Specificity of the Zn²⁺, Cd²⁺and Ni²⁺ion binding sites in the loop domain of the HypA protein

Abstract

The zinc binding loop domain of the HypA protein of Helicobacter pylori consists of two CXXC motifs with flanking His residues. These motifs bind metal ions, and thus they are crucial for the functioning of the whole protein. The N-terminal site, where His is separated from CXXC by Ser residue is more effective in binding Zn(2+) and Ni(2+) ions than the C-terminal site, in which His is adjacent to the CXXC motif. Studies on various modifications of the peptide sequence within the Ac-ELECKDCSHVFKPNALDYGVCEKCHS-NH2 loop show the role of the residues in the linker between the CXXC motifs and the effect of the length of the linker on the stability of the complexes it forms with Zn(2+), Cd(2+) and Ni(2+) ions. The proline residue in the linker between the two CXXC binding sites plays a distinct role in the metal ion binding ability of the loop, lowering the efficacy of the metal ion coordination. The deletion of the aliphatic residues from the linker between the CXXC motifs remarkably improves the binding efficacy of the loop.

Metal Binding Ability of Small Peptides Containing Cysteine Residues

“…The coordination of two or four thiolate-S donor atoms to Cd(II) in the [ML] and [ML 2 ] complexes, respectively, was confirmed by spectroscopic data (Table 3), which shows that the 2 × S À coordination can be characterized by 8000-11000 and 4 × S À coordination by 20000-27000 molar absorptivity. These parameters are in agreement with those published for CdL complex of Ac-ELECKDCSSV-NH 2 (ɛ = 11780 M À 1 cm À 1 ) [27] and CdL 2 complex of Ac-ELECKDCSHV-NH 2 (ɛ = 25670 M À 1 cm À 1 ) [26] and Ac-DYGVCEKCHS-NH 2 (ɛ = 24900 M À 1 cm À 1 ). [27] The suggested coordination mode of the Cd(II) complexes was further supported by the 113 Cd NMR spectroscopy.…”

“…These parameters are in agreement with those published for CdL complex of Ac-ELECKDCSSV-NH 2 (ɛ = 11780 M À 1 cm À 1 ) [27] and CdL 2 complex of Ac-ELECKDCSHV-NH 2 (ɛ = 25670 M À 1 cm À 1 ) [26] and Ac-DYGVCEKCHS-NH 2 (ɛ = 24900 M À 1 cm À 1 ). [27] The suggested coordination mode of the Cd(II) complexes was further supported by the 113 Cd NMR spectroscopy. In the spectrum registered in the Cd(II)-Ac-CSSC-NH 2 = 1 : 2 sample one peak can be observed at 648 ppm, which can be assigned to the 4 S À coordinated complex (Figure S1).…”

“…The enhancement of the stability of Ac-CGAD-NH 2 , Ac-CGAH-NH 2 and Ac-DAAC-NH 2 , [33] Ac-HAAC-NH 2 [34] is caused by Ac-CSSC-NH 2 Ac-ELECKDCSSV-NH 2 [27] Ac-CSSACS-NH 2 HS* [29,30] PS** [31] [ [33], [34] and [42]). The stoichiometries of complexes calculated from the potentiometric data are 1 : 1 and 1 : 2.…”

“…The results of the corresponding peptides show that in the Zn(II) and Cd(II) complexes the metal ion is coordinated by the thiol donors (2S À ) and as expected: the formed cadmium complexes are thermodynamically more stable than the zinc ones. [23,24] H. Kozlowski and co-workers studied the coordination behaviour of peptides containing CXXC motifs through the Ac-GCASCDNCRACKK-NH 2 , Ac-GCASCDNCRAAKK-NH 2 , Ac-GCASCD-NARAAKK-NH 2 [25] and the Ac-ELECKDCSHVFKPNALDYGVCEKCHS-NH 2 peptides [26] and its mutant [27] in the presence of the metals listed above. These latter peptides are fragments from the loop region of HypA which normally binds Zn(II).…”

The Eighth Central European Conference “Chemistry towards Biology”: Snapshot

“…Different numbers of repeats and patterns of Cys-rich regions can be involved in metal coordination (Zn(II), Cd(II) and Ni(II)) and specific relations between the binding sequence, thermodynamic stability and biological function can be found [33]. Studies on mutants of the poly-Cys sequence of the loop domain of HypA, a protein responsible for the homeostasis of Ni(II) in Helicobacter pylori , showed the role of these residues in the structure and the stability of Zn(II), Cd(II) complexes with Cys-rich domains in the proteins [34].…”