A Constrained Tetrapeptide as a Model of Cu(I) Binding Sites Involving Cu₄S₆ Clusters in Proteins

Abstract

Peptide design is an efficient strategy to create relevant models of natural metal binding sites found in proteins. The two short tetrapeptides Ac-Cys-dPro-Pro-Cys-NH (CdPPC) and Ac-Cys-Pro-Gly-Cys-NH (CPGC) were synthesized and studied as mimics of Cu(I) binding sites involved in Cu homeostasis. Both sequences contain β turn inducing motifs to rigidify the peptide backbone structure and thereby preorganize the metal-binding side chains. The more constrained structure of the peptide CdPPC with respect to CPGC was evidenced by the measurements of the temperature coefficients of the amide protons by H NMR, which suggest a solvent-shielded intramolecular hydrogen bond in CdPPC, and no H-bond in CPGC. The Cu(I) complexes were studied by UV, circular dichroism (CD), and NMR spectroscopies as well as electrospray ionization mass spectrometry (ESI-MS) experiments in aqueous solution at physiological pH. The complexes formed with CPGC showed a complicated speciation with the possible formation of many polymetallic species. By contrast, the better preorganization in CdPPC leads to the formation of a unique CuL complex involving a CuS core. The formation of this specific cluster was confirmed by ESI-MS and by diffusion-ordered NMR spectroscopy in solution. The affinity of CdPPC for Cu(I) (β = 10 calculated for a CuL complex) is more than 1 order of magnitude larger than the affinity measured for the less constrained peptide CPGC. Besides, this stability constant value is very similar to those reported with proteins. Therefore, the Cu(I) complex formed with the simple tetrapeptide CdPPC in water at physiological pH represents a very good model of Cu(I)-thiolate clusters found in proteins. The extremely large selectivity (10) in favor of Cu(I) with respect to Zn(II), an abundant competitor in cells, makes it a promising candidate to be targeted to the liver cells for the localized treatment of Cu overload in Wilson's disease.

Short oligopeptides with three cysteine residues as models of sulphur-rich Cu(i)- and Hg(ii)-binding sites in proteins

“…Cu(I) binding to the series of peptides at physiological pH revealed to be rather complicated, with the formation of a mixture of polymetallic species. In contrast, cysteine-rich highly structured peptides 77 or peptide-like ligands 39,76 are able to control the formation of well-defined Cu(I) complexes. Consequently, the complicated Cu(I)-complex speciation of the series of peptides, reported in this paper, has been assigned to their significantly larger flexibility.…”

“…39,76 More recently, the highly constrained tetrapeptide Ac-Cys-D-Pro-Pro-Cys-NH 2 with a strong turn was shown to form exclusively a Cu 4 S 6 core. 77 The larger flexibility of the peptides described here could be responsible for the lack of control of the speciation of the Cu(I) complexes and ultimately for the formation of a mixture of polymetallic species, with quite large stability. The determined apparent stability constants are similar in the whole peptide series, within the range of experimental errors, indicating that the structural differences have only a minor effect, if any, on the stability of the Cu(I) complexes.…”

Recent advances in uranyl binding in proteins thanks to biomimetic peptides

“…Therefore depending on the nature of the metal-binding site to model, namely residues involved in metal coordination, but also location with respect to the whole protein, solvent-exposed loop or highly constrained buried site, different types of peptides have been designed and studied for metal chelation. Peptide design includes simple linear sequences extracted from the protein structure, [39][40][41][42][43][44][45] short peptides with hairpin turns [46][47] and cyclic peptides. [48][49][50][51][52][53][54][55] Supramolecular peptide assemblies such as three-stranded coiled coils have also been used to produce functional metalloenzymes.…”

Separation of multiphosphorylated cyclopeptides and their positional isomers by hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization mass spectrometry (ESI-MS)

“…Peptides are efficient models to study metal-protein binding sites [1,2] and some of their derivatives have been proposed as specific metal chelators in detoxification applications [3,4] or as models of enzyme activity [5]. Peptides with sequences found in albumin [6], osteopontin [7] and calcium binding calmodulin [8] have been used to help identifying uranium (U) chelation sites in these proteins and to evaluate their affinity towards this cation, using spectroscopic techniques such as fluorescence spectroscopy, isothermal titration calorimetry (ITC), extended X-ray absorption fine structure (EXAFS) and circular dichroism.…”