Protamines and Other Biologically Important Proteins

Metal ions can be bound by a large number of proteins. Such protein-metal interactions occur within specific amino acid residues called metal-binding motifs. The motifs that putatively bind metal ions like Ni(II) preferentially contain clusters of Cys and/or His [132].

Nickel compounds are well-established as human carcinogens [133], but the responsible molecular events remain to be fully understood. DNA binds Ni(II) only weakly [134], leaving nuclear proteins as possible targets for Ni(II). Particularly, the abundance of histones inside the cell nuclei makes them the primary target for metal ions. The nucleosome is composed of a histone octamer (containing two copies of H2A, H2B, H3 and H4 histones) and 145-147 base pairs of DNA bundled around it [135]. Available information on the binding modes of Ni(II) to proteins [136] and data for nickel-peptide complexes [17] indicate that the imidazole of histidine and thiol of cysteine could be thermodynamically preferred by Ni(II) among the donor groups provided by protein-building amino acids. H1 histone does not have any histidine or cysteine residue. However, inspection of the available his-tone sequences revealed several histidine and cysteine residues in H2, H3, and H4.

Interactions of the TESHHK hexapeptide fragment representing the 34-amino acid residue C-terminus of histone H2A, resulted in hydrolysis of the peptide bond between the Glu and Ser residues under physiological conditions and formation of the albumin-like square planar Ni(II) complex with the SHHK sequence [137,138]. Studies on Ni(II) coordination with the N- and C-terminal protected hexapeptides TESHHK, TASHHK, TEAHHK, TESAHK, and TESHAK [139,140], in which Ala was inserted instead of Glu, Ser, or His-4 and His-5 in the TESHHK motif, were also performed. While substitution of His-4 or Glu by

Ala did not affect the hydrolysis reaction, substitution of the His-5 or Ser residues inhibited the reaction, supporting the important role of the last two residues in peptide bond hydrolysis [139,140]. Above pH 7 SHHK and SAHK (hydrolysis products) coordinate to Ni(II) equatorially through the imidazole of His-3, the N-terminal amino group, and the two amide nitrogens located between Ser-1 and His-3, {NH2, 2N", Nim}, forming 4N albumin-like square planar complexes [141]. Spectroscopic evidence and theoretical predictions suggest that the positioning of the free imidazole ring, in the Ni-SHHK complex, above the coordination plane, induces the extra stability of the complex [142].

The coordinating properties of the N- and C-protected peptide fragment of the C-terminal domain (102-107), Ac-Glu-Leu-Ala-Lys-His-Ala-amide, of his-tone H2B towards Ni(II) ions were studied [143]. Imidazole was proposed as an anchoring site and at high pH the diamagnetic species with a {Nim, 3N"} coordination mode was suggested.

The peptide sequence (110-113), Cys-Ala-Ileu-His, of the H3 histone is evo-lutionarily strictly conserved among animal species and it contains the very attractive set of Cys and His residues as far as metal ion coordination is concerned [144]. This sequence was suggested to be a potential binding site for Zn(II), resembling to some extent the zinc finger binding pattern [145], but it seems that no study follows this suggestion. Cys-110 is the only free thiol in H3, and as such has often been employed as a chemical labeling site [146]. The peptide CAIH as a minimal model for the H3 histone was synthesized allowing metal-ion-binding studies and thus, its complex formation with Ni(II) was characterized [147]: At the physiological pH, Ni(II) and the peptide yielded unusual macrochelate complexes, in which Ni(II) was bound through Cys and His side chains in a square planar arrangement.

Histidine (His-18) is also located in the histone H4 N-terminus that extends from the protein core, where it is accessible. Histone H4 is one of the most conserved proteins in nature, also for the N-terminal region (residues 1-22) [148]. This region features three repetitions of the sequence Gly-Lys-Gly and the unusual string of five basic residues, KRHRK. It was found that nickel is a potent inhibitor of histone H4 acetylation in yeast and in mammalian cells [149]. Studies with a minimal coordinating model of the H4 tail, AKRHRK [150], and of the larger N-terminal domain, SGRGKGGKGLGKGGAKRHRKVL (residues 1-22) [151,152], were performed. The spectroscopic data obtained for the Ni(II)-AKRHRK system have shown that histidine acts as an anchoring metal-binding site. The stability constant of the Ni(II) species with the H4 fragment was distinctly higher than that of the for Boc-AGGH peptide [150]. This may indicate that the positively charged side chains of Lys and Arg increase distinctly the stability of the 3N, 4N complexes [153]. Although the binding of Ni(II) in the physiological pH range is not very effective, the hydrophobic environment in the entire protein is expected to enhance the metal-binding capabilities, due to the multiple nonbonding interactions stabilizing the complex formed [154-156].

Protamines are small basic proteins which provide compact DNA binding in vertebrate sperm. Mammals possess two classes of protamines, P1 and P2. P1 rich in arginine and cysteine, but not histidine, is expressed in all mammals, while P2, which also contains histidines, has been detected in just a few mammalian species, including mice and humans [157,158]. Its presence at the levels of 50-70% of total protamine is, however, required for male fertility in humans [159]. HP2 contains the N-terminal tripeptide albumin-like sequence Arg-Thr-His. Analogous N-terminal sequences, containing histidine in the third position (Xx-Yy-His), are known for their specificity for Cu(II) and Ni(II) binding [18]. They are also present in several human peptide hormones and proteins, human serum albumin (HSA) being the most prominent [160]. The Asp-Ala-His sequence of HSA is the physiological carrier of Cu(II) [161], and it is also involved in nickel toxicity, providing the antigen for nickel allergy when coordinated to Ni(II) [162]. These facts strongly suggested that HP2 may contain a physiologically relevant Cu(II) and Ni(II) binding site at its N-terminus. The potentiometric and spectro-scopic results [153] indicate that the N-terminal tripeptide motif Arg-Thr-His is the exclusive binding site for nickel ions at the metal to HP21-15 molar ratio not higher than 1. A solution structure of the Ni(II) complex with the N-terminal pen-tadecapeptide of human protamine HP2 (HP21-15) was elucidated with the use of one- and two-dimensional 1H NMR techniques and molecular modeling [163].

Cap43 is a novel gene induced by a rise in free intracellular Ca2+ following nickel exposure [164,165]. No other metal compound significantly induced expression of this gene, indicating that it was expressed with a marked specificity to Ni(II) exposure [166]. Cap43 has a mono-histidine 10-amino acid-residue fragment (Thr-Arg-Ser-Arg-Ser-His-Thr-Ser-Glu-Gly) which is repeated three times in the C-terminal domain. It should be mentioned that such mono-histidine fragments, e.g., the octapeptide repeated regions in prion proteins, play a critical role in metal metabolism using a set of His imidazoles as the binding sites for metal ions [167]. The tetradecapeptide containing one 10-amino acid repeated sequence was analyzed for Ni(II) binding [168]. The 20- (Ac-TRSRSHTSEG-TRSRSHTSEG) and the 30-(Ac-TRSRSHTSEG-TRSRSHTSEG-TRSRSHTSEG) amino acid sequences were analyzed [169] as well. The 20-amino acid peptide can bind one or two metal ions, while the 30-amino acid fragment, binds up to three metal ions.

Fibrinogen is a dimer, the two halves being held together by disulfide bridges, and fibrinopeptides A and B, together with fibrin, are produced by the cleavage of fibrinogen by thrombin when there is some injury to the body. Once the fibrinopeptides have been released the fibrin residues form a clot by side-to-side and end-to-end aggregation [170]. Human fibrinopeptide A is a peptide containing 16 amino acid residues, Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg. Local concentrations of fibrinopeptides near the site of injury will be high and they would be competitive with other biopeptides in interaction with metal ions, particularly Cu(II). Complexes with Ni(II) of the amino-terminal tetrapeptide fragment of human fibrinopeptide A (Ala-Asp-Ser-Gly) have been studied [86].

The major difference compared to that of tetraalanine is the greater importance of the NiH_jL complex in the case of Ala-Asp-Ser-Gly. This suggests the formation of the Ni(II) chelation through the amino, amide, and P-carboxylate donor set.

Arginine8-vasopressin (AVP) and arginine8-vasotocin (AVT) are naturally occurring neurohormones with similar structural features, each possessing a 20-membered ring linked by a disulfide bridge with a tripeptide tail. Spectroscopic studies have shown that the similarities in their primary structure are accompanied by similar peptide backbone conformations, as well as the conformations around the disulfide bridge [171,172]. The complexes of vasopressin and vasotocin with Cu(II) are one of the most stable Cu-peptide complexes with 4N coordination yet reported [95]. The major stabilization factor is the favorable positioning of the binding donor atoms within the ring formed by the disulfide bridge of the peptide.

Secreted protein, acidic and rich in cysteine (SPARC) is a matricellular calcium-binding glycoprotein, which mediates the cell-matrix interactions without having any primarily structural role [173,174]. It has been reported as a product of some tumors and this result has been associated to the SPARC activity in angiogen-esis [175]. The human protein consists of 286 residues divided into three distinct domains [176]. The second domain is a Cys-rich, follistatin-like (FS) domain (residues 53-137), in which all Cys residues are disulfide-bonded, with a N-linked carbohydrate moiety at Asn 99. This domain is characterized by the presence of two copper-binding sites, the strongest of which contains the sequence Lys-Gly-His-Lys (KGHK) (residues 120-123). Both KGHK and the longer peptides containing this sequence have been recognized to regulate angiogenesis in vitro and in vivo [177]. The tetrapeptide KGHK binds Ni(II) ions in a tetradentate albumin-like fashion {NH2, 2N~, Niim}.

Angiotensin II, a peptide hormone which has the sequence Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, is involved in the regulation of blood pressure and has been shown to interact with metal ions in biological systems [178]. The study of the complexes of angiotensin II and two of its peptide fragments, Asp-Arg-Val-Tyr-and MeCO-Tyr-Ile-His, with Ni(II) shows that metal ions at high pH form 4N species with the metal ion bound at the N-terminus of angiotensin II, giving a complex closely similar to that formed by Asp-Arg-Val-Tyr [85]. There may be a role for the imidazole site at lower pH range, but comparison of the Ni(II) complexes of MeCO-Tyr-Ile-His with those of Asp-Arg-Val-Tyr shows the latter pep-tide to be more effective in 4N complex formation. The results suggest that, with Ni(II), the terminal amino nitrogen of the Asp residue is a more effective center to initiate coordination than is the imidazole nitrogen.

The thyrotropin releasing factor (TRF, L-pyroglutamyl-L-histydyl-L-prolina-mide, Pyr-His-Pro-NH2) and melanostatin (MIF, L-prolyl-L-leucyl-glycinamide, Pro-Leu-Gly-NH2) are both oligopeptide hormones containing among others a proline residue at the C- and N-terminal ends, respectively. The interactions of Ni(II) with Pyr-His-Pro-NH2 and the Pyr-His dipeptide analog have been studied [179]. In both systems, Ni(II)-TRF and Ni(II)-Pyr-His, at least two square planar complexes are formed with the metal-coordination sites at N3 imidazole, of the peptide linkage, and the deprotonated nitrogen of Pyr. The difference between these complexes is the pyrrole type nitrogen (N1) of imidazole, which may be protonated (pH < 10) or deprotonated (pH > 10). The drastic variations in the CD spectra in the d-d transition region due to deprotonation of the N1 imidazole, result most probably from a change in the chelate ring conformation. The N1 deprotonation of the imidazole ring changes its aromatic character and the coordinated N3 site alters its n acceptor properties. This may change the Ni(II)-N3 bond length slightly which is very sensitive to the geometry of the metal-imidazole interaction [180].

Melanostatin is a hypothalamic hormone and a therapeutic agent for Parkinson's disease [181]. Complex formation between Ni(II) and MIF was found to be exceedingly slow at 25°C, and 1:1 metal:ligand solutions tended to precipitate [58]. Above pH 8 a NiH_3L complex could be identified clearly as the planar complex. It is therefore reasonable to assume that this is a 4N complex comparable to the Cu(II) analog [182,183].

The gonadotropin-releasing hormone (GnRH) or luteinizing-hormone-releasing hormone (LHRH) is a decapeptide (pEHWSYGLRPG) and plays an essential role in mediating the neuroendocrine control of reproductive processes. It was shown [184] that Cu(II), Ni(II) and Zn(II), may distinctly change the biological activity of the LHRH, as was found in the case of TRF [185]. The complex of Cu(II) with GnRH brought about a high release of LH and an even higher release of FSH. The Ni(II) complex showed a similar although less distinct effect [186]. In the study of metal-GnRH complexes interacting with the rat pituitary receptor [187], the Cu(II) complex was shown to act more efficiently than native GnRH, whereas the activity of nickel or zinc complexes was slightly lower. The coordination of Ni(II) starts at a pyridine-like nitrogen and then adjacent amide nitrogens complete the coordination around the metal ion forming 3N of {Nim, 2N~} and 4N {Nim, 3N~} coordinated species [188,189]. The studies involving several metal ions have shown that LHRH is very specific ligand for Ni(II) and the coordination mode found for this metal ion is considerably different from that occurring for Cu(II). 1H NMR measurements have been performed for the free GnRH and its complexes with Ni(II) in DMSO (dimethyl sulfoxide) [190].

The examples mentioned above clearly indicate that Ni(II) ions are critical factors for oligopeptide structures and their biological activities. It is actually very likely that many natural oligopeptides are affected by metal ion coordination in their biological functions.

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