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Rational design of α-helical tandem repeat proteins with closed architectures

Nature volume 528, pages 585–588 (2015)Cite this article

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Abstract

Tandem repeat proteins, which are formed by repetition of modular units of protein sequence and structure, play important biological roles as macromolecular binding and scaffolding domains, enzymes, and building blocks for the assembly of fibrous materials1,2. The modular nature of repeat proteins enables the rapid construction and diversification of extended binding surfaces by duplication and recombination of simple building blocks3,4. The overall architecture of tandem repeat protein structures—which is dictated by the internal geometry and local packing of the repeat building blocks—is highly diverse, ranging from extended, super-helical folds that bind peptide, DNA, and RNA partners5,6,7,8,9, to closed and compact conformations with internal cavities suitable for small molecule binding and catalysis10. Here we report the development and validation of computational methods for de novo design of tandem repeat protein architectures driven purely by geometric criteria defining the inter-repeat geometry, without reference to the sequences and structures of existing repeat protein families. We have applied these methods to design a series of closed α-solenoid11 repeat structures (α-toroids) in which the inter-repeat packing geometry is constrained so as to juxtapose the amino (N) and carboxy (C) termini; several of these designed structures have been validated by X-ray crystallography. Unlike previous approaches to tandem repeat protein engineering12,13,14,15,16,17,18,19,20, our design procedure does not rely on template sequence or structural information taken from natural repeat proteins and hence can produce structures unlike those seen in nature. As an example, we have successfully designed and validated closed α-solenoid repeats with a left-handed helical architecture that—to our knowledge—is not yet present in the protein structure database21.

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Figure 1: Designed monomeric repeat architectures.
Figure 2: Overview of the repeat module design process.
Figure 3: Superposition of designed toroids (purple) and their refined crystallographic structures (green).
Figure 4: Crystal packing geometries of designed toroids.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Crystal structures determined in this study have been deposited in the RCSB Protein Data Bank under accession numbers 4YXX (dTor_6x35L), 4YY2 (dTor_3x33L_2-2a), 4YY5 (dTor_3x33L_2-2b), 4YXY (dTor_9x31L_sub), 4YXZ (dTor_9x31L), and 5BYO (dTor_12x31L).

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Acknowledgements

The authors thank Scientific Computing at the Fred Hutchinson Cancer Research Center for providing the computational infrastructure necessary for this project. This research was supported by the following research grants: National Institutes of Health R21GM106117 to P.B. and R01GM49857 to B.L.S., and Swiss National Science Foundation Postdoc Fellowship PBZHP3-125470 and Human Frontier Science Program Long-Term Fellowship LT000070/2009-L to F.P.

Author information

Authors and Affiliations

  1. Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N., Seattle, 98109, Washington, USA

    Lindsey Doyle, Jazmine Hallinan, Jill Bolduc, Barry L. Stoddard & Philip Bradley

  2. Department of Biochemistry, University of Washington, Seattle, 98195, Washington, USA

    Fabio Parmeggiani & David Baker

  3. Institute for Protein Design, University of Washington, Seattle, 98195, Washington, USA

    Fabio Parmeggiani, David Baker & Philip Bradley

  4. Howard Hughes Medical Institute, University of Washington, Seattle, 98195, Washington, USA

    David Baker

  5. Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N., Seattle, 98019, Washington, USA

    Philip Bradley

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Contributions

L.D., J.H., J.B. and F.P. expressed, purified, and characterized designed constructs. L.D., J.H. and J.B. performed crystal screening, collected diffraction data, and solved crystal structures. P.B. developed and implemented the repeat design algorithms. P.B. performed sequence design calculations with feedback from F.P. P.B., B.L.S. and D.B. supervised the research. P.B. conceived of the toroid design project with input from B.L.S. and D.B. P.B. wrote the manuscript with input from the other authors.

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Correspondence to Philip Bradley.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Handedness of α-helical bundles and helical linkers.

a, Design dTor_12x31L, shown on the left, has a left-handed helical bundle. The native toroid on the right, which has a right-handed bundle, is taken from the Protein Data Bank structure 4ADY and corresponds to the PC repeat domain of the 26S proteasome subunit Rpn2 (ref. 46). b, The handedness of a helical bundle is determined by the twist direction of the polypeptide chain as it wraps around the axis of the helical bundle. c, Helical linkers characterized by a negative (positive) dihedral angle between the axes of the connected helices will, upon repetition, tend to impart a left-handed (right-handed) twist to the bundle. d, Geometrical properties of the most common short α-helical linkers in the structural database indicate that certain turn types (for example, ‘E’ and ‘GBB’) tend to form left-handed connections whereas others (for example, ‘GB’ and ‘BAAB’) are associated with right-handed connections. Turn types are classified by mapping their backbone torsion angles to a coarse-grained alphabet27 as shown in e.

Extended Data Figure 2 Unbiased 2Fo − Fc omit maps contoured around the side chains comprising the central pore regions for each crystallized toroid.

The constructs shown are in the same order as in Fig. 3.

Extended Data Figure 3 The crystallographic structures of highly symmetrical designed toroidal repeat proteins display rotational averaging in the crystal lattice.

a, Electron difference density for construct dTor_6x35L. Left: anomalous difference Fourier peaks calculated from data collected from a crystal of selenomethionine-derivatized protein. Although only one methionine residue (at position 168) is present in the construct, strong anomalous difference peaks (I/σI greater than 4.0) are observed at equivalent positions within at least three modular repeats. Right: difference density extending across the modelled position of the N and C termini in the refined model, indicating partial occupancy at that position by a peptide bond. The other five equivalent positions around the toroidal protein structure display equivalent features of density, indicating that each position is occupied by a mixture of loops and protein termini. b, Electron density for construct dTor_12x31L, again calculated at a position corresponding to the refined N and C termini in the crystallographic model. As was observed for the hexameric toroid in a, the electron density indicates a mixture of loops and protein termini.

Extended Data Figure 4 Size-exclusion chromatography elution profiles for the four designed toroids whose crystal structures were determined.

The elution profiles (blue traces) shown correspond to runs in high (750 mM) NaCl for dTor_3x33L_2-2 (a) and dTor_6x35L (b), while the elution profiles for dTor_9x31L (c) and dTor_12x31L (d) correspond to runs in lower (150 mM) NaCl. The superimposed elution profiles of standard protein size markers (brown traces) correspond to runs at those same salt concentrations, conducted on the same column and day. The inset in each panel displays the migration and relative purity of each construct used for the analysis.

Extended Data Figure 5 Purification and characterization of designed toroids.

a–g, CD wavelength scan from 260 to 190 nm of several designed toroids and a positive control protein at 22 °C (blue) and 80 °C (red). a, dTor_9x31L_sub; b, dTor_3x33L_2-2; c, dTor_6x33R_1; d, dTor_6x35L; e, dTor_9x31L; f, dTor_12x31L; g, positive control. h, Bis-Tris gel (4–12%) showing designed toroids immediately after metal affinity purification. Lane L, molecular mass protein standards (in kilodaltons); lane 1, dTor_9x31L_sub; lane 2, dTor_3x33L_2-2; lane 3, dTor_6x33R_1; lane 4, dTor_6x35L; lane 5, dTor_9x31L; lane 6, dTor_12x31L.

Extended Data Figure 6 Potential dimerization interfaces observed in crystal packing interactions.

a, Superposition of monomer–monomer packing interactions for the dTor_3x33L_2-2 design observed in two entirely different crystal forms. b, Stacking interactions between two dTor_6x35L subunits observed in the crystal structure; lysine residues interacting with backbone carbonyl groups in the partner monomer are shown in stick representation and coloured yellow along with their interaction partners.

Extended Data Table 1 Characterization of designed constructs
Full size table
Extended Data Table 2 Crystallographic statistics
Full size table

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Doyle, L., Hallinan, J., Bolduc, J. et al. Rational design of α-helical tandem repeat proteins with closed architectures. Nature 528, 585–588 (2015). https://doi.org/10.1038/nature16191

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