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.

Metal-Binding Ability of Leu-Enkephalin, Related Glycoconjugates and Peptidomimetics

“…This spectrum shows -sheet like features and it is not a typical "Ca-induced spectrum", characteristic for many peptide-Ca 2+ complexes, where the main features would be a weak positive band above 210 nm and a strong negative one at ~ 195 nm. [39] …”

Stable intrachain and interchain complexes of neurofilament peptides: a putative link between Al3+ and Alzheimer disease.

“…The In an attempt to find a molecular mechanism for the effect of aluminum to induce aggregation of neuronal proteins or their catabolic fragments, circular dichroism (CD) spectroscopic studies have been performed in our laboratories on synthetic fragments of human neurofilament mid-sized subunit protein (NF-M), KSPVPKSPVEEKG (NF-M13), and EEKGKSPVPKSPVEEKG (NF-M17), as well as on their alanine-substituted and/or serine-phosphorylated derivatives (15)(16)(17). CD spectra were recorded as described (18) and deconvoluted into secondary structural components by the convex constraint analysis method (19,20 and A13+ were found to induce the adoption of the (3-pleated sheet conformation in the C terminus in a low-dielectricconstant environment, in 2,2,2-trifluoroethanol (TEE).…”

“…§1734 solely to indicate this fact. (+) or 12 equivalents of Al3+ (C) (17). The spectra showed continuous shifts upon addition of metal ions.…”

“…CD-monitored cation binding studies on the alaninesubstituted derivatives (17) have indicated that neurofilament peptides having a free (nonphosphorylated) C terminus or two serine residues bind A13+ irreversibly, but the spectral change is not characteristic of a shift of the conformational equilibrium toward (-sheets (Fig. 1).…”

“…The spectra showed continuous shifts upon addition of metal ions. [Ser(P)11]NF-M17 and the other monoand diphosphorylated derivatives of NF-M13 and NF-M17 yielded similar shifts toward a-pleated sheet conformation upon addition of Ca2+ or AP3+ (12,17). (C) CD spectra of [Ala6'111NF-M17 (193 LM) in TFE in the absence of metal ion (o) or in the presence of 8 equivalents of Ca2+ (+) or 8 equivalents of A13+ (C) (17).…”