Membrane Interactions of α-Synuclein Revealed by Multiscale Molecular Dynamics Simulations, Markov State Models, and NMR

Abstract

α-Synuclein (αS) is a presynaptic protein that binds to cell membranes and is linked to Parkinson's disease (PD). Binding of αS to membranes is a likely first step in the molecular pathophysiology of PD. The αS molecule can adopt multiple conformations, being largely disordered in water, adopting a β-sheet conformation when present in amyloid fibrils, and forming a dynamic multiplicity of α-helical conformations when bound to lipid bilayers and related membrane-mimetic surfaces. Multiscale molecular dynamics simulations in conjunction with nuclear magnetic resonance (NMR) and cross-linking mass spectrometry (XLMS) measurements are used to explore the interactions of αS with an anionic lipid bilayer. The simulations and NMR measurements together reveal a break in the helical structure of the central non-amyloid-β component (NAC) region of αS in the vicinity of residues 65-70, which may facilitate subsequent oligomer formation. Coarse-grained simulations of αS starting from the structure of αS when bound to a detergent micelle reveal the overall pattern of protein contacts to anionic lipid bilayers, while subsequent all-atom simulations provide details of conformational changes upon membrane binding. In particular, simulations and NMR data for liposome-bound αS indicate incipient β-strand formation in the NAC region, which is supported by intramolecular contacts seen via XLMS and simulations. Markov state models based on the all-atom simulations suggest a mechanism of conformational change of membrane-bound αS via a dynamic helix break in the region of residue 65 in the NAC region. The emergent dynamic model of membrane-interacting αS advances our understanding of the mechanism of PD, potentially aiding the design of novel therapeutic approaches.

Figure 1

Interaction of αS with a lipid bilayer explored by multiscale simulations alongside biophysical measurements. (A) Flow diagram of the use of CG and AT MD simulations combined with biophysical measurements and MSMs of the AT simulations. (B) Successive snapshots from a CG simulation of the interaction of αS with a PG bilayer. Monomeric αS is initially positioned distal to the membrane. During 10 replicate simulations, each of the 1 μs duration αS bound to the membrane. Colors are based on the initial starting model (PDB id 1XQ8): two helices (helix 1 in green and helix 2 in gray/purple) separated by an interhelical loop (in pink) and followed by a C-terminal disordered region (in orange). (C) Total number of simulations across the ensemble where each residue of αS is in contact with the membrane. (D) Total number of individual contacts of αS with PG summed over the 10 simulation replicates. A residue is considered in contact with the lipid if the residue backbone particle is within 0.7 nm of any lipid particle. (E) Side view of αS bound to the surface of a PG membrane from the endpoint of a simulation. Red particles indicate the top 14 residues making contact with PG lipid headgroups shown in gray.

Figure 2

CG MD simulations of the interaction of αS with a PC/PE/PS (2:5:3) lipid bilayer. Simulations were of 10 replicates each for 2 μs. (A) Contacts for all lipids for each residue of αS summed over the 10 simulation replicates. (B) Total contacts shown separately for the three lipid species. Colors on the histogram indicate the structural regions defined in Figure 1B (helix 1 in green, helix 2 in gray/purple, interhelical loop in pink, C-terminal disordered region in orange). (C) Total contacts for each lipid species (PC = red; PE = blue; PS = green) shown as a function of time.

Figure 3

AT MD simulations of the interaction of αS with a POPG membrane. (A) Three binding poses taken from the CG simulations are shown, chosen to capture initial interactions at the C-terminus (conformation 1-left), at the N-terminus (conformation 2-center) and at the inter-helical region (conformation 3-right). In each case, the CG binding pose was converted to the corresponding AT models shown, initiating AT simulations (of duration 100–250 ns with 10 replicates) and resulting in an aggregated simulation time of 5.5 μs. (B) Number of contacts to lipids within a 1 nm cutoff of the Cα atom of each residue is shown as a function of time and residue number for each starting model, averaged across replicates.

Figure 4

Secondary structure of αS, comparing AT simulations when bound to a POPG bilayer with NMR data. (A) Secondary structure (as defined by DSSP; blue = α-helix; red = β-sheet; green = bend, yellow = turn, white = coil) as a function of residue number and time for a representative trajectory starting from conformation 1 (see Figure 3 above). Loss of helical structure in the middle of helix 2 (in the region of residue 70) is observed. The locations of helices 1 and 2 in the NMR structure (PDB id 1XQ8) are indicated to the right of the diagram. (B) Comparable secondary structure plot for a representative trajectory starting from conformation 2. The red ellipse highlights formation of a small region of β-sheet around residue 70. (C) Frequency of an α-helical secondary structure as a function of residue averaged across all simulations starting from conformation 1. The fraction of α-helix is reduced in favor of random coil conformations between residues 60 and 70. (Similar profiles are seen for simulations starting from conformations 2 and 3). (D) NMR chemical shift data showing secondary structure propensities. Chemical shift indexing shows field-shifted atoms in the region between residues 60 and 75, indicating a reduction in the propensity of the α-helical structure. The values obtained are the average of the shift observed versus random coil expected shifts, weighted by their sensitivity to α-helical or extended conformations. Data are shown for αS bound to bicelles composed of DHPC, DMPG, and PIP₂ (see Methods for details). The extent of the two helices in the SDS-bound structure (PDB id 1XQ8) is again indicated by arrows.

Figure 5

Residue–residue distances of the αS monomer binding to a POPG bilayer, comparing the results of XLMS studies with contacts in simulations. (A) XLMS crosslink-pattern observed in αS when bound to POPG liposomes. The XLMS data are used to determine a sum of all PSMs found between two positions of the protein. This is shown as a pattern of PSMs determined with binwidth = 1, indicating the number (red = higher, blue = lower) of PSMs for each individual cross-link pair. (B) XLMS-pattern mapped onto the sequence of αS with K (blue), E and D (red) residues indicated. (C) Residue–residue distances generated from the AT simulation ensembles from conformations 1, 2, and 3. Off-diagonal elements are seen clearly in both the experimental data and the simulations, corresponding to contacts between helix 1 and the N-terminal segment of helix 2.

Figure 6

MSM of the conformational dynamics of residues 60–70 based on the AT simulations. (A) MSM microstate transitions and representative structures. Yellow circles indicate the two lowest energy macrostate centers from which representative structures (B,C) were extracted. (B) Residues 60–7 in an α-helical conformation and generated from a region that is structurally close to the conformation at the start of the trajectory. (C) Break in the helix such that the peptide backbone around residues 60–70 folds back on itself. (D) Schematic representation of the dynamic structure of αS bound to an anionic membrane showing helix 1 in blue, the interhelix loop around residue 40 in green, helix 2 in cyan, the dynamic break/possible β-sheet region centered around residue ∼65 in yellow, and the C-terminal disordered region in red. The potential seed region for subsequent amyloid formation is indicated by the yellow schematic labeled β.