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Phosphorylation of α-synuclein at the Serine-129 site (α-syn Ser129P) is an established pathologic hallmark of synucleinopathies, and also a therapeutic target. In physiologic states, only a small fraction of total α-syn is phosphorylated at this site, and consequently, almost all studies to date have focused on putative pathologic roles of this post-translational modification. We noticed that unlike native (total) α-syn that is widely expressed throughout the brain, the overall pattern of α-syn Ser129P is restricted, suggesting intrinsic regulation and putative physiologic roles. Surprisingly, preventing phosphorylation at the Ser-129 site blocked the ability of α-syn to attenuate activity-dependent synaptic vesicle (SV) recycling; widely thought to reflect its normal function. Exploring mechanisms, we found that neuronal activity augments α-syn Ser-129P, and this phosphorylation is required for α-syn binding to VAMP2 and synapsin - two functional binding-partners that are necessary for α-syn function. AlphaFold2-driven modeling suggests a scenario where Ser129P induces conformational changes in the C-terminus that stabilizes this region and facilitates protein-protein interactions. Our experiments indicate that the pathology-associated Ser129P is an unexpected physiologic trigger of α-syn function, which has broad implications for pathophysiology and drug-development.

Identifying subtle conformational fluctuations underlying the dynamics of bio macromolecules is crucial for resolving their free energy landscape. We show that a collective variable, originally proposed for crystalline solids, is able to filter out essential macromolecular motions more efficiently than other approaches. While homogenous or 'affine' deformations of the biopolymer are trivial, biopolymer conformations are complicated by the occurrence of in-homogenous or 'non-affine' displacements of atoms relative to their positions in the native structure. We show that these displacements encode functionally relevant conformations of macromolecule and, in combination with a formalism based upon time-structured independent component analysis, quantitatively resolve the free energy landscape of a number of macromolecules of hierarchical complexity. The kinetics of conformational transitions among the basins can now be mapped within the framework of a Markov state model. The non-affine modes, obtained by projecting out homogenous fluctuations from the local displacements, are found to be responsible for local structural changes required for transitioning between pairs of macro states.

Apolipoprotein E (apoE), a major determinant protein for lipid metabolism, actively participates in lipid transport in the central nervous system via high-affinity interaction with the low-density lipoprotein receptor (LDLR). Prior evidences indicate that the phospholipids first need to assemble around apoE before the protein can recognize its receptor. However, despite multiple attempts via spectroscopic and biochemical investigations, it is unclear what are the impacts of lipid assembly on the globular structure of apoE. Here, using a combination of all-atom and coarse-grained molecular dynamics simulations, we demonstrate that an otherwise compact tertiary fold of monomeric apoE3 spontaneously unwraps in an aqueous phospholipid solution in two distinct stages. Interestingly, these structural reorganizations are triggered by an initial localized binding of lipid molecules to the C-terminal domain of the protein, which induce a rapid separation of the C-terminal domain of apoE3 from the rest of its tertiary fold. This is followed by a slow lipidinduced interhelix separation event within the N-terminal domain of the protein, as seen in an extensively long coarse-grained simulation. Remarkably, the resultant complex takes the shape of an "open conformation" of the lipid-stabilized unwrapped protein, which intriguingly coincides with an earlier proposal by a small-angle X-ray scattering (SAXS) experiment. The lipid-binding activity and the lipid-induced protein conformation are found to be robust across a monomeric mutant and wild-type sequence of apoE3. The "open" complex derived in coarse-grained simulation retains its structural morphology after reverse-mapping to the all-atom representation. Collectively, the investigation puts forward a plausible structure of currently elusive conformationally activated state of apoE3, which is primed for recognition by the lipoprotein receptor and can be exploited for eventual lipid transport.

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