De novo design of bioactive protein switches

Abstract

Allosteric regulation of protein function is widespread in biology, but is challenging for de novo protein design as it requires the explicit design of multiple states with comparable free energies. Here we explore the possibility of designing switchable protein systems de novo, through the modulation of competing inter- and intramolecular interactions. We design a static, five-helix ‘cage’ with a single interface that can interact either intramolecularly with a terminal ‘latch’ helix or intermolecularly with a peptide ‘key’. Encoded on the latch are functional motifs for binding, degradation or nuclear export that function only when the key displaces the latch from the cage. We describe orthogonal cage–key systems that function in vitro, in yeast and in mammalian cells with up to 40-fold activation of function by key. The ability to design switchable protein functions that are controlled by induced conformational change is a milestone for de novo protein design, and opens up new avenues for synthetic biology and cell engineering.

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Fig. 1: Design of the LOCKR system.
Fig. 2: BimLOCKR design and activation.
Fig. 3: Testing functionality of degronLOCKR in live cells.
Fig. 4: Controlling gene expression using degronLOCKR in yeast.
Fig. 5: Controlling protein localization using nesLOCKR in yeast.

Data availability

All data that support the findings of this study are available within the Article and its Supplementary Information. The original data that support the findings are available from the corresponding author upon reasonable request. Plasmids that encode the LOCKR scaffolds (non-functional switches and keys), BimLOCKR, degronLOCKR and nesLOCKR can be found on Addgene (plasmids 127416-127424, 127200–127206 and 127246).

Code availability

Python scripts, bash scripts, and Rosetta Design XML files are available for download at https://github.com/BobbyLangan/DeNovoDesignofBioactiveProteinSwitches.

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Acknowledgements

R.A.L. was supported by Bruce and Jeannie Nordstrom, thanks to the Patty and Jimmy Barrier Gift for the Institute for Protein Design Directors Fund. S.E.B. is supported by a Career Award at the Scientific Interface from Burroughs Wellcome Fund. A.H.N. was supported by the Department of Defense (DoD) through the National Defense Science & Engineering Graduate Fellowship (NDSEG) Program. M.J.L. was supported by a Washington Research Foundation Innovation Postdoctoral Fellowship and a Cancer Research Institute Irvington Fellowship from the Cancer Research Institute. SAXS data were collected at the Advanced Light Source (ALS) at LBNL, supported by the following grants from NIH (P30 GM124169-01, ALS-ENABLE P30 GM124169 and S10OD018483), NCI SBDR (CA92584) and DOE-BER IDAT (DE-AC02-05CH11231). This work was supported by the Defense Advanced Research Projects Agency, contract no. HR0011-16-2-0045 to H.E.-S and by Open Philanthropy to D.B. The content and information does not necessarily reflect the position or the policy of the government, and no official endorsement should be inferred. H.E.-S. is a Chan-Zuckerberg investigator. We thank L. Carter and the Protein Production Facility in the Institute for Protein Design for protein used in this study, and L. Stewart, S. Bermeo, A. Quijano Rubio, B. Basanta, M. Chevalier, A. Bonny and J. Pedro-Fonseca for help and advice.

Reviewer information

Nature thanks Vincent J. Hilser and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Author information

R.A.L., A.H.N. and S.E.B. contributed equally to this publication. R.A.L., S.E.B., Z.C., W.R.P.N. and D.B. conceived the idea and initial steps for designing protein switches from de novo-designed helical bundles. R.A.L. and D.B. developed the thermodynamic model and the code upon which it works. R.A.L., S.E.B. and W.R.P.N. designed and biophysically characterized LOCKR scaffolds and BimLOCKR. R.A.L. performed mutagenesis and BLI experiments. S.B. characterized Bim interactions to Bcl2 homologues, and aided with experimental design. R.A.L. performed design calculations for orthogonal LOCKR designs using code from S.E.B. and V.K.M. A.H.N. and R.A.L. conceived caging cODC. R.A.L. performed design calculations to cage cODC and tune degronLOCKR. A.H.N. conceived and contributed to all experiments with degronLOCKR. T.H.N. performed dynamic measurement of degronLOCKR. A.M.W. tested degronLOCKR in HEK293T cells. M.J.L., S.E.B. and R.A.L. performed design calculations for asymmetric LOCKR. G.D. performed experiments with degronLOCKR and dCas9. G.D. contributed to plasmid and strain construction. R.A.L., S.E.B. and M.J.L. conceived caging sequences to control subcellular location and R.A.L. performed design calculations for nesLOCKR. J.A.S. and A.H.N. performed all experiments for nesLOCKR. D.B., H.E.-S. and J.E.D. supervised research. R.A.L., S.E.B., A.H.N., H.E.-S. and D.B. wrote the manuscript, all authors edited and approved.

Correspondence to David Baker.

Ethics declarations

Competing interests

: R.A.L., S.E.B., D.B., W.R.P.N. and M.J.L. have filed a provisional patent describing the design, composition and function of LOCKR switches, keys and scaffolds. R.A.L., A.H.N., S.E.B., M.J.L., D.B. and H.E.-S. have filed a provisional patent application describing the design, composition and function of degronLOCKR constructs. D.B., R.A.L., S.E.B. and M.J.L. hold equity in Lyell Immunopharma. D.B. holds equity in Sana Biotechnology.

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Extended data figures and tables

Extended Data Fig. 1 Biophysical data from LOCKR design.

a, Size-exclusion chromatography for the monomer, truncation and LOCKR designs on Superdex 75. Peaks indicated by vertical dashed lines represent monomeric protein used in downstream characterization and functional assays. Size-exclusion chromatography was repeated three times with similar results. b, Circular dichroism spectroscopy to determine protein stability upon heating and treatment with the chemical denaturant guanidinium chloride. Top, full wavescan at 25 °C (blue), 75 °C (orange) and 95 °C (red), then cooled to 25 °C (cyan). Middle, guanidinium chloride melts (also shown overlapped in Fig. 1d). Bottom, fraction of folded protein was converted to the equilibrium constant, and then to the conformational stability for protein unfolding (ΔGunfolding) value. The linear unfolding region, marked by vertical lines in the middle panels, was fit to determine the ΔGfolding for each design. The experiment was repeated four times with similar results. c, SAXS spectra (black; referenced in Fig. 1e) fit to the Rosetta design models (red) using FoXS with χ-values referenced in the top right.

Extended Data Fig. 2 GFP pulldown assay finds mutations for LOCKR.

Different putative LOCKR constructs were adhered via the 6×His tag to a Ni-coated 96-well plate, key–GFP was applied and excess was washed away. The resulting mean fluorescence values represent key–GFP bound to LOCKR constructs. The truncation was used as a positive control, because the key binds to the open interface. The monomer was used as a negative control, because it does not bind the key. Error bars represent the s.d. of three technical replicates (key–GFP was not purified from bacterial lysate. which led to minor technical variability).

Extended Data Fig. 3 Caging Bim-related sequences.

a, Three Bcl2-binding sequences were grafted onto the latch. aBcl2 is a single helix from a designed Bcl2 binder (RCSB Protein Data Bank code (PBD) 5JSN) in which non-Bcl2-interacting residues were reverted back to the standard LOCKR latch sequence (shown as dashes). pBim is the partial Bim sequence in which only Bcl2-interacting residues are grafted onto the latch. Bim is the full consensus sequence of the BH3 domain. b, LOCKR (left) with the latch in dark blue. The helical Bim sequence is taken from the Bim–Bcl2 interaction and grafted onto the latch. c, Left, Bcl2 (tan) binding to Bim (orange) from PDB 2MV6, with pBim residues shown as sticks. Centre, a well-caged graft in which important binding residues are caged. Right, a poor graft in which Bcl2-binding residues are exposed and polar surface residues are against the cage interface. d, Tuning BimLOCKR. aBcl2, pBim and Bim were caged to varying degrees of success. Early versions of the switch (with aBcl2 and pBim) did not efficiently cage Bcl2 binding in the off state. These version also bound the key only weakly, which led to a small dynamic range. The cage and key were extended by 5, 9 and 18 residues in an attempt to provide a larger interface to tightly hold the latch in the off state, and to provide a larger interface for binding of key to increase the dynamic range of activation. Mutations on the latch (identified in Extended Data Fig. 2) and providing toeholds for binding of key were the two strategies used to tune the switch. In graphs, ‘off’ refers to 250–310-nM switch and an absence of key, whereas ‘on’ refers to excess key added. The height of the bar graph shows the equilibrium response (Req) as measured by BLI.

Extended Data Fig. 4 Validation of model.

This is a validation of the model shown in Fig. 1a. a, Measurement of Bim–Bak affinity. BLI at three concentrations gave the on and off rates for Bim–Bak binding, which yielded the constants shown on right. Mean shown with s.d. of four technical replicates (to account for variability in drift on the BLI instrument). b, BLI measurement of BimLOCKRa (400 nM) binding to Bcl2 (gold), BclB (yellow) and Bak (lighter yellow, BimLOCKR at 1 μM) as key is added to the solution. Data are normalized, owing to differences in Rmax for Bcl2 and BclB on the tip. c, BLI measurement of BimLOCKRa binding to keya immobilized on the tip. Open circles, no Bcl2 present; gold points, Bcl2 present at 500 nM.

Extended Data Fig. 5 Caging cODC sequences.

a, Three variations of the cODC degron to cage. Variations meant to tune the Kopen value, by removing the destabilizing proline (no Pro) and minimizing mutations to the latch (Cys-Ala (CA) only). b, Predicted models of the full and no-Pro cODC sequences (orange) threaded onto the latch (dark blue). Thread position were chosen such that the cysteine residue needed for degradation is sequestered against the cage (light blue). Proline highlighted in red in the full cODC was mutated to an isoleucine in the no-Pro variant. c, Comparing the stability of YFP fused to cODC variants caged in switcha to an empty switcha, and to BimSwitcha. The dual-inducible system from Fig. 3a was used to express the various YFP–switcha fusions (solid lines and dots) via pGAL1 and oestradiol, and keya–BFP via pZ3 and progesterone. YFP (Venus) alone, YFP fused to the wild-type cODC (cODC) or YFP fused to the proline-removed cODC (cODC no Pro), were also expressed using pGal1 and oestradiol (dashed lines). Cells were induced with a saturating dose of oestradiol (50 nM) and progesterone was titrated in from 0–200 nM. Fluorescence was measured at steady state using a flow cytometer; data represent mean ± s.d. of three biological replicates. Lines connecting data are a guide for the eye. A moderate decrease in YFP fluorescence was observed as a function of progesterone for the full cODC variant, whereas only a small decrease was observed for the proline-removed and Cys-Ala only. No decrease in fluorescence was observed as a function of induction of key for YFP alone, empty switcha or BimSwitcha. d, Tuning toehold lengths of degronLOCKRa. The dual-inducible system from Fig. 3a was used to express the various YFP–switcha fusions via pGal1 and oestradiol, and keya–BFP via pZ3 and progesterone. YFP fused to the proline-removed cODC (cODC no Pro) was also expressed using pGal1 and oestradiol (dashed line). Cells were induced with a saturating dose of oestradiol (50 nM) and progesterone was titrated in from 0–200 nM. Fluorescence was measured at steady state using a flow cytometer; data represent mean ± s.d. of three biological replicates. Lines connecting data are a guide for the eye. Left, cODC variants alone to show the dynamic range of full cODC. Right, extending toehold on proline-removed version from 9 to 12 and 16 amino acids. Proline-removed cODC with 12-amino-acid toehold shows the greatest dynamic range of all the switches that we tested.

Extended Data Fig. 6 YFP and BFP expression.

a, b, YFP (a) and BFP (b) expression, corresponding to Fig. 3b. We used 0–50 nM oestradiol and 0–200 nM progesterone to induce expression of YFP–degronSwitcha and keya (full-length or truncated)–BFP, respectively. Fluorescence was measured at steady state using a flow cytometer. Heat maps depict mean fluorescence and are a representative sample of three biological replicates. The oestradiol dose (50 nM) depicted in Fig. 3b is indicated with the black rectangle on the heat maps. YFP fluorescence was normalized to the maximum fluorescence (50 nM oestradiol or 0 nM progesterone). BFP expression was not dependent on expression of the switch, which suggests that the key does not co-degrade with the switch.

Extended Data Fig. 7 degronLOCKRa–d orthogonality.

All combinations of pTDH3–YFP–degronSwitch and pTDH3–key–CFP were tested. Fluorescence was measured at steady state using a flow cytometer. YFP fluorescence was averaged across three biological replicates. The percentage degradation was calculated by subtracting the mean YFP–degronSwitch fluorescence with the given key–CFP coexpressed from the YFP–degronSwitch fluorescence without any key expressed, and normalizing by the YFP–degronSwitch fluorescence without any key expressed. degronSwitcha is activated strongly by keya and activated weakly by keyb. degronSwitchc is activated strongly by keyc and activated weakly by keyb. Because degronSwitcha and degronSwitchc are not activated by keyc and keya, respectively, we consider these two to be an orthogonal pair.

Extended Data Fig. 8 Comparison of variants of degronSwitch in HEK293T cells.

Fluorescence of variants of RFP–degronSwitch in the presence and absence of key–BFP were measured using flow cytometry. The original symmetric design was compared against an asymmetric design. Two toehold lengths were tested for each variant. Data in the bar graph represent the geometric mean ± s.d. of three biological replicates. Histograms are depicted for a representative sample. Asymmetric cage with an 8-residue toehold (signified by t8) demonstrates the largest dynamic range.

Extended Data Fig. 9 YFP and RFP expression for synTF and dCas9–VP64.

a, b, Assay for synTF (a) and dCas9–VP64 (b), corresponding to Fig. 4, as a function of oestradiol (E2) (0–125 nM) and progesterone (Pg) (0–100 nM). YFP fluorescence represents the transcriptional output of either synTF or dCas9–VP64 and RFP fluorescence represents fluorescence of either synTF or dCas9–VP64. Fluorescence was measured at steady state using flow cytometry. Heat maps depict mean fluorescence, and are a representative sample of three biological replicates. The oestradiol dose (31.25 nM) depicted in Fig. 5 is indicated with the black rectangle on the heat maps.

Extended Data Fig. 10 Design and characterization of nesLOCKR.

a, Nuclear export sequence used in this Article. b, The nuclear export sequence (orange) caged on the helical latch (dark blue, cartoon) with hydrophobic residues sequestered against the cage (light blue, surface). c, Left, schematic of cytosolic YFP–nesSwitcha and key–BFP with nuclear marker HTA2–RFP. Right, YFP fluorescence shows the expected cytosolic distribution when YFP–nesSwitcha is expressed with no NLS (left), but punctae of YFP fluorescence are observed when both YFP–nesSwitcha and key–BFP are expressed in the cytosol—which we assume is due to aggregation of the nesSwitcha. Key–BFP fluorescence is co-localized with YFP–nesSwitcha fluorescence. d, Left, schematic of NLS–YFP–nesSwitcha with key–BFP–NLS, and with nuclear marker HTA2–RFP. Right, YFP–nesSwitcha is localized to the nucleus when expressed with the strong (SV40) NLS. When key–BFP is expressed with a moderately strong NLS, the same pattern of cytosolic YFP punctae formation is observed as when key–BFP is expressed without a NLS (Fig. 5b), which indicates that uncaging of the nuclear export sequence is independent of NLS on key–BFP localization. Key–BFP–NLS fluorescence is co-localized to NLS–YFP–nesSwitcha fluorescence e, YFP and RFP expression for synTF assay (corresponding to Fig. 5c) as a function of oestradiol (0–125 nM) and progesterone (0–500 nM). Fluorescence was measured at steady state using flow cytometry. Heat maps depict mean fluorescence, and are a representative sample of three biological replicates. The oestradiol dose (31.25 nM) depicted in Fig. 5c is indicated with the black rectangle on the heat maps.

Supplementary information

Supplementary Information

This file contains computational methods for the results described in the main text: the Fig. 1 thermodynamic model, Rosetta design methods, and computation of the half-life for degronLOCKR. It also contains Supplementary Figures 1-3 to clarify computational methodology, and the original main text figures that passed peer-review but not length constrictions of Nature Article format.

Reporting Summary

Supplementary Tables

This file contains Supplementary Tables 1-7. Supplementary Table 1: SAXS statistics for the spectra summarized in Fig. 1e. Supplementary Table 2: Functional peptides used in LOCKRs presented in this report. Supplementary Table 3: All protein sequences screened in this report, including all efforts at tuning BimLOCKR and degronLOCKR. Supplementary Table 4: Yeast base strains used to construct strains used in this report. Supplementary Table 5: Yeast strains expressing LOCKR components in various combinations for data collected here. Supplementary Table 6: All plasmids used for testing degronLOCKR in mammalian cells. Supplementary Table 7: Parameters for computing the half-life of degronLOCKR.

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