Unravelling the Structure of the Tetrahedral Metal-Binding Site in METP3 through an Experimental and Computational Approach

Abstract

Understanding the structural determinants for metal ion coordination in metalloproteins is a fundamental issue for designing metal binding sites with predetermined geometry and activity. In order to achieve this, we report in this paper the design, synthesis and metal binding properties of METP3, a homodimer made up of a small peptide, which self assembles in the presence of tetrahedrally coordinating metal ions. METP3 was obtained through a redesign approach, starting from the previously developed METP molecule. The undecapeptide sequence of METP, which dimerizes to house a Cys4 tetrahedral binding site, was redesigned in order to accommodate a Cys2His2 site. The binding properties of METP3 were determined toward different metal ions. Successful assembly of METP3 with Co(II), Zn(II) and Cd(II), in the expected 2:1 stoichiometry and tetrahedral geometry was proven by UV-visible spectroscopy. CD measurements on both the free and metal-bound forms revealed that the metal coordination drives the peptide chain to fold into a turned conformation. Finally, NMR data of the Zn(II)-METP3 complex, together with a retrostructural analysis of the Cys-X-X-His motif in metalloproteins, allowed us to define the model structure. All the results establish the suitability of the short METP sequence for accommodating tetrahedral metal binding sites, regardless of the first coordination ligands.

Interactions of an Artificial Zinc Finger Protein with Cd(II) and Hg(II): Competition and Metal and DNA Binding

“…The structure of a ZF motif is stabilised by the tetrahedral coordination of Zn(II) and by the formation of a hydrophobic core [14]. Similar tetrahedral coordination was found in self-assembling peptides offering a Cys2His2 binding site [15]. Cys2His2-type proteins form the most populous family of ZFPs [16].…”

Designed Rubredoxin miniature in a fully artificial electron chain triggered by visible light

“…Others have installed the tetrahedral FeCys4 site in structurally different natural and de novo proteins [25][26][27] . We, and others, have focused on the Rd prototypical structural unit, making use of its intrinsic symmetry to build a miniaturized peptide scaffold [28][29][30][31] .…”

Designed Rubredoxin miniature in a fully artificial electron chain triggered by visible light

“…Others have installed the tetrahedral FeCys 4 site in structurally different natural and de novo proteins [28][29][30] . We, and others, have focused on the Rd prototypical structural unit, making use of its intrinsic symmetry to build a miniaturized peptide scaffold [31][32][33][34] . In particular, we previously recognized the designed protein METP (Miniaturized Electron Transfer Protein) as a minimal unit needed to reproduce Rds by retrostructural analysis 33 .…”