| alpha-Synuclein (alpha-syn) is the major protein component of the insoluble fibrils that make up Lewy Bodies, the hallmark lesions of Parkinson's Disease. Its C-terminal region contains motifs of charged amino acids that potentially bind metal ions, as well as several identified phosphorylation sites. We have investigated the metal-binding properties of synthetic model peptides and phosphopeptides that correspond to residues 119--132 of the C-terminal, polyacidic stretch of human alpha-syn, with the sequence Ac-Asp-Pro-Asp-Asn-Glu-Ala-Tyr-Glu-Met-Pro-Ser-Glu-Glu-Gly (alpha-syn119--132 ). The peptide pY125 replaces Tyr with phosphotyrosine, whereas pS129 replaces Ser with phosphoserine. By using Tb 3+ as a luminescent probe of metal binding, we find a marked selectivity of pY125 for Tb3+ compared to pS129 and alpha-syn119--132, a result confirmed by isothermal titration calorimetry. Truncated or alanine-substituted peptides show that the phosphoester group on tyrosine provides a metal-binding anchor that is supplemented by carboxylic acid groups at positions 119, 121 and 126 to establish a multidentate ligand, while two Glu residues at positions 130 and 131 contribute to binding additional Tb3+ ions. The interaction of other metal ions was investigated by electrospray ionization mass spectrometry (ESIMS), which confirmed that pY125 is selective for trivalent metal ions over divalent metal ions, and revealed that Fe3+ and Al3+ induce peptide dimerization through metal ion cross-links. Circular dichroism shows that Fe3+ can induce a partially folded structure for pY125, whereas no change was observed for pS129 or the unphosphorylated analog. We then studied the fusion peptides containing a known fibril-prone domain (Fib) plus the metal-binding domain (YNle), in which the native methionine has been replaced with norleucine, with the sequence Ac-Val-Thr-Gly-Val-Thr-Ala-Val-Ala-Gln-Lys-Thr-Val-Asp-Pro-Asp-Asn-Glu-Ala-Tyr-Glu-Nle-Pro-Ser-Glu-Glu-Gly (Fib-YNIle). The peptide Fib-pYNle replaces Tyr with phosphotyrosine. Thioflavin T assay was done to monitor fibril formation. Fib fibrillized immediately after incubation at 37°C, while Fib-pYNle and Fib-YNle significantly slowed down the initiation of fibril formation by 30 and 40 hours respectively. The presence of metal ions did not cause a detectable difference in the rate of fibril formation for these fusion peptides, although Fib-pYNle was more prone to fibrillize than Fib-YNle. The results of this study show that the type and location of a phosphorylated amino acid influences a peptide's metal-binding specificity and affinity, its overall conformation, as well as its aggregation behavior. |
