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.

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Fig. 1: Ribbon diagrams of Pb(ii)A3 (PDB: 6EGP) and Pb(ii)C3 (PDB: 6MCD) 3SCCs with coordinated water residues.
Fig. 2: Ball-and-stick models of the Cys and adjacent Ala/Leu layers for the 3 Ala and 2 Ala:1 Leu trimers.
Fig. 3: 207Pb NMR of T and G peptides with a-site Cys residues.
Fig. 4: pH titration curves of Pb(ii)AnB3 − n scaffolds at λmax for homo- and heterotrimers.

Data availability

Protein crystallographic datasets are available from the Protein Data Bank under accession codes 6EGP and 6MCD. The authors declare that all other data supporting the findings of this study are available within the Article and its Supplementary Information.

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Acknowledgements

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).

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A.E.T. and C.S.E. performed experiments and contributed equally to manuscript preparation. L.R. and J.A.S. performed crystallography work and L.R. contributed to manuscript preparation. T.J.P., V.M.J.-A. and R.P. performed QM/MM calculations and R.P. contributed to manuscript preparation. L.R. and T.J.P. contributed equally to this manuscript. K.P.N. performed experiments. V.L.P. directed research and contributed to manuscript preparation.

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Correspondence to Vincent L. Pecoraro.

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Supplementary Information

Experimental methods, Supplementary Figs. 1–10 and Table 1.

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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

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