The NMR Study On The Interaction Of α-Synuclein With Mitochondrial Membrane

Parkinson’s disease(PD)is the second most common neurodegenerative disease characterized by Lewy bodies,which is caused by the loss of dopaminergic neurons in the substantia nigra pars compacta.The central component of Lewy bodies is αsynuclein(α-syn)insoluble aggregates,companied by broken organelles like mitochondria.Mitochondrial dysfunction plays a central role in PD,and a large amount of α-syn has been observed to be localized in mitochondrial inner membrane in the brains of PD patients.α-syn associates with membranes through its N-terminal domain,changes in the interaction of α-syn with mitochondrial membrane affects mitochondrial membrane integrity,indicating that α-syn’s membrane binding is closely related to mitochondrial dysfunction.However,the mechanism underlying the interaction of αsyn with mitochondrial membrane as well as mitochondrial dysfunction implicated in PD are not yet clear.Here,we use nuclear magnetic resonance(NMR)to study the interaction of α-syn with mitochondrial membrane and the molecular mechanism of its cytotoxicity.1.The molecular mechanism of toxicity.C-terminally truncated α-syn(CT-α-syn)is found in brains of PD patients,which tends to aggregate and reduces cell viability.To investigate the mechanism behind its toxicity,we used paramagnetic relaxation enhancement to show that the C-terminal truncation disrupted the compact conformation of full-length(FL-α-syn),and CT-α-syn adopted a more extended conformation in which the N-terminus was exposed,resulting in the enhanced interaction of the N-terminus with membrane.We further performed functional experiments and found that CT-α-syn increased mitochondrial membrane permeability through enhanced association with mitochondrial membrane,thereby leading to mitochondrial damage.Based on these findings,we proposed the molecular mechanism by which C-terminal truncation enhanced α-syn’s cytotoxicity.2.Studying the interactions of α-syn in intact mitochondria by in situ NMR.We isolated intact mitochondria from mouse liver and studied the interactions of α-syn and its variants after being delivered into mitochondria through electroporation.The results showed that the N-and C-terminus of α-syn were involved in interactions in mitochondria,whereas the N-terminus mainly bound to membranes and molecular chaperones.Then we attempted to separate mitochondrial membrane from matrix,and we found a stronger interaction between the N-terminus of α-syn with membrane than with matrix.PD-related mutant Ala30Pro(A30P)has a well-established ability to impair membrane binding,but we didn’t observe the reduced affinity for mitochondrial chaperone tumor necrosis factor receptor-associated protein 1(TRAP1).Besides,the N-terminus of A30P showed the decreased interactions in mitochondria.These results suggested that in mitochondria,the membrane interaction predominated over the interaction of α-syn with molecular chaperones.When α-syn’s C-terminus was truncated,a significant leakage from mitochondria was detected due to the enhanced interaction with mitochondrial membrane,which further corroborated with the disruptive ability of C-terminal truncations to mitochondrial membrane.In Lewy bodies of PD,approximately 90%of α-syn is phosphorylated at Ser129.We found that phosphorylated α-syn displayed weaker affinity for mitochondrial membrane and chaperones in mitochondria,indicating that the C-terminal modification modulated the N-terminal interactions.In addition,we studied the interactions of methionine-oxidizedα-syn in mitochondria.The results showed that in mitochondria,the redox state of αsyn was preserved,and the oxidation weakened Met5(M5)-involved interactions.In vitro experiments showed that the oxidation disrupted the interaction of M5 with mimic mitochondrial membrane,but it had no effect on binding to TRAP1.These findings indicated that the oxidative modification was able to impair the interaction of M5 with mitochondrial membrane.In conclusion,we employed NMR to study the interactions of α-syn with mitochondrial membrane in membrane-mimic environments and in-situ mitochondria,and showed that the membrane binding was the dominant interaction regarding the Nterminus of α-syn in mitochondria.The enhanced membrane interaction resulted from C-terminal truncation would induce mitochondrial damage.Our work reveals the molecular mechanism of α-syn interacting with mitochondrial membrane,and benefits to understand the role of α-syn in the alteration of mitochondrial functions leading to PD.