Apoprotein Structure and Metal Binding Characterization of a <i>de Novo</i> Designed Peptide, α₃D<b>IV</b>, that Sequesters Toxic Heavy Metals

Abstract

<i>De novo</i> protein design is a biologically relevant approach that provides a novel process in elucidating protein folding and modeling the metal centers of metalloproteins in a completely unrelated or simplified fold. An integral step in <i>de novo</i> protein design is the establishment of a well-folded scaffold with one conformation, which is a fundamental characteristic of many native proteins. Here, we report the NMR solution structure of apo α₃DIV at pH 7.0, a <i>de novo</i> designed three-helix bundle peptide containing a triscysteine motif (Cys18, Cys28, and Cys67) that binds toxic heavy metals. The structure comprises 1067 NOE restraints derived from multinuclear multidimensional NOESY, as well as 138 dihedral angles (ψ, φ, and χ₁). The backbone and heavy atoms of the 20 lowest energy structures have a root mean square deviation from the mean structure of 0.79 (0.16) Å and 1.31 (0.15) Å, respectively. When compared to the parent structure α₃D, the substitution of Leu residues to Cys enhanced the α-helical content of α₃D<b>IV</b> while maintaining the same overall topology and fold. In addition, solution studies on the metalated species illustrated metal-induced stability. An increase in the melting temperatures was observed for Hg­(II), Pb­(II), or Cd­(II) bound α₃D<b>IV</b> by 18–24 °C compared to its apo counterpart. Further, the extended X-ray absorption fine structure analysis on Hg­(II)-α₃D<b>IV</b> produced an average Hg­(II)–S bond length at 2.36 Å, indicating a trigonal T-shaped coordination environment. Overall, the structure of apo α₃D<b>IV</b> reveals an asymmetric distorted triscysteine metal binding site, which offers a model for native metalloregulatory proteins with thiol-rich ligands that function in regulating toxic heavy metals, such as ArsR, CadC, MerR, and PbrR