Cyclic Peptides Bearing a Side‐Chain Tail: A Tool to Model the Structure and Reactivity of Protein Zinc Sites

Abstract

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...

Cooperative Metal Binding and Helical Folding in Model Peptides of Treble‐Clef Zinc Fingers

“…We observed that a cyclic peptide was indeed more efficient than its non-cyclic analogue to control the coordination properties and to constrain the backbone in a unique conformation around zinc. [16] The work described herein validates this approach, which allows the correct fold of the treble-clef zinc fingers to be reproduced with a minimal set of amino acids. Moreover, as observed for the models of the Hsp33 zinc site, the hydrogen-bond network between the backbone amides and the zinc-binding cysteinate sulfur atoms is excellently reproduced in these model complexes.…”

“…[16] This design allows the metallated peptides to fold in a defined conformation suitable to NMR structural characterisation, which is rarely the case for small, linear, peptide-based models. These peptides almost perfectly reproduce both the first coordination sphere and the hydrogen-bond network of the ZnA C H T U N G T R E N N U N G (Cys) 4 site of Hsp 33.…”

Oxidation of Zn(Cys)4 Zinc Finger Peptides by O2 and H2O2: Products, Mechanism and Kinetics

“…The fold of the backbone, the relative orientation of the cysteine side-chains and the hydrogen bonds involving the cysteine sulfur atoms in the protein PerR are reproduced by this model. The second peptide, Zn·L HSP , [28] was designed to mimic the ZnA C H T U N G T R E N N U N G (Cys) 4 site of Hsp33. [24] Here again, the model complex reproduces almost perfectly the fold of the protein around the zinc ion.…”

“…The evolution of the signal could be perfectly fitted by a single exponential. [28] The observed first-order rate constant k obs varies linearly with [H 2 O 2 ], which shows that the oxidation follows a second-order kinetic law,…”

“…We have previously described Zn·L TC and Zn·L HSP , two model peptides for the ZnA C H T U N G T R E N N U N G (Cys) 4 sites of the structural treble-clef zinc fingers [27] and Hsp33, [28] respectively. In this article we report the reactivity towards H 2 O 2 and O 2 of these two models and of Zn·CP1A C H T U N G T R E N N U N G (CCCC), [29] the Cys 4 variant of the classical bba zinc finger consensus peptide.…”