Structural Comparisons of Apo- and Metalated Three-Stranded Coiled Coils Clarify Metal Binding Determinants in Thiolate Containing Designed Peptides

Abstract

Over the past two decades, designed metallopeptides have held the promise for understanding a variety of fundamental questions in metallobiochemistry; however, these dreams have not yet been realized because of a lack of structural data to elaborate the protein scaffolds before metal complexation and the resultant metallated structures which ultimately exist. This is because there are few reports of structural characterization of such systems either in their metallated or nonmetallated forms and no examples where an apo structure and the corresponding metallated peptide assembly have both been defined by x-ray crystallography. Herein we present x-ray structures of two de novo designed parallel three-stranded coiled coils (designed using the heptad repeat (a→g)) CSL9C (CS = Coil Ser) and CSL19C in their non-metallated forms, determined to 1.36 Å and 2.15 Å resolutions, respectively. Leucines from either position 9 (a site) or 19 (d site) are replaced by cysteine to generate the constructs CSL9C and CSL19C, respectively, yielding thiol-rich pockets at the hydrophobic interior of these peptides, suitable to bind heavy metals such as As(III), Hg(II), Cd(II) and Pb(II). We use these structures to understand the inherent structural differences between a and d sites to clarify the basis of the observed differential spectroscopic behavior of metal binding in these types of peptides. Cys side chains of (CSL9C) 3 show alternate † Current Address: Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation St, Worcester, Massachusetts 01605, USA ‡ Current Address: University of Birmingham, School of Chemistry, Edgbaston, Birmingham, UK B15 2TT Suporting Information Available: Helical wheel diagrams of (CSL9C) 3 , (CSL19C) 3 , location of Zn(II) ions at crystal packing interfaces of (CSL19C) 3 , orientation of Cys Sγ plane with respect to Leu 16 and Leu 23 layers of (CSL19C) 3 , the overlay of α-helical backbones of (CSL9C) 3 , (CSL19C) 3 , (CSL16LPen) 3 and (CSL16DPen) 3 , UV-Vis titrations of Zn(II) to CSL9C and Hg(II), UV-Vis titrations of Hg(II) to CSL9C in the presence and absence of Zn(II) and 199 Hg NMR spectra of CSL9C with 199 Hg(II) in the presence and absence of Zn(II). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public AccessAuthor Manuscript J Am Chem Soc. Author manuscript; available in PMC 2011 September 29. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript conformations and are partially preorganized for metal binding, whereas cysteines in (CSL19C) 3 are present as a single conformer. Zn(II) ions, which do not coordinate or influence Cys residues at the designed metal sites but are essential for forming x-ray quality crystals, are bound to His and Glu residues at the crystal packing interfaces of both structures. These "apo" structures are used to clarify the changes in metal site organization between metallated As(CSL9C) 3 and to speculate on the differential basis of Hg(II) binding in a versus d peptid...

Natural and Artificial Proteins Containing Cadmium

“…In contrast, the thiol goups in a d site were directed towards the helical interface, suggesting that reorganization was required in order to coordinate to a metal ion [38]. More recently crystal structures have been obtained of related three-stranded coiled coils based on CoilSer [52] with Cys in an a site (9 position) or a d site (19 position) which are in reasonable agreement with the original findings [53]. The major rotamer of Cys side chains in an a site are directed towards the interior of the coiled coil with sulfur-sulfur distances of 3.3-3.4 Å , forming a pre-organized metal binding site ( Figure 5, left).…”

“…In this structure, two of the Cys side chains are pointed towards the coiled coil interior, and the third is directed away from the interior towards the helical interface. That latter is likely to require reorganization to position itself for metal binding [53].…”

Structural Characteristics of the Redox-sensing Coiled Coil in the Voltage-gated H+ Channel

“…There have been only minimal successes in the structural determination of the disulfide bond in the coiled coil. Crystal structures of de novo designed parallel trimeric coiled coils were determined, showing that the Cys in the core of the coiled coil forms a binding pocket suitable for heavy metals (25). In another case, the crystal structure of a bacterial DNA-binding protein shows a disulfide bond on the dimerization interface in the core of a short antiparallel dimeric coiled coil (26).…”

Control of enzyme reaction by a designed metal-ion-dependent α-helical coiled-coil protein

“…, and As 2? [46][47][48][49], and their metal complexes were determined by the X-ray crystal studies [7,[50][51][52][53]. Although the binding affinity should be further improved, one application of our construct may be the development of transcription system induced by a specific metal ion.…”