Release of long-range tertiary interactions potentiates aggregation of natively unstructured alpha-synuclein

Abstract

In idiopathic Parkinson's disease, intracytoplasmic neuronal inclusions (Lewy bodies) containing aggregates of the protein alpha-synuclein (alphaS) are deposited in the pigmented nuclei of the brainstem. The mechanisms underlying the structural transition of innocuous, presumably natively unfolded, alphaS to neurotoxic forms are largely unknown. Using paramagnetic relaxation enhancement and NMR dipolar couplings, we show that monomeric alphaS assumes conformations that are stabilized by long-range interactions and act to inhibit oligomerization and aggregation. The autoinhibitory conformations fluctuate in the range of nanoseconds to micro-seconds corresponding to the time scale of secondary structure formation during folding. Polyamine binding and/or temperature increase, conditions that induce aggregation in vitro, release this inherent tertiary structure, leading to a completely unfolded conformation that associates readily. Stabilization of the native, autoinhibitory structure of alphaS constitutes a potential strategy for reducing or inhibiting oligomerization and aggregation in Parkinson's disease.

Fig. 1.

PRE of amide protons in spin-labeled αS. Three cysteine mutants of αS (A18C, A90C, and A140C) were labeled with MTSL. HSQC spectra in the presence (paramagnetic) and absence (diamagnetic) of spin label were recorded at 15°C, and the intensity ratio (I_param/I_diam) of the resonance peaks was determined. Dashed lines indicate paramagnetic effects expected for a random coil polypeptide (34). (a-c) αS in buffer A. (d-f) αS in buffer A plus 8 M urea. (g-i) αS in buffer A plus 6 mM spermine.

Fig. 3.

Native-state conformations of αS. Representation of the native state of αS calculated from PRE data. Shown are the seven most populated clusters containing 80, 75, 46, 39, 25, 24, and 20 structures representing 50% of all calculated conformations. The 10 lowest-energy structures of each cluster within an atomic density map (32) calculated from all conformations contained in each cluster are shown. RDCs were mapped onto the structures with the use of a continuous color scale.

Fig. 4.

Long-range interactions in αS. Average contact map for the center structures of the seven most populated clusters representing 50% of all calculated conformations. A continuous gray scale from 3 Å (black) to 22 Å (white) is used. RDCs as a function of residue number are indicated by a color bar.

Fig. 2.

RDCs in αS. N-H dipolar couplings at 15°C. (a) Functional domains of αS (7, 11, 19, 44). (b) RDC profile of αS aligned with Pf1 (red). (c) RDC profile of αS aligned with C8E5 in buffer A (red), plus 4 M urea (blue), or 8 M urea (cyan).

Fig. 5.

Influence of aggregation prone conditions on long-range structure in αS. (a) Influence of polyamines on dipolar couplings. ¹d_NH couplings were measured at 15°C, for αS aligned in Pf1 phage (red) and in the presence of 3 mM putrescine (+2) (orange), 3 mM spermidine (+3) (green), and 3 mM spermine (+4) (cyan). (b) Influence of temperature on RDCs. C-terminal RDC profile of αS aligned with Pf1 at 15°C (red) and at 37°C (green). (c) Influence of temperature on PRE effects for the C terminus of αS A90C labeled with MTSL at 15°C (red), 37°C (green), and 47°C (cyan).