Inhibition of α-Synuclein Aggregation by the Interaction Between Protein Disulfide Isomerase and α-Synuclein
PEI Yun-shan1,2, ZHANG Cai1,2, LIU Xiao-li1, CHENG Kai1, ZHANG Ze-ting,1, LI Cong-gang1
1. CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan 430071, China
2. University of Chinese Academy of Sciences, Beijing 100049, China
Abnormally misfolded and aggregated α-synuclein (αsyn) is the hallmark of Parkinson's disease (PD). Molecular chaperone protein disulfide isomerase (PDI) has been shown to interact with αsyn and inhibit its aggregation in vitro, but the mechanism for the recognition of αsyn by PDI is not yet clear. Herein, we used nuclear magnetic resonance (NMR) spectroscopy to identify that human PDI b'xa' bound with the N-terminal domain of αsyn, and thioflavin T (ThT) fluorescence assay revealed that b'xa' domain of PDI significantly inhibited αsyn aggregation. Furthermore, by using NMR titration, we observed that PDI bound to αsyn mainly through its hydrophobic cavity of the b' domain. Based on these findings, a docking model of PDI binding with αsyn was established and a possible mechanism of how PDI inhibits αsyn aggregation was proposed. Our work provides experimental evidences for understanding the inhibitory role of PDI in αsyn aggregation.
Keywords：nuclear magnetic resonance (NMR)
;
α-synuclein (αsyn)
;
protein disulfide isomerase
;
interaction
;
fibrillation
PEI Yun-shan. Inhibition of α-Synuclein Aggregation by the Interaction Between Protein Disulfide Isomerase and α-Synuclein. Chinese Journal of Magnetic Resonance[J], 2022, 39(4): 381-392 doi:10.11938/cjmr20222974
Fig.1
The b'xa' domain of PDI interacts with N-terminal domain of αsyn. (a) Model for PDI (red) primary sequence, PDI ab domain (pink) and PDI b'xa' domain (blue) are shown underneath. (b) Surface crystal structure of human PDI (PDB ID: 4EKZ). (c) Protein purities analyzed by SDS-PAGE. M, Marker; lane 1, αsyn1-60; lane 2, αsyn; lane 3, PDI ab; lane 4, PDI b'xa'; lane 5, PDI. (d) and (e) Spectral overlay of αsyn in the absence (black) and presence of equivalent amount of PDI (red) and PDI b'xa' (blue). Residues showing significant signal attenuation or shifts were marked. (f) Residue-resolved attenuation (I/I0) of αsyn in the presence of PDI (red), PDI ab (pink) and PDI b'xa' (blue). (g) Hydrophobic residues of αsyn predicted by OMH method, the binding and hydrophobic areas are colored gray
Fig.2
The effect on αsyn aggregation of PDI, PDI ab and PDI b'xa' detected by ThT experiment. (a) Aggregations kinetics of αsyn in the absence (black) and presence of equimolar PDI ab (pink), PDI b'xa' (blue) and PDI (red). (b) The fitted half-time (t1/2) values of aggregation and their statistical analysis. ns: not significant, ****: p < 0.0001; the dots represent the values obtained from four parallel experiments
Fig.3
NMR results showing the interaction between PDI b'xa' with the N-terminal domain of αsyn. Overlaid 1H-15N TROSY spectra of PDI b'xa' (red) with (blue) αsyn 1-19 (a), αsyn 30-42 (d) or αsyn 1-60 (g). E239 were selected as representative residues for global chemical shift perturbations (CSPs) during NMR titration. Residue-resolved CSPs of PDI b'xa' during titrations with αsyn 1-19(b), 30-42(e) and 1-60 (h). KD values were fitted from curves of residues with significant CSPs, as a function of αsyn 1-19(c), αsyn 30-42(f), and αsyn 1-60 (i) concentration. Residues showing significant CSPs were marked in spectra and mapped red on the crystal structure surface of PDI b'xa'. Threshold level was indicated as 'mean+standard deviation'. Disperse residues were verified and removed by the threshold of 'mean +2×standard deviation'
Fig.4
Hydrophobic binding mode of PDI with the N-terminal domain of αsyn. (a) Docking model of PDI binding αsyn1-40 (left panel) and a local zoom view of binding area (right panel). The relevant residues of αsyn1-40 involving in binding PDI were depicted as magenta sticks. HDOCK experiments were performed on the web server (http://hdock.phys.hust.edu.cn/) by uploading human PDI crystal structure, amino acid sequences of αsyn1-40 and their binding sites identified by NMR titration. Optimal docking results were plotted with PyMol software, PDI molecular surface was colored gray and its hydrophobic pocket was marked in red, the magenta cartoon represented αsyn1-40 structure. (b) Alignment of amino acid sequence of human PDI b'xa' from HiPDI b'xa'. Sequence identity and similarity were calculated by Clustalw[52] and ESPript 3.0[53]. Similar amino acids were showed as black letters in yellow boxes and identical amino acids were showed as white letters in red boxes. The secondary structural domains were indicated on the top of the sequences. Blue and green asterisks represented the key hydrophobic residues of αsyn interacting with human PDI and HiPDI, respectively
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Hydrophobic binding mode of PDI with the N-terminal domain of <i>α</i>syn. (a) Docking model of PDI binding <i>α</i>syn1-40 (left panel) and a local zoom view of binding area (right panel). The relevant residues of <i>α</i>syn1-40 involving in binding PDI were depicted as magenta sticks. HDOCK experiments were performed on the web server (http://hdock.phys.hust.edu.cn/) by uploading human PDI crystal structure, amino acid sequences of <i>α</i>syn1-40 and their binding sites identified by NMR titration. Optimal docking results were plotted with PyMol software, PDI molecular surface was colored gray and its hydrophobic pocket was marked in red, the magenta cartoon represented <i>α</i>syn1-40 structure. (b) Alignment of amino acid sequence of human PDI b'xa' from <i>Hi</i>PDI b'xa'. Sequence identity and similarity were calculated by Clustalw<sup>[<xref ref-type="bibr" rid="b52">52</xref>]</sup> and ESPript 3.0<sup>[<xref ref-type="bibr" rid="b53">53</xref>]</sup>. Similar amino acids were showed as black letters in yellow boxes and identical amino acids were showed as white letters in red boxes. The secondary structural domains were indicated on the top of the sequences. Blue and green asterisks represented the key hydrophobic residues of <i>α</i>syn interacting with human PDI and <i>Hi</i>PDI, respectivelyFig.4
... [52] and ESPript 3.0[53]. Similar amino acids were showed as black letters in yellow boxes and identical amino acids were showed as white letters in red boxes. The secondary structural domains were indicated on the top of the sequences. Blue and green asterisks represented the key hydrophobic residues of αsyn interacting with human PDI and HiPDI, respectively Fig.4
Hydrophobic binding mode of PDI with the N-terminal domain of <i>α</i>syn. (a) Docking model of PDI binding <i>α</i>syn1-40 (left panel) and a local zoom view of binding area (right panel). The relevant residues of <i>α</i>syn1-40 involving in binding PDI were depicted as magenta sticks. HDOCK experiments were performed on the web server (http://hdock.phys.hust.edu.cn/) by uploading human PDI crystal structure, amino acid sequences of <i>α</i>syn1-40 and their binding sites identified by NMR titration. Optimal docking results were plotted with PyMol software, PDI molecular surface was colored gray and its hydrophobic pocket was marked in red, the magenta cartoon represented <i>α</i>syn1-40 structure. (b) Alignment of amino acid sequence of human PDI b'xa' from <i>Hi</i>PDI b'xa'. Sequence identity and similarity were calculated by Clustalw<sup>[<xref ref-type="bibr" rid="b52">52</xref>]</sup> and ESPript 3.0<sup>[<xref ref-type="bibr" rid="b53">53</xref>]</sup>. Similar amino acids were showed as black letters in yellow boxes and identical amino acids were showed as white letters in red boxes. The secondary structural domains were indicated on the top of the sequences. Blue and green asterisks represented the key hydrophobic residues of <i>α</i>syn interacting with human PDI and <i>Hi</i>PDI, respectivelyFig.4
... [53]. Similar amino acids were showed as black letters in yellow boxes and identical amino acids were showed as white letters in red boxes. The secondary structural domains were indicated on the top of the sequences. Blue and green asterisks represented the key hydrophobic residues of αsyn interacting with human PDI and HiPDI, respectively Fig.4
