Mercury Trithiolate Binding (HgS₃) to a de Novo Designed Cyclic Decapeptide with Three Preoriented Cysteine Side Chains

Abstract

Mercury(II) is an unphysiological soft ion with high binding affinity for thiolate ligands. Its toxicity lies in the interactions with low molecular weight thiols including glutathione and cysteine-containing proteins that disrupt the thiol balance and alter vital functions. However, mercury can also be detoxified via interactions with Hg(II)-responsive regulatory proteins such as MerR, which coordinates Hg(II) with three cysteine residues in a trigonal planar fashion (HgS coordination). The model cyclodecapeptide P, c(GCTCSGCSRP) was designed to promote Hg(II) chelation in a HgS coordination environment through the parallel orientation of three cysteine side chains. The binding motif is derived from the dicysteine P cyclodecapeptide validated previously as a model for d metal transporters containing the binding sequence CxxC. The formation of the mononuclear HgP complex with a HgS coordination is demonstrated using electrospray ionization mass spectrometry, UV absorption, and Hg NMR. Hg L-edge extended X-ray absorption fine structure (EXAFS) spectroscopy indicates that the Hg(II) coordination environment is T-shaped with two short Hg-S distances at 2.45 Å and one longer distance at 2.60 Å. The solution structure of the HgP complex was refined based on H-H NMR constraints and EXAFS results. The cyclic peptide scaffold has a rectangular shape with the three binding cysteine side chains pointing toward Hg(II). The HgPH complex has a p K of 4.3, indicating that the HgS coordination mode is stable over a large range of pH. This low p K value suggests that the preorientation of the three cysteine groups is particularly well-achieved for Hg(II) trithiolate coordination in P.

Short oligopeptides with three cysteine residues as models of sulphur-rich Cu(i)- and Hg(ii)-binding sites in proteins

“…The synthesis of P 3C has been published earlier. 40 For the other peptides analytical HPLC and ESI-MS characterization is available in Table S1 and Fig. S1 in the ESI.…”

“…For peptide P 3C , initial coordinates were either generated as for other peptides or taken from the solution NMR structure of the HgP 3C complex. 40 Model parameters. For the holo peptides, a bonded model of the mercury site was used.…”

“…38,39 In a recently published article, the Hg(II) binding of the cyclic peptide P 3C incorporating a three Cys variant of the metal binding loop of Atx1 was discussed. 40 This cyclic solvent accessible peptide was considered as a predisposed structure that could pre-orient the Cys-sidechains for an easy accommodation of metal ions in a tri-coordinate fashion. The results confirmed the formation of a mononuclear Hg(II) complex with properties indicative of an HgS 3 coordination.…”

Molecular Dissection of dH3w, A Fluorescent Peptidyl Sensor for Zinc and Mercury

“…Only in the presence of a large excess of ligand or inside protein complexes can a third ligand bind to Hg(II), but even in those cases the three ligands are not necessarily equivalent. An interesting example is given by a family of cyclic peptides with three cysteine residues (arranged in the motif CXCXXC), which show several similarities to dH3w [27]. These peptides form 1:1 (peptide/metal) complexes at low mercury concentrations and, at higher concentrations, 1:2 and 2:3 (peptide/metal) complexes.…”

Uranyl-chelating peptides to help understanding uranium toxicity at a molecular level

“…At the CIBEST lab, we propose a biomimetic approach to help understanding the interactions of metals with biological molecules, relevant to their regulation and toxicity. [1,2] This strategy was applied for uranium, which most stable form in vivo is the uranyl cation, UO 2…”