PhD Defence - Mahdi Lavasani

NMR-Guided investigation of Membrane-Regulated Conformational Plasticity and LC3B Recognition of α-synuclein: Implications for understanding protein-membrane interactions, Aggregation and Neurodegeneration 

Abstract

Parkinson’s disease (PD) and related synucleinopathies are characterized by the accumulation of α-synuclein (α-syn) aggregates in neurons. Although α-syn is intrinsically disordered in solution, it undergoes major conformational transitions upon interacting with lipid membranes and protein partners. These interactions influence α-syn aggregation, membrane remodelling, cellular trafficking, and toxicity. This thesis investigates two interconnected aspects of α-syn biology: how membrane electrostatics regulate α-syn conformational dynamics and aggregation, and how α-syn interacts with the autophagy-related protein LC3B.  

The first part of this work examines how negatively charged lipid membranes regulate α-syn membrane association and fibrillogenesis. Using solid-state nuclear magnetic resonance (ssNMR) spectroscopy and chemical exchange saturation transfer experiments, we show that decreasing membrane electrostatic attraction progressively releases regions of α-syn from the lipid surface, particularly within the central Non-amyloid beta component (NAC) domain, which is critical for aggregation, and parts of the N-terminal region. These released segments predominantly adopt random-coil conformations and show no evidence of transient rebinding to the membrane. Thioflavin T (ThT) fluorescence assays further demonstrate that reduced membrane charge and progressive release of the NAC and N-terminal regions correlate with enhanced nucleation rates during fibril formation. Together, these findings support a model in which membrane charge modulates the balance between membranebound and aggregation-prone α-syn conformations, thereby coupling membrane interactions to aggregation kinetics.  

The second part of this thesis focuses on the interaction between α-syn and LC3B, a member of the ATG8 protein family that associates with autophagosomal membranes and has been implicated in the processing and propagation of α-syn pathology. Using solution NMR titration experiments, we mapped residue-specific interaction interfaces on both α-syn and LC3B and identified a non-canonical LC3-interacting region-like (LIR-like) motif, Y125-E126-M127-P128, within the α-syn terminal region. This motif engages the canonical LIR-binding site of LC3B, which is involved in interactions with autophagy receptors. Structural models generated using High Ambiguity Driven protein-protein DOCKing (HADDOCK) and AlphaFold3 (AF3) support the formation of a dynamic α-syn/LC3B complex involving both electrostatic and hydrophobic interactions. Isothermal titration calorimetry and mutational analysis further validated the interaction model and confirmed the importance of key interface residues.  

Collectively, this thesis demonstrates that α-syn conformational plasticity is strongly regulated by interactions with membranes and autophagy-associated proteins. Membrane electrostatics influence the balance between membrane-bound and aggregation-prone conformations, while direct recognition of LC3B establishes a mechanistic link between α-syn pathology and autophagic pathways. These findings advance our understanding of the molecular determinants governing α-syn aggregation, membrane remodelling, and cellular clearance mechanisms in PD and related neurodegenerative disorders.  

Examination Committee

• Dr. Robert Wickham, Chair 
• Dr. Vladimir Ladizhansky, Advisor 
• Dr. Scott Ryan, Advisor  
• Dr. Rui Huang, Graduate Faculty 
• Dr. Kalle Gehring, External Examiner (McGill University)