Apoprotein Structure and Metal Binding Characterization of a de Novo Designed Peptide, α₃DIV, that Sequesters Toxic Heavy Metals

Abstract

De novo 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 de novo 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 de novo 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 α3DIV 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 α3DIV by 18–24 °C compared to its apo counterpart. Further, the extended X-ray absorption fine structure analysis on Hg(II)-α3DIV produced an average Hg(II)–S bond length at 2.36 Å, indicating a trigonal T-shaped coordination environment. Overall, the structure of apo α3DIV 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

Development of a Rubredoxin-Type Center Embedded in a de Dovo-Designed Three-Helix Bundle

“…Previous studies have demonstrated that α 3 D IV , α 3 D IV -H72C, and α 3 D IV -L21C are well-folded, three-helix bundle proteins. 7,8,27 Furthermore, circular dichroism spectra indicated that the structure of the peptide is not drastically altered by the presence of iron and that iron binding stabilizes the peptide slightly (Figure S1). Fe(II) binding titrations as monitored by UV–vis spectroscopy showed that the peptide binds Fe(II) in a pH-dependent fashion with a K D of 6.3 μ M at pH 8.5 (Figure S2).…”

Clarifying the Copper Coordination Environment in a de Novo Designed Red Copper Protein

“…45 However, additional mutations such as those in the α 3 DChC2 sequence led to proteins of markedly inferior thermodynamic stability. It was also considered that, for this reason, the α 3 DChC2-designed hydrophobic box was imperfectly constraining the metal binding site, causing the red copper spectral properties.…”

De Novo Design and Characterization of Copper Metallopeptides Inspired by Native Cupredoxins

“…The preassembled fold of α 3 D offers facile incorporation of a mixed-ligand site within a de novo designed scaffold. Utilizing the structure of α 3 DIV 16 as a foundation, we designed three distinct constructs, designated as α 3 D-core (CR), -chelate (CH), and -chelate-core (ChC) (Table 1). Our goal was to assess if the physical properties (e.g., absorption λ max at 600 nm, compressed A ∥ , short Cu–S(Cys) bond, and E ° value >180 mV) of a metal center that is naturally observed in a β -barrel fold can be achieved in the antiparallel three-helix bundle fold of α 3 D. Ultimately, the lessons learned from this work will provide the foundation to study long-rate ET reactions within a de novo designed framework and develop bifunctional/bimetallic constructs that contain a catalytic and an ET center, such as in copper nitrite reductase.…”