Metal-binding properties and structural characterization of a self-assembled coiled coil: Formation of a polynuclear Cd-thiolate cluster

This paper describes the design, characterization, and metal-binding properties of a 32-residue polypeptide called AQ-C16C19. The sequence of this peptide is composed of four repeats of the seven residue sequence Ile-Ala-Ala-Leu-Glu-Gln-Lys but with a Cys-X-X-Cys metal-binding motif substituted at positions 16-19. Size exclusion chromatography with multiangle light scattering detection (SEC-MALS) and circular dichroism (CD) spectroscopy studies showed that the apo peptide exhibits a pH-dependent oligomerization state in which a three-stranded alpha-helical coiled coil is dominant between pH 5.4 and 8.5. The Cd2+-binding properties of the AQ-C16C19 peptide were studied by ultraviolet-visible spectroscopy (UV-vis), electrospray ionization mass spectrometry (ESI MS), and Cd-113 NMR techniques. The holoprotein was found to contain a polynuclear cadmium-thiolate center formed within the hydrophobic core of the triple-stranded alpha-helical coiled-coil structure. The X-ray crystal structure of the Cd-loaded peptide, resolved at 1.85 angstrom resolution, revealed an adamantane-like configuration of the polynuclear metal center consisting of four cadmium ions, six thiolate sulfur ligands from cysteine residues and four oxygen-donor ligands. Three of these are from glutamic acid residues and one is from an exogenous water molecule. Thus, each cadmium ion is coordinated in a distorted tetrahedral S3O geometry. The metal cluster was found to form cooperatively at pH 5.4 but in a stepwise fashion at pH>7. The results demonstrate that synthetic coiled-coils can be designed to incorporate multinuclear metal clusters, a proof-of-concept for their potential use in developing synthetic metalloenzymes and multi-electron redox agents. (C) 2012 Elsevier Inc. All rights reserved.