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.
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We thank J. Meager for her help with crystal data collection. We also thank The CCP4/APS School in Macromolecular Crystallography, from data collection to structure refinement and beyond 2016 for their help with crystal data processing. We acknowledge funding from NIH grant no. R01 ES012236, NSF grant no. CHE-1664926 and the Skill Development Grant from King Mongkut’s University of Technology, Thonburi, Thailand. Use of the Advanced Photon Source, an Office of Science User Facility operated for the US Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the US DOE under contract no. DE-AC02-06CH11357. Use of LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant no. 085P1000817).
The authors declare no competing interests.
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Tolbert, A.E., Ervin, C.S., Ruckthong, L. et al. Heteromeric three-stranded coiled coils designed using a Pb(ii)(Cys)3 template mediated strategy. Nat. Chem. 12, 405–411 (2020). https://doi.org/10.1038/s41557-020-0423-6