Structure and membrane binding of alpha-synuclein

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In dopaminergic neurons, a-synuclein (aS) partitions between a disordered cytosolic state and a lipid-bound state. Binding of aS to membrane phospholipids is implicated in its functional role of synaptic regulation, but also impacts fibril formation associated with Parkinson's disease. A 2011 study by Selkoe et al reported that if aS is expressed in mammalian cells and purified without a heat denaturation step, it adopts a stable tetrameric helical structure. We developed this expression system but were unable to duplicate their findings. However, we found by high-resolution NMR spectroscopy and circular dichroism (CD) measurements, that the N-terminal acetylation which occurs in mammalian cells impacts the protein's structure and dynamics in free solution and also affects the protein's membrane binding properties. While no tetrameric form of acetylated aS could be isolated, N-terminal acetylation resulted in chemical shift perturbations of the first 12 residues of the protein which progressively decreased with distance from the N-terminus. The directions of the chemical shift changes and small changes in backbone 3JHH couplings are consistent with an increase in alpha-helicity of the first six residues of aS, although a high degree of dynamic conformational disorder remains and the helical structure is sampled less than 20%. Chemical shift and 3JHH data for the intact protein are virtually indistinguishable from those recorded for the corresponding N-terminally acetylated and non-acetylated 15-residue synthetic peptides. An increase in alpha-helicity at the N-terminus of aS is supported by CD data on the acetylated peptide, and by weak medium-range NOE contacts indicative of alpha-helical character. The remainder of the protein has chemical shift values that are very close to random coil values and indistinguishable between the two forms of the protein. No significant difference in the fibrillation kinetics were observed between acetylated and non-acetylated aS. However, the lipid binding properties of aS are strongly impacted by acetylation, and exhibit distinct behavior for the first 12 residues, indicative of an initiation role for the N-terminal residues in an "initiation-elongation" process of binding to the membrane. Using a diverse set of NMR parameters, including backbone chemical shifts, homo- and heteronuclear J couplings, as well as short range NOEs, we have developed a detailed description of the backbone torsion angle distribution at the residue-specific level for this intrinsically disordered protein. Although the deviations from classical coil libraries are small, they are statistically quite significant and provide a first detailed view of the backbone torsion angles sampled by an intrinsically disordered protein. These backbone torsion angle distributions cross validate considerably better than ensemble model descriptions of the full chain, an approach developed in several other laboratories.