Auto-inhibitory long range interactions in the native state of αS

The protein α-synuclein (αS) is implicated in the deposition of pertinacious aggregates in many neurodegenerative disorders, including Parkinson´s disease. αS is expressed in high levels in the substantia nigra of healthy individuals, and locus triplication, point mutations, or as yet elusive environmental conditions are able to cause the self-association of the protein in the form of toxic oligomers and amyloid like fibrilar structures (Cookson, 2005). What is intriguing from the biophysical point of view, is that αS belongs to the class of natively unfolded proteins with no apparent ordered secondary structure detectable by far-UV CD, FTIR or NMR spectroscopy (Uversky, 2003). Thus, it is not evident how this protein is able to avoid self-association and amyloid deposition in the neuronal cytoplasm.

The challenge of this thesis work has been to rationalize in structural terms the inactive state of the soluble, unstructured protein, searching how the enhancement of aggregation is achieved by point mutations, ligand binding or changes in solution conditions.

However, the conformational flexibility inherent to natively unfolded proteins places them beyond the reach of classical structural biology, and a special set of NMR-based experiments had to be implemented in order to study the native soluble state of αS, namely paramagnetic relaxation enhancement and residual dipolar couplings.

When we employed paramagnetic relaxation enhancement from site-directed spin-labeling, we were able to detect a complex network of long range interactions involving an N- to terminal contact plus a shielding of the hydrophobic central NAC region by the C-terminus. Experiments conducted under chemical denaturation suggest that the contacts between the NAC region and the C-terminus are of hydrophobic nature. On the contrary, the interaction between the positively charged N-terminus and the acidic C-terminus are electrostatic in origin. Furthermore, distance restraints were obtained from the paramagnetic experiments and an ensemble of conformations was obtained for the native state of αS (Figure 8.1.).

The model that we present here can be interpreted as either an ensemble of unfolded structures, where each structure is satisfying the experimental data, or as an ensemble of unfolded structures where as an average the ensemble satisfy the experimental data (Teilum et al., 2002). Despite these uncertainties, it is evident from our experiments and structure calculations that αS is not a random coil as was described before, but rather populates a

defined set of conformations that, despite lacking secondary structure, are stabilized by long range tertiary contacts.

Figure 8.1. Summary of structural features in the ensemble of αS conformations derived by PRE. The native state of αS, although deprived of persistent secondary structure, should not be viewed as a random coil, rather specific long range (tertiary) contacts promote population of compact conformations, which aid the protein to remain soluble.

We attempted to determine the importance of these long range contacts in maintaining the protein in its soluble state by assaying different conditions known to enhance self- association and aggregation of the protein. We observed a significant impairment of long range interactions upon addition of a natural polycation, spermine, which specifically bind to the C-terminus of the protein. This effect was evidenced as a retirement of the C-terminus from the NAC region, causing a solvent exposure of the central hydrophobic domain.

Similarly, raising the temperature to 47 °C caused a release of this protective effect. In the case of the polyamine-bound αS we further obtained evidenced that the hydrodynamic shape of the ensemble was increased, supporting the achievement of a more extended conformation.

Furthermore, when we studied the A30P or A53T familial mutants of αS, known to cause early on-set PD, likely due to an increased oligomerization propensity, we also observed a slight perturbation of the key long range interaction between the C-terminus and the NAC region. However, strong N-terminal and C-terminal compactions remain unaltered in these proteins, and a slight increase of the electrostatic interaction is suggested. Thus, the general shape of the ensemble of conformations populated by the mutants is somewhat collapsed respect the wt protein.

Interestingly, this mechanism of losing key tertiary contacts rendering a more unfolded state is proposed as the trigger mechanism for the misfolding of proteins that account for defined secondary structure (Jahn and Radford, 2005). The similar situation that

is found in our studies for αS would imply that even for natively unfolded proteins, the general mechanisms proposed for amyloid formation are still applicable.

Examination of the homologous protein βS, which lacks the central NAC region and consequently does not readily oligomerize, did not evidence long range interactions. Neither electrostatic N- to C-terminal contacts nor C-terminal compaction occur in βS, as probed by the PRE strategy. This is in line with the determination of the size of the ensemble, which showed to be considerably more expanded than αS. While the positively charged N-terminus of αS and βS is almost identical, the very acidic C-terminus is highly divergent and more negatively charged in βS, thus electrostatic long-range interactions would be favored. The absence of such contacts could be then rationalized either by the requirement of the flip-back of the C-terminus on the NAC region for the electrostatic interaction to occur or by particular conformational states adopted by the domains imply in the interaction which disfavors such contacts. Since in the genetic mutants we evidenced persistent electrostatic interactions in the absence of the hydrophobic core, it is very likely that conformational restrictions at the C-terminus of βS are responsible for inhibiting the formation of such interactions. As discussed afterwards, we found at the C-terminus of βS a strong amount of polyproline II (PII) extended conformations which opposes to the compact and flexible nature of the C-terminus in αS.

At the same time our studies were reported, a similar work on αS was published by the Dobson laboratory (Dedmon et al., 2005). The authors employed the same PRE-based strategy to investigate the nature of the ensemble of conformations populated by αS, arriving to the same conclusions as the one presented in this work. They investigated 5 different Cys-containing mutants and obtained by restrained molecular dynamic simulations an ensemble of conformations which reproduces the compact dimensions experimentally observed in the protein. A close comparison of their contact map with ours shows a striking correlation, validating our studies, and supporting the occurrence of tertiary interactions in αS.

Spin-labeling techniques have been previously employed in the study of the unfolded states of otherwise normally folded proteins, but have never been used in natively unfolded proteins. From the methodological point of view, this study represents the first of such application of long range restraints to determine a low resolution model of a natively unfolded protein. The number of intrinsically unstructured proteins is growing rapidly (Uversky et al., 2000), and it is becoming clear that these proteins participate in a vast range of biochemical processes (Dyson and Wright, 2005). We thus believe that our results will

encourage future high resolution structural studies of dynamic ensembles of interconverting conformers.