Binding of Nickel(II) and Copper(II) to the N-Terminal Sequence of Human Protamine HP2

Abstract

A potentiometric and spectroscopic (UV/vis and CD) study of Cu(II) and Ni(II) binding to the N-terminal pentadecapeptide of human protamine HP2 (HP2(1-15)) was performed. The results indicate that the N-terminal tripeptide motif Arg-Thr-His is the exclusive binding site for both metal ions at a metal to HP2(1-15) molar ratio not higher than 1. The very high value of protonation-corrected stability constant (log *K) for Ni(II)-HP2(1-15) complex, -19.29, indicates that HP2 has the potential to sequester Ni(II) from other peptide and protein carriers, including albumin. The same is likely for Cu(II) (log *K = -13.13). The CD spectra of Cu(II) and Ni(II) complexes of HP2(1-15) indicate that the N-terminal metal binding affects the overall conformation of the peptide that, in turn, may alter interaction of HP2 with DNA. These results imply HP2 as a likely target for the toxic metals Ni(II) and Cu(II).

Lead effects on protamine-DNA binding

“…It is known that several cysteine-zinc-containing proteins have shown af®-nity for Pb 2 [Waalkes et al, 1984;Wetmur, 1994], but lead also has high af®nity for carboxyl-rich proteins [Habermann et al, 1983;Quintanilla-Vega et al, 1995;Smith et al, 1998]. HP2 binding to other metals has been reported for Cd 2 [Gatewood et al, 1990], and for Ni 2 and Cu 2 which are able to bind to the N-terminal motif of HP2 [Bal et al, 1997]. HP2 may be a common target for toxic metals.…”

Investigations of the Copper Peptide Hepcidin-25 by LC-MS/MS and NMR

“…It is a metal binding site specific for the coordination of Cu 2+ and Ni 2+ , present at the amino terminus of several naturally occurring proteins [18][19][20]. It consists of four nitrogen atoms in the first three N-terminal amino acids, involving the free α-NH2 nitrogen, two following amide nitrogen atoms and the imidazole nitrogen of a histidine residue in the third position.…”

“…In case of Cu 2+ , a paramagnetic complex is formed with the ATCUN motif leading to severe line broadening of residues in close proximity to the coordination site. When Cu 2+ is added in sub-stoichiometric amounts, two sets of signals arise with one species corresponding to the metal-free peptide showing sharp NMR resonances and on species [18][19][20]. Figure 5 illustrates that Ni 2+ forms a stable complex with hepcidin's ATCUN motif, both with the DTHFPI hexapeptide (Fig.…”

“…In the Nterminal part, we applied the same distance and dihedral angle restraints that we had previously determined for the Ni 2+ complex of the hexapeptide. In addition, Ni 2+ was replaced by Cu 2+ , and the square planar coordination was imposed by restraining distances and dihedral angles as described [17][18][19][20][21]48]. Cu 2+ -N-distances were adjusted to standard values found in X-ray structures of ATCUN motifs.…”

The Involvement of Amino Acid Side Chains in Shielding the Nickel Coordination Site: An NMR Study

“…The involvement of remote residues in the coordination sphere contributes to a further stabilization of the complex itself, again through the phenolic oxygen of Tyr 28 and the approach of Lys 27 side chain to the binding site, as suggested by its α-proton up-field shift together with a down-field shift for all the remaining aliphatic protons (β > γ > δ > ε). It is thus evident the participation of different side chains to the stabilization of the complex, starting from the residues close to the central ion, Ala 17 and Val 18 , which build up a hydrophobic fence above the plane, and the bulky aliphatic group of Leu 13 below it; the approach of the distant Lys 27 -Tyr 28 -Thr 29 -Ser 30 -Ser 31 -Lys 32 segment, covering up the whole coordination centre, contributes to form an effective shield against the bulk of the solution.…”