Solution structure of the circular γ-domain analog from the wheat metallothionein E(c)-1

Abstract

The first cyclic analog of a metallothionein (MT) was prepared and analyzed by UV and (magnetic) circular dichroism spectroscopy, ESI-MS as well as NMR spectroscopy. Results reveal that the evaluated cyclic γ-E(c)-1 domain of the wheat MT E(c)-1 retains its ability to coordinate two Zn(II) or Cd(II) ions and adopts a three-dimensional structure that is highly similar to the one of the linear wild-type form. However, the reduced flexibility of the protein backbone facilitates structure solution significantly and results in a certain stabilization of metal binding to the protein.

Figure 1

Sequence and purity of cyc-γ-E_c-1. (a) Amino acid sequence of cyc-γ-E_c-1. The sequence of the wild-type linear form is given in capital letters, Cys residues are highlighted with bold letters, amino acids of the additional linker region are given in lower case letters. (b) Deconvoluted ESI-MS spectrum of cyc-Zn₂γ-E_c-1 (calculated mass of molecular ion 2,897.9 Da). (c) Deconvoluted ESI-MS spectrum of cyc-Cd₂γ-E_c-1 (calculated mass of molecular ion 2,991.9 Da). Peaks with a higher mass correspond to the Na-adducts of the respective species.

Figure 2

Comparison of (a) UV, (b) CD, and (c) MCD spectra of circular metal-free, Zn₂-, and Cd₂γ-E_c-1 with the respective linear wild-type forms. See legend in (a) for assignment of spectra.

Figure 3

Plots of molar absorptivity against pH values for the pH titrations of (a) cyc-Zn₂- and (b) cyc-Cd₂γ-E_c-1 and the respective linear forms including the data fits using Equation (1) or (2) (see Table 1). For assignment of data and curves see the legends.

Figure 4

NMR solution structure of cyc-γ-E_c-1. (a) Protein backbones of the 20 lowest energy structures of cyc-Cd₂γ-E_c-1 in ribbon presentation. The structure closest to the average structure is depicted in dark grey and also the Cd^II-thiolate cluster arrangement is shown (Cd^II ions as blue spheres, Cys thiolates as yellow sticks); (b) Backbone overlay of cyc- (blue) and linear (grey) Cd₂γ-E_c-1 structures that are closest to the respective mean structure (Cd^II ions as spheres, Cd^II-thiolate connectivities are indicated with dotted lines); (c) Protein backbone overlays of cyc-Cd₂γ-E_c-1 with Cys3 and Cys9 (blue) or Cys21 and Cys9 (yellow) as bridging residues between the two Cd^II ions of the cluster and the structure that was calculated without any metal restraints (grey). Cd^II ions are shown as larger spheres and the thiolate groups of the Cys residues as smaller spheres.

Figure 5

Selected NMR spectra of Cd₂γ-E_c-1. (a) Comparison of the amide regions of the 2D [¹H,¹H] TOCSY NMR spectra of cyc- (left) and linear (right) Cd₂γ-E_c-1. Evidently, most of the resonances have similar chemical shifts in the two species; (b) [¹¹³Cd,¹H]-HSQC spectra of cyc-Cd₂γ-E_c-1 including cross peak assignments.

Figure 6

Structure bundles of the 20 lower energy structures of (a) cyc- and (b) linear Cd₂γ-E_c-1 calculated without any metal restraints.