Sam, A. | bioRxiv Science

bioRxiv2025

Is synuclein aggregation a derived or ancestral trait? Ancestral sequence reconstruction uncovers stepwise evolution of synuclein aggregation

Protein aggregation drives many neurodegenerative diseases, including Parkinsons disease, where misfolded -synuclein (Syn) forms fibrillar assemblies that accumulate as Lewy bodies. Although Syn aggregation has been extensively characterized, its evolutionary origins and sequence determinants remain unresolved. Here, we use ancestral sequence reconstruction (ASR) to trace the emergence of fibril-forming ability in the synuclein family. We inferred synuclein phylogeny and experimentally resurrected common ancestors, including ROOT synuclein, the last common ancestor of all synucleins, and key intermediates along the Syn lineage. Strikingly, ROOT synuclein is non-aggregating, demonstrating that fibril formation is an evolved, rather than ancestral property. Aggregation first emerges at the ancestral {beta} node, is retained in Syn, and suppressed in {beta}-synuclein. Biophysical analyses including mass spectrometry and NMR reveal that aggregation aligns with greater complexity and heterogeneity in the monomer conformational ensemble, suggesting that evolutionary sequence changes progressively remodel monomer landscapes to favor fibril formation. Complementing these insights, comparative sequence analysis reveals that the transition from ROOT to -WT is marked by the stepwise acquisition of residues critical for stabilizing the fibril core. Early mutations stabilized the {beta}-arch core, enabling the onset of fibril formation, followed by substitutions that reinforce protofilament-protofilament interactions. Together, ASR defines an evolutionary framework for synuclein aggregation linking progressive sequence evolution and conformational complexity to the molecular origins of Syn fibril formation.

biophysics↗

bioRxiv2021

Molecular Dynamics Analysis of a Flexible Loop at the Binding Interface of the SARS-CoV-2 Spike Protein Receptor-Binding Domain

Since the identification of the SARS-CoV-2 virus as the causative agent of the current COVID-19 pandemic, considerable effort has been spent characterizing the interaction between the Spike protein receptor-binding domain (RBD) and the human angiotensin converting enzyme 2 (ACE2) receptor. This has provided a detailed picture of the end point structure of the RBD-ACE2 binding event, but what remains to be elucidated is the conformation and dynamics of the RBD prior to its interaction with ACE2. In this work we utilize molecular dynamics simulations to probe the flexibility and conformational ensemble of the unbound state of the receptor-binding domain from SARS-CoV-2 and SARS-CoV. We have found that the unbound RBD has a localized region of dynamic flexibility in Loop 3 and that mutations identified during the COVID-19 pandemic in Loop 3 do not affect this flexibility. We use a loop-modeling protocol to generate and simulate novel conformations of the CoV2-RBD Loop 3 region that sample conformational space beyond the ACE2 bound crystal structure. This has allowed for the identification of interesting substates of the unbound RBD that are lower energy than the ACE2-bound conformation, and that block key residues along the ACE2 binding interface. These novel unbound substates may represent new targets for therapeutic design.

biophysics↗