Surveying the Conformational Landscape of α-Synuclein Using Native Crosslinking, Ion Mobility-Mass Spectrometry and Ensemble Modeling

Intrinsically disordered proteins (IDPs) contain highly flexible regions and bind to different protein partners to help maintain proper cellular function. Misfolded IDPs form higher order structures and aggregates that are linked to neurodegenerative diseases. In particular, α-Synuclein (αSN) is a major component of Lewy bodies, which are a defining characteristic of Parkinson’s disease. αSN adopts various conformers in solution and learning how the αSN conformational ensemble relates to aggregation is critical in understanding disease progression. Here, native protein crosslinking, ion mobility (IM) separations and tandem mass spectrometry (MS/MS) are combined to identify intramolecular crosslinks from multiple αSN monomer charge states (z = +8, +9, +11 and +13). IM provides overall size measurements of the different conformer subpopulations while crosslinks inform on localized interresidue distance constraints. Together with an initial αSN ensemble generated computationally by IDPConformerGenerator, our approach results in a realistic αSN conformational landscape comparable to a published ensemble obtained using distances derived from spin label nuclear magnetic resonance experiments as restraints in molecular dynamics simulations. The representative structures, resulting from structural clustering of the final αSN ensemble, contain characteristic features previously studied in the context of αSN aggregation. Overall, our findings validate the applicability of our approach in accurately describing the conformationally diverse ensemble of an IDP such as αSN.