α-Synuclein-Induced Membrane Remodeling Is Driven by Binding Affinity, Partition Depth, and Interleaflet Order Asymmetry

Abstract

We have investigated the membrane remodeling capacity of the N-terminal membrane-binding domain of α-synuclein (α-Syn100). Using fluorescence correlation spectroscopy and vesicle clearance assays, we show that α-Syn100 fully tubulates POPG vesicles, the first demonstration that the amphipathic helix on its own is capable of this effect. We also show that at equal density of membrane-bound protein, α-Syn has dramatically reduced affinity for, and does not tubulate, vesicles composed of a 1:1 POPG:POPC mixture. Coarse-grained molecular dynamics simulations suggested that the difference between the pure POPG and mixture results may be attributed to differences in the protein’s partition depth, the membrane’s hydrophobic thickness, and disruption of acyl chain order. To explore the importance of these attributes compared with the role of the reduced binding energy, we created an α-Syn100 variant in which we removed the hydrophobic core of the non-amyloid component (NAC) domain and tested its impact on pure POPG vesicles. We observed a substantial reduction in binding affinity and tubulation, and simulations of the NAC-null protein suggested that the reduced binding energy increases the protein mobility on the bilayer surface, likely impacting the protein’s ability to assemble into organized pretubule structures. We also used simulations to explore a potential role for interleaflet coupling as an additional driving force for tubulation. We conclude that symmetry across the leaflets in the tubulated state maximizes the interaction energy of the two leaflets and relieves the strain induced by the hydrophobic void beneath the amphipathic helix.

α-Synuclein Reduces Tension and Increases Undulations in Simulations of Small Unilamellar Vesicles

“…As expected, the headgroup density is higher in the inner leaflet due to compaction in the convex vesicle. The protein has very little effect on bilayer structure, partitioning to a similar depth as previous studies (6,9,10,17), with no notable change in bilayer thickness (Fig. S1 in the Supporting Material).…”

Computational ‘microscopy’ of cellular membranes

“…When sufficient chemical information is retained, coarse-grained models can discriminate between different lipids and distinguish protein residues, and thus form a useful bridge between atomistic and macroscopic data. For instance, one can now simulate the collective lipid-mediated selfassembly of membrane proteins (Benjamini and Smit, 2013;Hall et al, 2012;Johnston et al, 2012;Periole et al, 2012;van den Bogaart et al, 2011) and their sorting between different membrane domains (de Jong et al, 2013;Janosi et al, 2012;Schäfer et al, 2011), as well as large-scale protein-induced membrane remodelling -including fusion and fission events (Baoukina and Tieleman, 2010;Braun et al, 2014;Davies et al, 2012;Fuhrmans and Müller, 2015;Kawamoto et al, 2015;Pinot et al, 2014;Risselada et al, 2014;Simunovic et al, 2013).…”

Membrane Remodelling Activity of ?-Synuclein

“…Recently, it has been shown that all three human synucleins, α, β and γ 52 , induce the tubulation of vesicles composed of brain polar lipids, at concentrations estimated to be physiologically relevant 31 . In vitro, the remodelling activity is most efficient when pure negatively charged membranes are involved 27,30,32,53 . The fact that α-Syn shares common features with known curvature-inducing protein and with apolipoproteins suggests shared functionalities and a direct involvement of α-Syn in vesicle trafficking and lipid transport/storage.…”