Polyamine binding releases long-range interactions in αS

To address the functional relevance of these long-range, intramolecular interactions, we performed the same set of experiments as performed for the native state of the protein, but now for the previously examined conditions favoring aggregation, namely polycation binding, increased temperature and reduced pH.

The polycation spermine is a naturally occurring polyamine that increases the kinetic efficiency of αS aggregation by 105 due to a specific interaction with the C-terminus of the protein (Fernandez et al., 2004). There was a general change in the PRE profiles of the three Cys-containing mutants upon addition of spermine. In particular the PRE effects of residues 18 to 60 (A18C mutant) were considerably reduced (Figure 4.14A), demonstrating that binding of polyamines to the C-terminal domain causes a release of the N-terminus and suggesting an opening of the αS structure. This phenomenon is also consistent with the intensity increases of NMR signals of residues 22 to 93 resulting from polyamine binding (Figure 4.3) (Fernandez et al., 2004).

For the A90C mutant, binding of spermine led to a minimization of paramagnetic broadening in the C-terminus, achieving a profile similar to that of the denaturated state in 8 M urea (Figures 4.10E vs. Figure 4.13B). According to the A140C data, the presence of spermine also reduced the compactness of the region between residues 110 to 125 (Figure 4.13C). Broadening in other regions of the protein reflects the increased flexibility of the C-terminus and residual N-C-terminus/C-C-terminus interaction.

Figure 4.14. PRE on polyamine-liganded spin-labeled αS. Intensity ratios between paramagnetic and diamagnetic states (Iparam/Idiam) for amide resonance peaks corresponding to A18C (A), A90C (B) and A140C (C) cysteine mutants of αS labeled with MTSL and in the presence of 6mM Spermine (+4). Colored bars correspond to the intensity ratios measured in the experiment and are compared with the profile corresponding to the same mutants of αS in the absence of the polycation (bold black line). Dashed grey lines indicate paramagnetic effects expected for a random coil polypeptide with the paramagnetic tag in the same position as the αS mutant.

In line with these findings, the large RDCs observed for the C-terminus of the protein were reduced in the presence of polyamines (Figure 4.15). Greater polyamine charge – putrescine (+2) < spermidine (+3) < spermine (+4) – correlated with a stronger reduction in

1DNH values, as well as with an enhancement of fibrillation (Fernandez et al., 2004).

In addition, binding of spermine to the C-terminus reduced RDCs for residues 12 to 26 by about 60 % indicating a long-range effect with the N-terminus, in agreement with intensity increases of NMR signals of residues 22 to 93 upon from polyamine binding (Fernandez et al., 2004). Such a decrease was not observed upon addition of urea, pointing to an electrostatic interaction between the negatively charged C- and the positively charged N-terminus. The persistent paramagnetic broadening in the C-terminus for the A18C mutant (Figure 4.13D) indicates that the N-terminus/C-terminus interaction is not abolished upon addition of polyamines but only weakened, and that RDCs and PREs possess different sensitivities to the strength of long-range interactions.

Figure. 4.15. Residual dipolar couplings in polyamine-bound αS. A. ¹DNH

dipolar couplings measured on αS aligned with 5 mg/ml Pf1 phage solution in buffer A (dark red), and in the presence of 3 mM putrescine (+2) (orange), 3 mM spermidine (+3) (green) and 3 mM spermine (+4) (cyan). B. RDCs measured for in 5 % C8E5/octanol mixture for αS alone (dark red) and in the presence of 3mM spermine (+4) (cyan).

We further investigated whether hydrodynamic properties of the protein were affected upon polyamine binding, as suggested by the PRE profiles. By means of pulse-field gradient NMR it is possible to estimate the diffusion coefficient of polypeptides, and thus derive their hydrodynamic radius (Rh) by comparison with standards of known size (Wilkins et al., 1999). Empirical relationships have been derived by using these method, which relate the length of a polypeptide chain protein with its hydrodynamic radii at folded (Rn = 4.75 x N^0.29 Å) and unfolded (Rd = 2.21 x N^0.57 Å) states. Thus, according to its 140 amino acids, the expected size for a collapsed state of αS would be 18 Å, while the completely unfolded state would reach 34 Å.

Our determinations, showed on figure 4.16, demonstrated that αS populates relatively compact conformations at physiological pH (Rh of 29.3 Å), and that it radius increases upon addition of 100 mM NaCl (Rh of 31.9 Å), which is in accordance with a ~ 2-3 fold increase on the rate of aggregation of αS in the presence of salt (Hoyer et al., 2002; Hoyer et al.,

Figure 4.16. PFG-NMR measurement of hydrodynamic properties of αS under different solution conditions. A. Hydrodynamic radius (Rh) of αS was measured by PFG-NMR under the following conditions: pH 7.4, 100 mM NaCl, urea 8M and spermine 3mM. B. Comparison of Rh determinations for αS with the dimensions expected for a globular protein or a random coil polypeptide of 140 residues. Errors in the determination are within 2%.

Upon polyamine binding we evidenced a significant increase in the apparent size of the conformations populated by αS (Rg of 33.4 Å), dimensions similar to those determined for the urea denatured state of the protein (Rg of 33.9 Å). This suggests that upon conditions that significantly impair long range interactions there is a concurrent increase in the hydrodynamic dimensions of the ensemble of conformations populated by αS. This could be rationalized in terms of adoption of more extended structures, as probed by PREs (Figures 4.11 and 4.14) and due to an increment on the dynamics of the backbone of the polypeptide chain, as probed by RDCs (Figures 4.6 and 4.15).

Taking together, these data demonstrate that long-range interactions involving the C-terminus and the NAC region, presumably of hydrophobic nature, as well as with the N-terminus, acting via electrostatic interactions, protect αS from oligomerization. Docking of polycations to the C-terminus destabilizes these interactions. The high free energy of the extended conformation, in which the hydrophobic NAC region is exposed to the solvent, would favor the association of monomers, thereby increasing the extent of both the nucleation and propagation steps of aggregation (Fernandez et al., 2004).