Serine-129 phosphorylation of α-synuclein is an activity-dependent trigger for physiologic protein-protein interactions and synaptic function.

Phosphorylation of α-synuclein at the serine-129 site (α-syn Ser129P) is an established pathologic hallmark of synucleinopathies and a therapeutic target. In physiologic states, only a fraction of α-syn is phosphorylated at this site, and most studies have focused on the pathologic roles of this post-translational modification. We found that unlike wild-type (WT) α-syn, which is widely expressed throughout the brain, the overall pattern of α-syn Ser129P is restricted, suggesting intrinsic regulation. Surprisingly, preventing Ser129P blocked activity-dependent synaptic attenuation by α-syn-thought to reflect its normal function. Exploring mechanisms, we found that neuronal activity augments Ser129P, which is a trigger for protein-protein interactions that are necessary for mediating α-syn function at the synapse. AlphaFold2-driven modeling and membrane-binding simulations suggest a scenario where Ser129P induces conformational changes that facilitate interactions with binding partners. Our experiments offer a new conceptual platform for investigating the role of Ser129 in synucleinopathies, with implications for drug development.

Spontaneous transmembrane pore formation by short-chain synthetic peptide.

Amphiphilic ß-peptides, which are synthetically designed short-chain helical foldamers of ß-amino acids, are established potent biomimetic alternatives of natural antimicrobial peptides. An intriguing question is how the distinct molecular architecture of these short-chain and rigid synthetic peptides translates to its potent membrane-disruption ability. Here, we address this question via a combination of all-atom and coarse-grained molecular dynamics simulations of the interaction of mixed phospholipid bilayer with an antimicrobial 10-residue globally amphiphilic helical ß-peptide at a wide range of concentrations. The simulation demonstrates that multiple copies of this synthetic peptide, initially placed in aqueous solution, readily self-assemble and adsorb at membrane interface. Subsequently, beyond a threshold peptide/lipid ratio, the surface-adsorbed oligomeric aggregate moves inside the membrane and spontaneously forms stable water-filled transmembrane pores via a cooperative mechanism. The defects induced by these pores lead to the dislocation of interfacial lipid headgroups, membrane thinning, and substantial water leakage inside the hydrophobic core of the membrane. A molecular analysis reveals that despite having a short architecture, these synthetic peptides, once inside the membrane, would stretch themselves toward the distal leaflet in favor of potential contact with polar headgroups and interfacial water layer. The pore formed in coarse-grained simulation was found to be resilient upon structural refinement. Interestingly, the pore-inducing ability was found to be elusive in a non-globally amphiphilic sequence isomer of the same ß-peptide, indicating strong sequence dependence. Taken together, this work puts forward key perspectives of membrane activity of minimally designed synthetic biomimetic oligomers relative to the natural antimicrobial peptides.

Conformational Reorganization of Apolipoprotein E Triggered by Phospholipid Assembly.

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 lipid-induced 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.

Fundamental Challenges and Outlook in Simulating Liquid-Liquid Phase Separation of Intrinsically Disordered Proteins.

Intrinsically disordered proteins (IDPs) populate an ensemble of dynamic conformations, making their structural characterization by experiments challenging. Many IDPs undergo liquid-liquid phase separation into dense membraneless organelles with myriad cellular functions. Multivalent interactions in low-complexity IDPs promote the formation of these subcellular coacervates. While solution NMR, Förster resonance energy transfer (FRET), and small-angle X-ray scattering (SAXS) studies on IDPs have their own challenges, recent computational methods draw a rational trade-off to characterize the driving forces underlying phase separation. In this Perspective, we critically evaluate the scope of approximation-free field theoretic simulations, well-tempered ensemble methods, enhanced sampling techniques, coarse-grained force fields, and slab simulation approaches to offer an improved understanding of phase separation. A synergy between simulation length scale and model resolution would reduce the existing caveats and enable theories of polymer physics to elucidate finer details of liquid-liquid phase separation (LLPS). These computational advances offer promise for rigorous characterization of the IDP proteome and designing peptides with tunable material and self-assembly properties.

Nonaffine Displacements Encode Collective Conformational Fluctuations in Proteins.

Identifying subtle conformational fluctuations underlying the dynamics of biomacromolecules 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 homogeneous or "affine" deformations of the biopolymer are trivial, biopolymer conformations are complicated by the occurrence of inhomogeneous or "nonaffine" displacements of atoms relative to their positions in the native structure. We show that these displacements encode functionally relevant conformations of macromolecules, and in combination with a formalism based upon time-structured independent component analysis, they 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 nonaffine modes, obtained by projecting out homogeneous fluctuations from the local displacements, are found to be responsible for local structural changes required for transitioning between pairs of macrostates.