Two peptides, L(TC) and L(TC)(T) have been synthesised to model the treble-clef zinc fingers encountered in many Zn(Cys)(4)-site-containing proteins. Both are cyclic peptides with a linear tail grafted on a glutamate side chain of the cycle. They differ by the length of this tail, which lacks five amino acids in L(TC)(T) compared to L(TC). Both peptides bind Zn(2+) and Co(2+) in 1:1 metal/peptide ratio and the structure of these complexes have been characterised by NMR, UV/Vis and CD spectroscopy. Both peptides fold the same way around the metal ion and they fully reproduce the classical fold of treble-clef zinc fingers and display an extended hydrogen-bond network around the coordinating sulfur atoms. The structures of the ML(TC) complexes reveal that the linear tail forms a short two-turn alpha-helix, present in the metallated form only. The formation of this helix constitutes a rare example of metal-induced folding. The second turn of this helix is composed of the five amino acids that are absent in L(TC)(T). The study of the pH-dependence of the Zn(2+) binding constants shows that the metal ion is bound by four cysteinates above pH 5.2 and the binding constants are the highest reported so far. Interestingly, the binding constant of Zn x L(TC) is about tenfold higher than that of Zn x L(TC)(T). This difference clearly indicates that the helix, present in Zn x L(TC) only, stabilises the Zn(2+) complex by about 1.2 kcal mol(-1). The origin of this stabilisation is ascribed to an electrostatic interaction between the [ZnS(4)](2-) centre and the helix. This reveals a cooperative effect: zinc binding allows the folding of the tail into a helix which, in turn, strengthens the zinc complex.

A recent bioinformatics study has evaluated to about 1000 (or ca. 3 % of the total protein number) the number of human proteins possessing a tetracysteinate zinc site.[1] These sites were initially presumed to have a structural role because they were associated to zinc finger proteins, [2] where they fold the protein chain in a conformation suitable for its binding to DNA. They were later found in several proteins and enzymes involved in demethylation processes, such as the DNA repair protein Ada [3] and various transferases. [4] More recently, such sites were discovered in the heat shock protein Hsp33 [5] and the disulfide reductase Trx2, [6] where their interaction with reactive oxygen species (ROS) contributes to the oxidativestress response. This is of special interest as tetracysteinate zinc sites, especially in zinc finger proteins, have been considered to be likely targets of ROS. Free cysteines are commonly involved in peroxide sensing and response, [7] and their reactivity has been thoroughly studied over the past twenty years. A reasonable reactivity picture has emerged that points to the importance of hydrogen bonding in increasing the nucleophilic character of the cysteine sulfur atom. No such rationale is available for metal-bound cysteinates.To obtain a better understanding of the reactivity of tetracysteinate zinc sites with ROS, we are developing a biomimetic approach based on de novo peptide synthesis. This approach is particularly suited to mimicking these sites and the potentially important hydrogen-bonding interactions, which is not possible with metallo-organic complexes in organic solvents. The validity of this approach has been demonstrated by Berg and Shi in their modeling studies of zinc finger proteins with a mixture of cysteinate and histidine ligands, [8] and was further highlighted more recently by Gibney et al. [9] Both groups used linear 16-to 26-mer peptides incorporating two CX n C (n = 2-4) zinc-binding motifs. Regan and Clarke [10] relied on self-assembling peptides to constitute a four-helix bundle orienting the cysteinates in the proper way to bind zinc. Nevertheless, this approach is generally weakened by the difficulty of obtaining detailed structural characterization of metallopeptides. In addition, these two designs cannot reproduce the tetracysteinate arrangements that belong to b hairpins, such as that of Hsp33. [5] This prompted us to develop a totally new design based on introducing one CX n C motif into a cyclic peptide and another one into a linear chain connected to the cycle through a glutamate or lysine residue. Herein, we show that these peptides with limited size and flexibility, allow the almost perfect reproduction of both the structure and the reactivity of the tetracysteinate zinc site of the protein Hsp33. Figure 1 illustrates the tetracysteinate zinc site of Hsp33, which consists of a CXXC motif (C 263 KWC 266 ) located in a b-hairpin loop and a CXC motif (C 231 DC 233 ). To reproduce the topology of this site, we designed a cyclic peptide to mimic the b-h...

No abstract