Abstract
Increasingly, it is possible to design peptide and protein assemblies de novo from first principles or computationally. This approach provides new routes to functional synthetic polypeptides, including designs to target and bind proteins of interest. Much of this work has been developed in vitro. Therefore, a challenge is to deliver de novo polypeptides efficiently to sites of action within cells. Here we describe the design, characterisation, intracellular delivery, and subcellular localisation of a de novo synthetic peptide system. This system comprises a dual-function basic peptide, programmed both for cell penetration and target binding, and a complementary acidic peptide that can be fused to proteins of interest and introduced into cells using synthetic DNA. The designs are characterised in vitro using biophysical methods and X-ray crystallography. The utility of the system for delivery into mammalian cells and subcellular targeting is demonstrated by marking organelles and actively engaging functional protein complexes.
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Data availability
The CC database CC+ is open and publicly accessible. The coordinate and structure factor files for homomer-S, apCC-Di, apCC-Di-AB_var & apCC-Di-AB have been deposited in the Protein Data Bank with accession codes 7Q1Q, 7Q1R, 7Q1S and 7Q1T, respectively. All the raw data used in this publication has been deposited in the Zenodo repository (https://doi.org/10.5281/zenodo.6519961).
Code availability
The scripts used for bioinformatic analysis are available from a Zenodo repository (https://doi.org/10.5281/zenodo.6518524).
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Acknowledgements
G.G.R. is supported by the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement number 88899. J.A.C. is supported by the EPSRC-funded Bristol Centre for Doctoral Training in Chemical Synthesis (EP/G036764/1). H.F.T. is supported by the EPSRC- and BBSRC-funded Centre for Doctoral Training in Synthetic Biology (EP/L016494/1). M.P.D. is a Lister Institute of Preventative Medicine Fellow and work in his lab is supported by BBSRC (BB/S000917/1). We thank the University of Bristol School of Chemistry Mass Spectrometry Facility for access to the EPSRC-funded Bruker Ultraflex MALDI–TOF instrument (EP/K03927X/1), the BBSRC-funded BrisSynBio centre for access to peptide synthesis and a plate reader (BB/L01386X/1), and the Wolfson Bioimaging Facility for their assistance in this work. B.H. acknowledges financial support and allocation of beamtime by HZB and we thank the beamline staff at BESSY for assistance.
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G.G.R., J.A.C. and W.M.D. contributed equally to the project. G.G.R., W.M.D., B.H. and D.N.W. conceived and developed the project idea. G.G.R. conducted the bioinformatic analysis and designed the peptides. W.M.D. and G.G.R. synthesised and purified the peptides and conducted the biophysical analysis. W.M.D. conducted and analysed the fluorescence-quenching assays. H.F.T. and N.J.S. conducted and analysed the BiFC assay. S.S. and G.G.R. conducted the X-ray crystallography. J.A.C. and M.P.D. conducted and analysed all mammalian cell experiments. G.G.R, B.H. and D.N.W. wrote the manuscript with contributions from all authors.
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Extended data
Extended Data Fig. 1 Electron density map of apCC-Di crystal structure.
Fo-Fc difference map of the apCC-Di crystal structure represented as a grey wire mesh overlaid on a stick model. The map was contoured at 1 rmsd.
Extended Data Fig. 2 Electron density map of apCC-Di-AB crystal structure.
Fo-Fc difference map of the apCC-Di-AB crystal structure represented as a grey wire mesh overlaid on a stick model. The map was contoured at 1 rmsd.
Extended Data Fig. 3 The asymmetric unit of apCC-Di-AB_var is composed of 14 chains.
While there are minor differences between the surface-exposed residues of the seven dimer pairs, all the biological units are structurally similar (left). The high number of biological units in the asymmetric unit is a result of crystal packing. In the crystal form, apCC-Di-AB_var forms perpendicular layers of right-handed α-helical fibres (right). Between each dimer pair, the individual peptides self-associate to form KIH parallel interfaces, leading to alternating parallel and antiparallel interfaces.
Extended Data Fig. 4 Symmetry mates for the crystal structure of apCC-Di.
Four symmetry mates are depicted above that form a repeating pattern in the crystal structure. SOCKET 2 analysis of the structure reveals four antiparallel CC dimers. There are no inter-biological unit CC interactions.
Extended Data Fig. 5 Symmetry mates for the crystal structure of apCC-Di-AB.
Two symmetry mates are depicted above that form a repeating pattern in the crystal structure. SOCKET 2 analysis of the structure reveals two antiparallel CC dimers. There are no inter-biological unit CC interactions.
Extended Data Fig. 6 Cartoon representation of mutation positions in designed coiled-coil (CC) dimers for the fluorescence quenching assay.
For homomeric CCs, the fluorophore was incorporated near the N termini and a fluorescence quencher near the C termini. For parallel CCs (top left) this should lead to fluorescence, whereas for antiparallel CCs (top right) this should lead to quenching. For antiparallel heteromeric CCs, incorporating the fluorophore and fluorescence quencher near the same termini (bottom left) should lead to fluorescence, whereas incorporating at opposite termini should lead to quenching (bottom right).
Extended Data Fig. 7 Extended plots for the fluorescence-quenching assay for labelled apCC-Di, apCC-Di-AB containing additional controls.
(left) Fluorescence quenching assay for homomeric peptides. Controls include the parallel coiled coil CC-Di and swapping the fluorophore and the fluorescence quencher between the termini of apCC-Di. (right) Fluorescence quenching assay for the designed heteromeric peptides. The control peptides include labelling both apCC-Di-A and apCC-Di-B near the C-termini, which does not lead to quenching in an antiparallel orientation. Key: n and c indicate mutations near the N and C termini, respectively; 4CF, 4-Cyano-L-phenylalanine fluorophore; MSE, L-selenomethionine fluorescence quencher. Conditions: 100 μM concentration of each peptide, 50 mM sodium phosphate, pH 7.
Extended Data Fig. 8 Extended bimolecular fluorescence complementation (BiFC) assay containing controls for homomerisation of apCC-Di-A and apCC-Di-B, and controls for heteromerisation of CC-Di-A and CC-Di-B (a parallel heteromeric system).
BiFC assay for apCC-Di and apCC-Di-AB. V1 and V2 are N- and C-terminal fragments of the Venus yellow fluorescent protein, where V1 represents the V1 mutant. Peptide names that precede the Venus fragment name denote fusion to the C termini of the coiled coil, for example apCC-Di-B-V2. Values are normalised according to cell density (OD600) and are presented as mean values±1 SD from n ≥ 3 technical replicate measurements (dots).
Extended Data Fig. 9 Cell penetration of apCC-Di-B occurs at 4 °C and in the presence of endocytosis inhibitors.
a, Representative confocal images of HeLa cells treated for 1 h with 2 µM TAMRA-labelled apCC-Di-B at 37 oC and 4 °C. These representative images are taken from n = 3 biological replicates. b, The same for cells at 37 °C pre-incubated with 0.1% DMSO (as a control) and with endocytosis inhibitors 30 µM MiTMAB, 80 µM Dynasore, and 0.1% NaN3 in the DMEM before treatment with 2 µM TAMRA labelled apCC-Di-B peptide in media containing each of the inhibitors for 1 h. apCC-Di-B remains cell penetrating at 4 °C and in the presence of the inhibitors and at 37 °C, as shown by TAMRA fluorescence in the cytoplasm and nucleus. Scale bar 10 µm. These representative images are taken from n = 3 biological replicates.
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Rhys, G.G., Cross, J.A., Dawson, W.M. et al. De novo designed peptides for cellular delivery and subcellular localisation. Nat Chem Biol 18, 999–1004 (2022). https://doi.org/10.1038/s41589-022-01076-6
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DOI: https://doi.org/10.1038/s41589-022-01076-6
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