title: Phosphorylation-dependent metal binding by alpha-synuclein peptide fragments.
abstract: 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 tyrosine with phosphotyrosine, whereas pS129 replaces serine with phosphoserine. By using Tb(3+) as a luminescent probe of metal binding, we find a marked selectivity of pY125 for Tb(3+) compared with 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 glutamic acid residues at positions 130 and 131 contribute to binding additional Tb(3+) ions. The interaction of other metal ions was investigated by electrospray ionization mass spectrometry, which confirmed that pY125 is selective for trivalent metal ions over divalent metal ions, and revealed that Fe(3+) and Al(3+) induce peptide dimerization through metal ion cross-links. Circular dichroism showed that Fe(3+) can induce a partially folded structure for pY125, whereas no change was observed for pS129 or the unphosphorylated analog. The results of this study show that the type and location of a phosphorylated amino acid influence a peptide's metal-binding specificity and affinity as well as its overall conformation.
authors.affiliation: Department of Chemistry, Duke University, P.O. Box 90346, Durham, NC 27708, USA.
authors.forename: Lucy L
authors.initials: LL
authors.lastname: Liu
authors.forename: Katherine J
authors.initials: KJ
authors.lastname: Franz
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002151:Calorimetry / Q000379:methods; D002942:Circular Dichroism / Q000379:methods; D006801:Humans; D016631:Lewy Bodies / Q000737:chemistry* / Q000378:metabolism; D008670:Metals / Q000737:chemistry / Q000378:metabolism*; D010300:Parkinson Disease / Q000378:metabolism; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000378:metabolism*; D010748:Phosphopeptides / Q000378:metabolism*; D010766:Phosphorylation; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013329:Structure-Activity Relationship; D051844:alpha-Synuclein / Q000737:chemistry / Q000378:metabolism*
chemical_list: D008670:Metals; D010446:Peptide Fragments; D010748:Phosphopeptides; D051844:alpha-Synuclein