Structure of alpha-synuclein fibrils derived from human Lewy body dementia tissue.

The defining feature of Parkinson disease (PD) and Lewy body dementia (LBD) is the accumulation of alpha-synuclein (Asyn) fibrils in Lewy bodies and Lewy neurites. Here we develop and validate a method to amplify Asyn fibrils extracted from LBD postmortem tissue samples and use solid state nuclear magnetic resonance (SSNMR) studies to determine atomic resolution structure. Amplified LBD Asyn fibrils comprise a mixture of single protofilament and two protofilament fibrils with very low twist. The protofilament fold is highly similar to the fold determined by a recent cryo-electron microscopy study for a minority population of twisted single protofilament fibrils extracted from LBD tissue. These results expand the structural characterization of LBD Asyn fibrils and approaches for studying disease mechanisms, imaging agents and therapeutics targeting Asyn.

13C and 15N resonance assignments of alpha synuclein fibrils amplified from Lewy Body Dementia tissue.

Fibrils of the protein α-synuclein (Asyn) are implicated in the pathogenesis of Parkinson Disease, Lewy Body Dementia, and Multiple System Atrophy. Numerous forms of Asyn fibrils have been studied by solid-state NMR and resonance assignments have been reported. Here, we report a new set of 13C, 15N assignments that are unique to fibrils obtained by amplification from postmortem brain tissue of a patient diagnosed with Lewy Body Dementia.

Biologically Representative Lipid-Coated Gold Nanoparticles and Phospholipid Vesicles for the Study of Alpha-Synuclein/Membrane Interactions.

Alpha-synuclein is an intrinsically disordered protein whose formation of beta-sheet-rich protein aggregates in the brain is implicated in the development of Parkinson's disease. Due to its believed role in synaptic vesicle trafficking and neurotransmission, many studies have employed simple, synthetic model systems to investigate alpha-synuclein/membrane interactions in an attempt to gain a better understanding of the protein's native and pathogenic functions. Interestingly, these studies seem to suggest that alpha-synuclein interacts differently with rigid vesicle mimics in comparison to malleable vesicle mimics. However, the use of different mimic sizes and surface chemistries across existing studies makes it challenging to directly compare the effects of membrane mechanical properties on protein behavior observed thus far. In this work, we developed a synaptic vesicle mimic library comprising a range of both malleable and rigid synaptic vesicle mimics possessing the same size and biologically representative lipid surface chemistry. Limited proteolysis mass spectrometry experiments revealed distinct fragmentation patterns between rigid and malleable synaptic vesicle mimics. The N-terminal and C-terminal regions of alpha-synuclein were found to become less solvent-accessible upon binding to all synaptic vesicle mimics. Nevertheless, minor variations in digestion pattern were observed in the central region of the protein dependent upon mimic size, rigidity, and lipid composition. Higher binding affinities were observed for alpha-synuclein binding to rigid synaptic vesicle mimics compared to malleable synaptic vesicle mimics. Additionally, the binding affinity of alpha-synuclein toward small lipid vesicles and small lipid-coated gold nanoparticles without cholesterol was found to be lower than that of their respective malleable and rigid counterparts. Interestingly, the binding curves for the rigid synaptic vesicle mimics demonstrated a nontraditional peak and dip shape believed to arise from differences in alpha-synuclein orientation on the particle surface at different protein-to-particle incubation ratios.

Delineating Structural Propensities of the 4E-BP2 Protein via Integrative Modeling and Clustering.

The intrinsically disordered 4E-BP2 protein regulates mRNA cap-dependent translation through interaction with the predominantly folded eukaryotic initiation factor 4E (eIF4E). Phosphorylation of 4E-BP2 dramatically reduces the level of eIF4E binding, in part by stabilizing a binding-incompatible folded domain. Here, we used a Rosetta-based sampling algorithm optimized for IDRs to generate initial ensembles for two phospho forms of 4E-BP2, non- and 5-fold phosphorylated (NP and 5P, respectively), with the 5P folded domain flanked by N- and C-terminal IDRs (N-IDR and C-IDR, respectively). We then applied an integrative Bayesian approach to obtain NP and 5P conformational ensembles that agree with experimental data from nuclear magnetic resonance, small-angle X-ray scattering, and single-molecule Förster resonance energy transfer (smFRET). For the NP state, inter-residue distance scaling and 2D maps revealed the role of charge segregation and pi interactions in driving contacts between distal regions of the chain (∼70 residues apart). The 5P ensemble shows prominent contacts of the N-IDR region with the two phosphosites in the folded domain, pT37 and pT46, and, to a lesser extent, delocalized interactions with the C-IDR region. Agglomerative hierarchical clustering led to partitioning of each of the two ensembles into four clusters with different global dimensions and contact maps. This helped delineate an NP cluster that, based on our smFRET data, is compatible with the eIF4E-bound state. 5P clusters were differentiated by interactions of C-IDR with the folded domain and of the N-IDR with the two phosphosites in the folded domain. Our study provides both a better visualization of fundamental structural poses of 4E-BP2 and a set of falsifiable insights on intrachain interactions that bias folding and binding of this protein.

13C and 15N Resonance Assignments of Alpha Synuclein Fibrils Amplified from Lewy Body Dementia Tissue.

Fibrils of the protein α-synuclein (Asyn) are implicated in the pathogenesis of Parkinson Disease, Lewy Body Dementia, and Multiple System Atrophy. Numerous forms of Asyn fibrils have been studied by solid-state NMR and resonance assignments have been reported. Here, we report a new set of 13C, 15N assignments that are unique to fibrils obtained by amplification from postmortem brain tissue of a patient diagnosed with Lewy Body Dementia.

Structure of alpha-synuclein fibrils derived from human Lewy body dementia tissue.

The defining feature of Parkinson disease (PD) and Lewy body dementia (LBD) is the accumulation of alpha-synuclein (Asyn) fibrils in Lewy bodies and Lewy neurites. We developed and validated a novel method to amplify Asyn fibrils extracted from LBD postmortem tissue samples and used solid state nuclear magnetic resonance (SSNMR) studies to determine atomic resolution structure. Amplified LBD Asyn fibrils comprise two protofilaments with pseudo-21 helical screw symmetry, very low twist and an interface formed by antiparallel beta strands of residues 85-93. The fold is highly similar to the fold determined by a recent cryo-electron microscopy study for a minority population of twisted single protofilament fibrils extracted from LBD tissue. These results expand the structural landscape of LBD Asyn fibrils and inform further studies of disease mechanisms, imaging agents and therapeutics targeting Asyn.