Study of Al3+ binding and conformational properties of the alanine-substituted C-terminal domain of the NF-M protein and its relevance to Alzheimer's disease.

ABSTRACT

NF-M13 [H-(Lys-Ser-Pro-Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly)-OH], NF-M17 [H-(Glu-Glu-Lys-Gly-Lys-Ser-Pro-Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly) -OH], and their phosphorylated derivatives, representing the C-terminal phosphorylation domain of the neurofilament protein midsize subunit, have four possible binding sites for metal ions the COO- group of glutamate, the OH group of the serine residue, the PO3H- group of phosphoserine (when present), and the COO- at the terminus of the peptide chain. The CD titration of the phosphorylated neurofilament fragments with Al3+ and Ca2+ yielded a significant conformational change that resulted in conformations containing high beta-pleated-sheet contents, which precipitate on standing (intermolecular complex). Al3+ binding to the unphosphorylated NF-M13 and NF-M17 did not exhibit this behavior. Several alanine analogues of the parent NF-M17 peptide were synthesized in order to determine the relationship between metal ions and possible binding sites. CD titration of analogues with Ca2+ indicated that the critical residues of NF-M17 for Ca(2+)-induced conformational changes, from random to beta-pleated sheet, are the N-terminal serine or both phosphorylated serines. Al(3+)-induced conformational changes suggest that the critical sites of NF-M17 yielding the beta-pleated-sheet structure are the four glutamates or phosphorylated serines, especially the C-terminal SerP. On the basis of the titration data, it is very likely that analogues with a serine in position 11 form a stable intramolecular complex with Al3+ that, however, does not result in the adoption of the beta-conformation. Back-titration with citric acid fails to reverse the Al(3+)-induced conformational changes of the phosphorylated peptides. The above results, especially the possible formation of intramolecular and intermolecular Al3+ complexes, may have relevance to the molecular mechanism, through which the neurotoxin Al3+ gives rise to the formation of neurofilament tangles.