Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments

Abstract

Protein structures evolved through a complex interplay of cooperative interactions, and it is still very challenging to design new protein folds de novo. Here we present a strategy to design self-assembling polypeptide nanostructured polyhedra based on modularization using orthogonal dimerizing segments. We designed and experimentally demonstrated the formation of the tetrahedron that self-assembles from a single polypeptide chain comprising 12 concatenated coiled coil–forming segments separated by flexible peptide hinges. The path of the polypeptide chain is guided by a defined order of segments that traverse each of the six edges of the tetrahedron exactly twice, forming coiled-coil dimers with their corresponding partners. The coincidence of the polypeptide termini in the same vertex is demonstrated by reconstituting a split fluorescent protein in the polypeptide with the correct tetrahedral topology. Polypeptides with a deleted or scrambled segment order fail to self-assemble correctly. This design platform provides a foundation for constructing new topological polypeptide folds based on the set of orthogonal interacting polypeptide segments.

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Figure 1: Design of the self-assembling polypeptide tetrahedron.
Figure 2: Self-assembly of the tetrahedron-forming polypeptide TET12.
Figure 3: Imaging of the assembled TET12 reveals tetrahedral topology.
Figure 4: Determination of the sequence-dependent topology of the self-assembled polypeptide.

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Acknowledgements

This research was supported by grants from the Slovenian Research Agency (J2-2131, P4-0176 to R.J.) and the Excellent NMR–Future Innovation for Sustainable Technologies Centre of Excellence, which is financed in part by the European Union regional development funds. B.W. and A.Š. acknowledge US National Institutes of Health grants R01 GM083960 and R01 GM54762 (both to A.Š.). We thank the rest of the members of the 2009 Slovenian International Genetically Engineered Machine (iGEM) competition team (students M. Verce, A. Lukan, N. Debeljak, Š. Miklavič and U. Jelerčič and mentors O. Fekonja, J. Pohar, R. Bremšak and M. Benčina) for their pioneering work on the development of concatenated coiled coil–based nanostructures (http://2009.igem.org/Team:Slovenia), underlying the development of the polypeptide polyhedra; R. Bremšak for excellent technical support; J. Rus for calculations regarding the number of polyhedral topology; the Centre for Electron Microscopy at the Jožef Stefan Institute for the use of electron microscopes; and K. Djinović Carugo for comments on the manuscript.

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R.J. conceived the idea, designed the tetrahedral polypeptide, participated in the mathematical analysis of the polyhedral topology, coordinated the project, discussed the results and wrote the manuscript. H.G. performed TEM, DLS, CD and fluorescence experiments; coordinated the project; discussed the results; and wrote the manuscript. S.B. prepared, purified and analyzed polypeptides; performed TEM, CD and fluorescence experiments; discussed the results; and wrote the manuscript. T.D. prepared, purified, and analyzed polypeptides; performed AFM measurements; discussed the results; and wrote the manuscript. D.V. performed TEM measurements. I.H.-B. performed AFM measurements. A.M. performed DLS measurements. B.W. prepared the molecular model. A.Š. prepared the molecular model and wrote the manuscript. S.K. solved the mathematical analysis of the polyhedral topology and wrote the manuscript.

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Correspondence to Roman Jerala.

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Gradišar, H., Božič, S., Doles, T. et al. Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments. Nat Chem Biol 9, 362–366 (2013). https://doi.org/10.1038/nchembio.1248

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