Structural and spectroscopic studies of heavy metal binding to de novo designed coiled -coil peptides.

Abstract

Structural characterization of both apo and As(III) bound cysteine-containing designed three-stranded coiled coil peptides and spectroscopic studies to determine Pb(II) and Cd(II) binding specificity to peptides with two thiolate metal binding sites were performed to better understand the interactions of these toxic metals with cysteine rich biomolecules. Crystallographic determination of the structures of the peptides CSL19C [Ac-EWEALEKKLAALESKLQACEKKLEALEHG-NH₂] and As(CSL9C)₃ the As(III) complex of CSL9C, [Ac-EWEALEKKCAALESKLQALEKKLEALEHG-NH₂]. UV-Vis, CD, <super>113</super>Cd NMR, and NOESY 2D NMR spectroscopic techniques used to evaluate metal binding to the disubstituted peptides. The parallel structure obtained for the Coil Ser derivative CSL19C differs from the antiparallel orientation obtained for its parent peptide. The orientation of cysteine side chains in the <bold>d</bold> position away from the center of the coiled coil experimentally confirms hypotheses from modelling studies and provides a basis on which to understand metal binding to these sites. Additionally, the discovery of Zn(II) ions at crystal packing interfaces provides a tool to facilitate crystallization of other derivatives. The first crystallographic characterization of As(III) in a biologically relevant tristhiolate coordination environment in the interior of a three-stranded coiled coil reveals the surprising pyramidal coordination of As(III) <bold> below</bold> the plane of the beta-methylene protons of the three cysteine residues, not above, as was predicted. The parallel orientation of the coiled coil in the presence of metal and the necessity of Zn(II) for crystal packing were confirmed in this 1.8 A structure. Analysis of preferences of Cd(II) and Pb(II) for <bold>a</bold> vs. <bold> d</bold> position cysteines in the heptad repeat were evaluated with both monosubstituted and disubstituted peptides. Peptides with one <bold>a</bold> and one <bold>d</bold> or two <bold>d</bold> binding sites were both shown to be able to discriminate between Cd(II) and Pb(II). Fascinatingly, the coordination of Pb(II) was seen to perturb the coordination of Cd(II) to a less favored three-coordinate species from mixed 3- and 4-coordinate species. The investigation of Pb(II) and Cd(II) binding to a peptide containing two <bold>a</bold> binding sites that was previously shown to have no Cd(II) discrimination resulted in the discovery of a preference for one mixed Pb(II)-Cd(II) species. Results of this work give a structural explanation of spectroscopic and thermodynamic observations, the evaluation of specificity of metal binding for Cd(II) and Pb(II) systems adds to our understanding of the coordination preferences of these toxic metals in biological systems.