Structural Characterization of the Intrinsically Unfolded Protein β-Synuclein, a Natural Negative Regulator of α-Synuclein Aggregation

The synuclein family of intrinsically unfolded proteins is composed of three highly homologous members, α-synuclein (αS), β-synuclein (βS) and γ-synuclein (γS), which are linked to neurodegenerative disorders and cancer. αS has been studied intensively after its identification as the major protein component of amyloid-like deposits in Parkinson's disease and dementia with Lewy bodies. βS, on the other hand, was found to act as a potent inhibitor of αS amyloid formation, and it is proposed as a natural regulator of its neurotoxicity. It is then of particular interest to elucidate the structural and dynamic features of the soluble state of βS as a first step to understand the molecular basis of its anti-amyloidogenic effect on αS. We present here the characterization of natively unstructured βS by high resolution heteronuclear NMR techniques. A combination of pulse-field gradient, three-dimensional heteronuclear correlation, residual dipolar couplings, paramagnetic relaxation enhancement and backbone relaxation experiments were employed to characterize the ensemble of conformations populated by the protein. The results indicate that βS adopts extended conformations in its native state, characterized by the lack of the long-range contacts as previously reported for αS. Despite the lack of defined secondary structure, we found evidence for transient polyproline II conformations clustered at the C-terminal region. The structuring of the backbone at the C terminus is locally encoded, stabilized by the presence of eight proline residues embedded in a polypeptide stretch rich in hydrophilic and negatively charged amino acids. The structural and functional implications of these findings are analyzed via a thorough comparison with its neurotoxic homolog αS.