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Heteromeric three-stranded coiled coils designed using a Pb(II)(Cys)₃ template mediated strategy.

ABSTRACT: Three-stranded coiled coils are peptide structures constructed from amphipathic heptad repeats. Here we show that it is possible to form pure heterotrimeric three-stranded coiled coils by combining three distinct characteristics: (1) a cysteine sulfur layer for metal coordination, (2) a thiophilic, trigonal pyramidal metalloid (Pb(II)) that binds to these sulfurs and (3) an adjacent layer of reduced steric bulk generating a cavity where water can hydrogen bond to the cysteine sulfur atoms. Cysteine substitution in an a site yields Pb(II)A₂B heterotrimers, while d sites provide pure Pb(II)C₂D or Pb(II)CD₂ scaffolds. Altering the metal from Pb(II) to Hg(II) or shifting the relative position of the sterically less demanding layer removes heterotrimer specificity. Because only two of the eight or ten hydrophobic layers are perturbed, catalytic sites can be introduced at other regions of the scaffold. A Zn(II)(histidine)₃(H₂O) centre can be incorporated at a remote location without perturbing the heterotrimer selectivity, suggesting a unique strategy to prepare dissymmetric catalytic sites within self-assembling de novo-designed proteins.

SUBMITTER: Tolbert AE

PROVIDER: S-EPMC7169047 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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 metalated structures which ultimately exist. This is because there are few reports of structural characterization of such systems either in their metalated or nonmetalated forms and no examples where an apo structure and the corresponding metalated 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 nonmetalated forms, determined to 1.36 and 2.15 A 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 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 metalated As(CSL9C)(3) and to speculate on the differential basis of Hg(II) binding in a versus d peptides. Thus, for the first time, one can establish general rules for heavy metal binding to Cys-rich sites in designed proteins which may provide insight for understanding how heavy metals bind to metallochaperones or metalloregulatory proteins.

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Heteromeric three-stranded coiled coils designed using a Pb(II)(Cys)₃ template mediated strategy.

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OmicsDI is part of the ELIXIR infrastructure

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Heteromeric three-stranded coiled coils designed using a Pb(II)(Cys)3 template mediated strategy.

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OmicsDI is part of the ELIXIR infrastructure

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Heteromeric three-stranded coiled coils designed using a Pb(II)(Cys)₃ template mediated strategy.