Differential copper binding to alpha-synuclein and its disease-associated mutants affect the aggregation and amyloid formation

• Published in: Biochimica et biophysica acta. General subjects 2017-02, Vol.1861 (2), p.365-374
• Main Authors: Ranjan, Priyatosh, Ghosh, Dhiman, Yarramala, Deepthi S., Das, Subhadeep, Maji, Samir K., Kumar, Ashutosh
• Format: Article
• Language: English
• Subjects: alpha-Synuclein - metabolism; Amyloid - metabolism; Amyloidogenic Proteins - metabolism; Amyloidosis - metabolism; Amyloidosis - pathology; Binding Sites; Cell Line; Copper; Copper - metabolism; Humans; Kinetics; Long-range contacts; Non-amyloid-β component; Parkinson Disease - metabolism; Parkinson Disease - pathology; Protein Aggregation, Pathological - metabolism; Protein Aggregation, Pathological - pathology; α-Syn G51D; α-Syn H50Q
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2016.11.043

Copper is an essential trace element required for the proper functioning of various enzymes present in the central nervous system. An imbalance in the copper homeostasis results in the pathology of various neurodegenerative disorders including Parkinson’s Disease. Hence, residue specific interaction of Cu2+ to α-Syn along with the familial mutants H50Q and G51D needs to be studied in detail. We investigated the residue specific mapping of Cu2+ binding sites and binding strength using solution-state NMR and ITC respectively. The aggregation kinetics, secondary structural changes, and morphology of the formed fibrils in the presence and absence of Cu2+ were studied using fluorescence, CD, and AFM respectively. Copper binding to α-Syn takes place at three different sites with a higher affinity for the region 48-53. While one of the sites got abolished in the case of H50Q, the mutant G51D showed a binding pattern similar to WT. The aggregation kinetics of these proteins in the presence of Cu2+ showed an enhanced rate of fibril formation with a pronounced effect for G51D. Cu2+ binding results in the destabilization of long-range tertiary interactions in α-Syn leading to the exposure of highly amyloidogenic NAC region which results in the increased rate of fibril formation. Although the residues 48-53 have a stronger affinity for Cu2+ in case of WT and G51D, the binding is not responsible for enhancing the rate of fibril formation in case of H50Q. These findings will help in the better understanding of Cu2+ catalyzed aggregation of synucleins. The binding of Cu2+ to α-Syn occurs at three binding sites with a higher affinity for region 48-53. In case of the familial mutant H50Q, the single point mutation resulted in the abolishment of Cu2+ binding site in the region 48-53. However, binding at the N- (3-11) and C- (115-123) terminus was found to be similar to the WT. The binding of Cu2+ to the familial mutant G51D was found to be comparable to the WT. In all the synucleins, an increased rate of fibril formation was observed in the presence of Cu2+ with the release of long-range contacts between the N- and C- terminus. Even though the binding of Cu2+ to the mutant H50Q was less, it showed the formation of the fibrils at an early stage suggesting that the involvement of region 48-53 in Cu2+ binding is not responsible for enhancing the fibril formation. [Display omitted] •Residue specific mapping of Cu2+ binding sites along with the strength and order of binding was