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

Abstract

In idiopathic Parkinson's disease, intracytoplasmic neuronal inclusions (Lewy bodies) containing aggregates of the protein ␣-synuclein (␣S) are deposited in the pigmented nuclei of the brainstem. The mechanisms underlying the structural transition of innocuous, presumably natively unfolded, ␣S to neurotoxic forms are largely unknown. Using paramagnetic relaxation enhancement and NMR dipolar couplings, we show that monomeric ␣S 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 microseconds corresponding to the time scale of secondary structure formation during folding. Polyamine binding and͞or 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 ␣S constitutes a potential strategy for reducing or inhibiting oligomerization and aggregation in Parkinson's disease.is the second most common neurodegenerative disease and the most common movement disorder, affecting 1-2% of the population over 65 years of age (1). The cause of PD is as yet unclear due in part to a complex etiology involving a combination of genetic susceptibility and numerous environmental factors (2). Proteinaceous aggregates in motor neurons of the substantia nigra and locus coeruleus are characteristic of idiopathic PD. An abundant component of these so-called Lewy bodies is the presynaptic protein ␣-synuclein (␣S) (3). Three genetic mutations in ␣S (A30P, E46K and A53T) have been identified in autosomal-dominantly inherited early-onset PD (4, 5). In vitro, different conditions such as increased temperature, lower pH, and naturally occurring polyamines accelerate ␣S aggregation (6, 7). Compelling evidence now supports a cytotoxic role in PD for protofibrils, early oligomers of ␣S (8).In other amyloid-related neurological disorders, such as Creutzfeldt-Jakob disease, protein oligomerization͞aggregation requires destabilization of a soluble monomeric protein followed by the formation of highly ordered, ␤-sheet-like fibrillar structures (9). ␣S, however, belongs to the class of natively unfolded proteins with no apparent ordered secondary structure detectable by far-UV CD, Fourier transform IR, or NMR spectroscopy (6, 10, 11), although recent evidence indicates the existence of distinct, functionally relevant intramolecular interactions (refs. 7 and 12 and this work). The challenge is to rationalize in structural terms the inactive state of the soluble, unstructured protein and the potentiation of aggregation by point mutations, ligand binding, or changes in solution conditions. During the past decade numerous NMR techniques have been developed for elucidating the unfolded states of proteins in atomic detail (13). 15 N-relaxation time measurements and paramagnetic relaxation enhancement (PRE) from site-directed spin labeling allo...

Charge neutralization and collapse of the C‐terminal tail of alpha‐synuclein at low pH

“…Observation of C-to-N terminal contacts in WT aS, 31,32 and data suggesting such contacts are abrogated in aggregation prone mutants 50 supported a proposed model in which long-range contacts prevented the formation of fibrilization pathway intermediates by occluding the hydrophobic NAC region of the protein. This model, however, was not supported by more recent studies of other synuclein family members, where the predicted correlation between long-range contacts and aggregation propensity was not observed.…”

“…49 For aS, it has also been suggested that large amplitude RDCs in the C-terminal tail of the protein may report on long-range transient contacts between the C-terminus and regions in the N-terminal lipid-binding domain. 32 We measured RDCs for aS at pH 3.0 aligned in C8E5 bicelles and compared them to previous measurements at pH 7.4 43 (see Fig. 5).…”

“…Long-range C-to N-terminal contacts are largely unperturbed at low pH Transient long-range contacts between the C-and Nterminal domains of aS were originally detected using PRE measurements, 31,32 which revealed a broadening effect on resonances near the N-terminus of the protein and in the NAC region for spin-label sites in the C-terminal tail. The PRE data presented here indicate that such long-range contacts are largely preserved in aS at pH 3.0.…”

“…aS exists in a number of conformations. When isolated in dilute solution at neutral pH, the protein shows a primarily disordered conformation without a well defined secondary or tertiary structure, 29 although both nascent secondary structure preferences 30 and transient tertiary interactions 31,32 are detectable. At Low pH, however, aS has been reported to adopt a more compact conformation with altered secondary structure content, 33 and the formation of this low pH intermediate conformation is maximal at pH 3.0.…”

α-Synuclein interacts with the switch region of Rab8a in a Ser129 phosphorylation-dependent manner

“…Intra-and intermolecular contacts can be detected in monomeric, unfolded αS and are implicated in modulating the aggregation propensity (Bertoncini et al, 2005;Dedmon et al, 2005;Outeiro et al, 2008;Wu and Baum, 2010). The negatively charged C-terminus remains disordered in several conformational states such as monomeric, fibrillar and membrane-bound αS (Del Mar et al, 2005;Eliezer et al, 2001;Qin et al, 2007;Ulmer et al, 2005;Vilar et al, 2008).…”