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Engineering proteins for allosteric control by light or ligands

Abstract

Control of protein activity in living cells can reveal the role of spatiotemporal dynamics in signaling circuits. Protein analogs with engineered allosteric responses can be particularly effective in the interrogation of protein signaling, as they can replace endogenous proteins with minimal perturbation of native interactions. However, it has been a challenge to identify allosteric sites in target proteins where insertion of responsive domains produces an allosteric response comparable to the activity of native proteins. Here, we describe a detailed protocol to generate genetically encoded analogs of proteins that can be allosterically controlled by either rapamycin or blue light, as well as experimental procedures to produce and test these analogs in vitro and in mammalian cell lines. We describe computational methods, based on crystal structures or homology models, to identify effective sites for insertion of either an engineered rapamycin-responsive (uniRapR) domain or the light-responsive light–oxygen–voltage 2 (LOV2) domain. The inserted domains allosterically regulate the active site, responding to rapamycin with irreversible activation, or to light with reversible inactivation at higher spatial and temporal resolution. These strategies have been successfully applied to catalytic domains of protein kinases, Rho family GTPases, and guanine exchange factors (GEFs), as well as the binding domain of a GEF Vav2. Computational tasks can be completed within a few hours, followed by 1–2 weeks of experimental validation. We provide protocols for computational design, cloning, and experimental testing of the engineered proteins, using Src tyrosine kinase, GEF Vav2, and Rho GTPase Rac1 as examples.

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Fig. 1: Outline of the procedure.
Fig. 2: Strategies for engineered control of protein activity.
Fig. 3: An approach to the design of allosteric protein switches.
Fig. 4: QuikChange strategy to clone iFKBP, uniRapR, or LOV2 into a target gene.
Fig. 5: An expected result for PI-Vav2 generated using high-content imaging.
Fig. 6: Testing of uniRapR and PI constructs with biochemical and imaging assays.

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Acknowledgements

This work was supported by NIH grants R35GM122596 (K.M.H.), and R01-GM114015, R01-GM064803, and R01-GM123247 (N.V.D).

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Contributions

O.D. developed and optimized the protocol with input from N.V.D and K.M.H. O.D. wrote the protocol with input from K.M.H.

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Correspondence to Klaus M. Hahn.

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The authors declare no competing interests.

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Related links

Key references using this protocol

Dagliyan, O. et al. Science 354, 1441–1444 (2016): https://doi.org/10.1126/science.aah3404

Karginov, A. V. et al. Nat. Chem. Biol. 10, 286–290 (2014): https://doi.org/10.1038/nchembio.1477

Dagliyan, O. et al. Proc. Natl Acad. Sci. USA 110, 6800–6804 (2013): https://doi.org/10.1073/pnas.1218319110

Dagliyan, O. et al. ACS Synth. Biol. 6, 1257–1262 (2017): https://doi.org/10.1021/acssynbio.6b00359

Karginov, A. V., Ding, F., Kota, P., Dokholyan, N. V. & Hahn, K. M. Nat. Biotechnol. 28, 743–747 (2010): https://doi.org/10.1038/nbt.1639

Chu, P.-H. et al. PNAS 111, 12420–12425 (2014): https://doi.org/10.1073/pnas.1404487111

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Dagliyan, O., Dokholyan, N.V. & Hahn, K.M. Engineering proteins for allosteric control by light or ligands. Nat Protoc 14, 1863–1883 (2019). https://doi.org/10.1038/s41596-019-0165-3

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