Mapping Cu(II) binding interfaces in αS by heteronuclear NMR

Due to the paramagnetic nature of Cu(II), we could explore the details of Cu(II) binding to αS by NMR spectroscopy. Similarly to the paramagnetic effect that we exploited with

nitroxide spin labels in the previous chapters, the electron spin relaxation from the Cu(II) ion provokes differential broadening of the amide resonances linked to the paramagnetic center.

Thus, through-bond or through-space interactions (up to ~ 10Å) will cause a reduction in the intensity of the peaks in the NMR spectrum, providing means for mapping the metal binding interfaces in the protein.

We first recorded a series of ¹H-¹⁵N HSQC spectra of 100 μM αS at pH 6.5 in the presence of increasing concentrations of Cu(II) (0-60 μM) and determined the extent of paramagnetic broadening by evaluating the ratio of peak intensities in the presence (Ipara) and absence (Idiam) of the metal ion. As shown on figure 7.8, significant changes in cross-peak intensities occurred in well-defined regions of αS.

Figure 7.8. NMR spectra of Cu(II) binding to αS. Overlaid ¹H-¹⁵N-HSQC spectra of αS(100 μM) in the absence (red) and presence of 40 μM Cu(II) (blue, left) and 100 μM Cu(II) (blue, right).

The strongest broadening effects at 20 μM Cu(II) were centered on the amide group of His50 in the N-terminal region, whereas little or no broadening was observed for the amide groups of residues located in the NAC region or belonging to the C-terminal domain (Figure

7.9A). Thus, the N-terminal region was the most affected by Cu(II) binding under this condition.

The broadening was further pronounced at 40-60 μM Cu(II), as reflected in values of Ipara/Idiam < 0.2 for the resonances corresponding to residues 3-9 and 49-52 (Figure 7.9B, 7.9C). Residues 15-45 were also involved to a lesser extent, but significant changes were evidenced at the C-terminal region, the strongest effect centered on Asp¹²¹. Interestingly, the amide resonances assigned to the NAC region remained unaltered, even at higher Cu(II) concentrations. All NMR spectral changes induced by Cu(II) were abolished upon EDTA addition, confirming the reversibility of Cu(II) binding.

Figure 7.9. Cu(II) binding to αS investigated by NMR. Ipara/ Idiam profiles corresponding to 100 μM αS at pH 6.5. A. 20 μM Cu(II). B. 40 μM Cu(II). C. 60 μM Cu(II).

The NMR titration experiment showed that the residues encompassing His50 were the most perturbed, suggesting that the imidazole ring plays a critical role in anchoring the Cu(II) ion to the N-terminal domain. In order to prove this assertion we tested whether modification of the sole His residue in αS by DEPC affects Cu(II) binding to the protein.

Exposure to DEPC shifted the paramagnetic influence of Cu(II) from the N-terminal to the C-terminal domain (Figure 7.10A), manifested by substantial changes in cross-peak intensities at Cu(II) concentrations as low as 20 μM. The strongest effects were centered on the amide group of Asp¹²¹, whereas resonances in the NAC domain remained unaffected under these conditions. The decrease in intensity of the signals corresponding to residues 49-52 was likely due to residual Cu(II) coordination capability of His50, e.g. through the imidazole N atom unmodified by DEPC. In summary, Cu(II) binding to DEPC-treated αS clearly shows the existence of two different copper binding interfaces in the protein, one at the N-terminus and another at the C-terminus.

Figure 7.10. Modulation of copper binding to αS studied by NMR. Peak intensity ratios corresponding to Cu(II) binding to modified and C-terminal truncated αS. A DEPC-modified αS, 20 μM Cu(II). B. 20 μM Cu(II), pH 5.0. C. Wild type (Δ) and 1-108 (•) αS, 20 μM Cu(II).

Because protonation of His50 should reduce Cu(II) binding, we determined the pH dependence of Cu(II) binding to αS in the range of 5.0-6.5 (Figure 7.11). The Ipara/Idiam

profile measured at pH 5.0 and 20 μM Cu(II) is shown in figure 7.10B. The strongest broadening effects corresponded to the C-terminal domain, again centered on Asp121, with a smaller effect evident in the N-terminal domain, located at His50. The cross-peaks of residues in the NAC region remained insensitive to Cu(II) at pH 5.0.

Figure 7.11. Protonation of His⁵⁰ modulates Cu(II) binding to αS. pH titration of 100 μM αS in the presence of 40 μM Cu(II). Selected regions of the ¹H-¹⁵N HSQC spectra are shown, and the most affected signals by the binding of Cu(II) are identified.

It is noteworthy that even when the most affected cross-peaks in the N-terminal region were severely broadened under conditions favoring Cu(II) binding (pH 6.5); signals were still detected at 40-60 μM Cu(II) (Figure 7.9B, 7.9C). In contrast, upon DEPC modification or protonation of His50, Cu(II) concentrations as low as 20 μM were sufficient

to broaden beyond detection the resonances of the most affected residues in the C-terminal region (Figure 7.10A, 7.10B). This behavior presumably reflects differences in the exchange dynamics and residence times of the two Cu(II) binding sites in αS and supports the assignment of the N-terminal region as the high affinity interface.

The interaction of the C-terminal truncated species of the protein with Cu(II) was studied with the aim of understanding further the structural contribution of the C-terminus to the overall binding process. The Ipara/Idiam profiles for αS1-108 in the concentration range of 0-60 μM of Cu(II) were almost identical to those obtained with wild-type αS (Figure 7.10C), confirming that the binding of Cu(II) to the N-terminal region of αS is independent of the presence of the C-terminus.

Conditions that were effective in triggering αS aggregation were shown previously to induce changes in the general properties of the ensemble populated by the protein (Chapter 1). In addition, mM concentrations of metal ions have shown to cause a reduction in the radio of gyration (Rg) of the protein, and effect that was attributed to the formation of a partially folded intermediate (Uversky et al., 2001b; Uversky et al., 2001c). We sought evidence for the formation of metal-induced misfolded intermediates of αS by determining the hydrodynamic properties of the αS-Cu(II) complexes employing PFG-NMR.

Measurement of Rh for αS (100 μM) in the absence and presence of Cu(II) (40 to 200 μM) did not provide evidences of a partially collapsed intermediate or an extended species. While the values observed for the protein in its native state (Rh of 32.0 Å) were consistent with previous determinations (Morar et al., 2001; Uversky et al., 2001b) no changes were detected upon addition of Cu(II) (Rh of 32.1 Å), suggesting that the interaction neither affects the size of native αS ensemble nor causes a significant collapse to a more compact conformation.