Structural characterization of alpha-synuclein in an aggregation prone state

Abstract

The relation of alpha-synuclein (alphaS) aggregation to Parkinson's disease has long been recognized, but the pathogenic species and its molecular properties have yet to be identified. To obtain insight into the properties of alphaS in an aggregation-prone state, we studied the structural properties of alphaS at acidic pH using NMR spectroscopy and computation. NMR demonstrated that alphaS remains natively unfolded at lower pH, but secondary structure propensities were changed in proximity to acidic residues. The ensemble of conformations of alphaS at acidic pH is characterized by a rigidification and compaction of the Asp and Glu-rich C-terminal region, an increased probability for proximity between the NAC-region and the C-terminal region and a lower probability for interactions between the N- and C-terminal regions.

Figure 1

The aggregation-prone state of αS at acidic pH is natively unfolded. A: Primary sequence of αS. αS consists of three regions based on the distribution of charged residues. Residues that are positively and negatively charged at pH 7.4 are color-coded red and blue, respectively. B: ¹H-¹⁵N HSQC spectra of αS at pH 7.4 (left) and pH 3.0 (right). Both spectra show only a limited dispersion of chemical shifts typical of a natively unfolded protein. Selected cross peaks are labeled with their assignment.

Figure 2

Secondary structure propensities of αS at acidic pH. A: SSP analysis using C^α and C^β chemical shifts. Positive SSP values are indicative of residual helical structure, negative values of residual β-structure. Note that all Glu and Asp residues were excluded in this analysis. B: Difference in SSP scores at pH 3.0 and pH 7.4. Most difference values are positive suggesting a preferentially population of helical and turn conformations at pH 3.0, in particular in the C-terminal region. C: ³J(H^NH^α) scalar couplings at pH 3.0 (black) and pH 7.4 (gray). On top, the region organization of αS is shown. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 3

The C-terminal region of αS is more rigid at acidic pH. A: Comparison of normalized peak intensities observed in ¹H-¹⁵N HSQC spectra of αS at pH 3.0 and pH 7.4. At pH 7.4, the peak intensities observed for residues in the C-terminal region are higher than those observed for residues located in the N-terminal region. At pH 3.0, however, residues 105–130 and 134–137 have lower peak intensities than residues in the N-terminal region, indicating that residues in the C-terminal region are less flexible at pH 3.0. B: Steady-state heteronuclear ¹⁵N{¹H}-NOEs of αS at pH 7.4 (gray) and at pH 3.0 (black). B: ¹⁵N R₂ and R_1ρ spin relaxation rates of backbone resonances of αS at pH 3.0.

Figure 4

Residual dipolar couplings probe structural changes in C-terminal region. A: One-bond H—N RDCs of αS at pH 7.4 (gray line) and at pH 3.0 (black bars) as a function of residue number. B, C: One-bond H—N RDCs of residues 100–140 in detail.

Figure 5

The C-terminal region of αS is more compact at acidic pH. A: Hydrodynamic radius values R_h of full-length αS, αS(1–108), and αS(105–136) as estimated by pulsed-field gradient NMR. The left panel shows R_h values at pH 7.4, the right panel at pH 3.0. Error bars are based on two independent measurements.

Figure 6

Transient long-range interactions in αS. A: Comparison of experimental PRE profiles (black bars for pH 3.0, orange dashed line for pH 7.4) with the PRE profiles calculated from the FM simulation at pH 3.0 (green line). The nitroxide label MTSL (red mark) was attached to the single-cysteine mutants A18C, A76C, A90C, and A140C (from top to bottom). B: Long-range contacts in the representative ensemble as a function of pH. Contacts are plotted as log(〈d_ij〉/〈d_ij,ref〉), where d_ij,ref refers to the distance in the reference ensemble of 10,000 structures and d_ij refers to the distance in the selected ensemble.

Figure 7

Schematic representation of the conformational changes that occur in αS, when the pH is lowered to pH 3.0.