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

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 (HgS₃ coordination). The model cyclodecapeptide P^3C, c(GCTCSGCSRP) was designed to promote Hg(II) chelation in a HgS₃ coordination environment through the parallel orientation of three cysteine side chains. The binding motif is derived from the dicysteine P^2C cyclodecapeptide validated previously as a model for d¹⁰ metal transporters containing the binding sequence CxxC. The formation of the mononuclear HgP^3C complex with a HgS₃ coordination is demonstrated using electrospray ionization mass spectrometry, UV absorption, and ¹⁹⁹Hg NMR. Hg L_III-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^3C complex was refined based on ¹H-¹H 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 HgP^3CH complex has a p K_a of 4.3, indicating that the HgS₃ coordination mode is stable over a large range of pH. This low p K_a value suggests that the preorientation of the three cysteine groups is particularly well-achieved for Hg(II) trithiolate coordination in P^3C.