drastic effect on copper binding due to capping of the anchoring free amine that was structurally available in the non-acetylated αS variant (Chart 1). Baum and coworkers verified this through advanced NMR analyses of the CuII coordination modes revealing that two independent CuII subpopulations exist around H50 and D121,17 while Fernandez and coworkers reported that the CuI binding site remains at the N-terminus, yet coordinated by the thioethers of M1 and M5.26 Through theoretical structural modeling, Ramis et al. demonstrated that there are seven possible low energy representations for CuII binding within the N-terminus and seven other representations of lower stability within the C-terminus.27 In this latter study, the favored coordination mode within the N-terminus included H50 and V49 while the C-terminal site included E123, D121, and D119 (Chart 1); additional amino acid contributors to these optimized structures such as N122 or water remain unconfirmed.27
Chart 1. Structural representation highlighting the different Cuᴵᴵ binding sites that have been reported for αS, illustrated on a model based on PDB entry 1XQ828.
In our work described herein, we experimentally verify the nature of these theoretically derived coordination sites through electron paramagnetic resonance (EPR) spectroscopy and site-specific tryptophan quenching. The majority of prior αS research has focused on the non-acetylated protein, despite increasing evidence that the physiologically relevant N-acetylation imparts both structural and biochemical consequences on this dynamic protein. Our work represents the first EPR characterization of the full-length NAcαS-CuII protein as well as the CuII binding site of the disease relevant NAcH50Q missense mutation. Biophysical characterization methods enable the description of the quaternary and secondary structures of NAcαS and NAcH50Q following CuII coordination and subsequent fibrillization, revealing a very
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different trend than that previously reported for the analogous non-acetylated variants. Specifically, we now show that C-terminal CuII binding greatly enhances the aggregation propensity of NAcαS in a statistically significant manner compared to binding at the preferential N-terminal CuII coordination site. Hence, CuII coordination further augments aggregation of PD-relevant NAcH50Q.
RESULTS AND DISCUSSION Confirmation of CuII binding to N-acetylated α-Synuclein (NAcαS). The native form of human NAcαS was prepared recombinantly by exploiting NatB, a eukaryotic acetylase complex from fission yeast,29 to install the N-terminal acetyl group in situ during co-expression in Escherichia coli. Consistent with this yeast derived acetylase, the human NatB complex acetylates proteins in vivo that contain a Met-Asp starting sequence, including αS.30 NAcαS was isolated from the harvested cells and further purified via ion exchange chromatography as previously reported.31 Following treatment with CuII, high-resolution mass spectrometry (HRMS) was utilized to verify the successful installation of the N-terminal acetyl group. Our HRMS analysis also indicates that copper-bound NAcαS represents the major species present. By deconvoluting the charge state masses, we determined an average molecular weight of 14567.00838 Da, which is within 1.2 Da of the expected value for NAcαS-CuII and thus verifies both acetylation of αS and copper binding (Figure 1A). We have previously utilized trypsin digestion in combination with LC-MS/MS to validate acetylation occurring on the starting methionine residue, as expected for the activity of the NatB complex.32 The observed average molecular weight derived from the charge state distribution shown in Figure 1A is consistent with NAcαS-CuII, but we elected to additionally determine the experimental monoisotopic mass because of the increased potential for the incorporation of heavy atom isotopes in a molecule this size.33 Using the resolved isotopic distributions of the +14 charge envelope and taking into account the expected 13C incorporation pattern based on natural 13C/12C relative abundances, the observed monoisotopic mass was determined as 14556.2609 Da (Figure 1B). This value is in excellent agreement (< 7ppm) with the theoretical value for NAcαS-CuII, further supporting our assignment.
Figure 1. High resolution mass spectrometric (HRMS) characterization of NAcαS-CuII. A) Mass spectrum of the charge state distribution used to determine the average molecular weight. B) Mass spectrum of the +14 charge envelope used to calculate the monoisotopic mass.
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Characterization of CuII binding to NAcαS. In order to delineate the nature of the CuII coordination site, the magnetic properties of NAcαS-CuII were determined by electron paramagnetic resonance (EPR) spectroscopy. The dominant EPR splitting pattern for NAcαS-CuII was measured as g‖ = 2.229 and gꓕ = 2.025 (Figure 2A), exhibiting axial symmetry as is consistent with a tetragonal or square planar geometry. It has been demonstrated by Peisach and Blumberg that the g‖ region can be utilized as a diagnostic tool for copper coordination sites among both proteins and synthetic model complexes.34, 35 By plotting the hyperfine splitting values against the g‖-tensor, the nature of the donor atom contributors (N-, O-, S-) to the CuII coordination site can be determined. Based on the Peisach-Blumberg correlation diagram, the principal coordination site of NAcαS-CuII is within the N3O1 and N2O2 region (Figure 3) considering the hyperfine coupling pattern of A‖ = 192.5 G. This is consistent with the lowest energy structure calculated by Ramis et al. as N3O1 based on density functional theory calculations that includes water as the O-atom donor, the H50 imidazolyl residue and associated deprotonated amide backbone, as well as the backbone nitrogen of V49 (Figure 2B, top).27 This CuII coordination mode was originally suggested by KowalikJankowska et al. as a secondary site within non-acetylated αS protein based on potentiometric measurements.36
Figure 2. A) EPR spectrum of ᴺᴬᶜαS-Cuᴵᴵ (magenta) depicting two populations of coordination spheres. Both the principal binding site (dark blue) and the secondary site (orange) are denoted. B) Schematic representation of the preferential Cuᴵᴵ/N3O1 binding site of ᴺᴬᶜαS (top) and the postulated secondary site (bottom).
A lower population contributor to the CuII hyperfine structure is also resolved with a g‖ = 2.304 and A‖ = 169.6 G (Figure 2A). After simulating the EPR data for NAcαS-CuII (Figure S1) using EasySpin,37 the ratio of each population was calculated as 68% (major) and 32% (minor). A shift of the g‖-tensor to higher values is typically associated with an increase in O-atom donation character to the binding mode (Figure 3). Baum and coworkers have previously hinted that the Asp121 C-terminal residue may additionally contribute to the CuII coordination sphere based on heteronuclear single-quantum coherence (HSQC) NMR measurements,17 and hence we consider this mode as a potential minor contributor (Figure 2B, bottom).
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Figure 3. Peisach-Blumberg plot depicting the correlation between A‖ and g‖ parameters for Cuᴵᴵ adducts of different proteins, peptides, and/or metallocomplexes (triangles): N4 – four nitrogen donors (blue), N3O1 – three nitrogen donors and one oxygen donor (purple), N2O2 – two nitrogen and two oxygen donors (proteins, light green; coordination complexes, dark green), N1O3 – one nitrogen and three oxygen donors (yellow), O4 – four oxygen donors (red). Previously reported Cuᴵᴵ-bound non-acetylated αS protein variants and peptide models follow the same color scheme and are demonstrated by open circles (see also Supporting Information Table S1). Data corresponding to this work are signified for the principal binding sites of ᴺᴬᶜαS-Cuᴵᴵ (site 1 - magenta; site 2 - black) and ᴺᴬᶜH50Q-Cuᴵᴵ (teal) by closed squares and dotted lines. In order to confirm that the N3O1 subpopulations are not resulting from N-terminal contributions, we measured CuII tryptophan quenching, as the intrinsic fluorescence of tryptophan can serve as a reporter of changes in the local environment.38, 39 An N-terminally acetylated F4W variant (NAcF4W) was prepared through site-directed mutagenesis and compared to the non-acetylated F4W variant for contrast. Identification of the N-terminal amine as the anchoring residue for CuII coordination to non-acetylated αS (Figure 4A) was previously established based on full emission quenching of W4 with addition of 1 equivalent of CuII (Figure 4B), while tryptophan insertions in other regions of the protein exhibited only minimal quenching (
