Engineering orthogonal signalling pathways reveals the sparse occupancy of sequence space
Gene duplication is a common and powerful mechanism by which cells create new signalling pathways1,2, but recently duplicated proteins typically must become insulated from each other and from other paralogues to prevent unwanted crosstalk3. A similar challenge arises when new sensors or synthetic si...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Springer Science and Business Media LLC
2020
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| Online Access: | https://hdl.handle.net/1721.1/125163 |
| _version_ | 1811073939472908288 |
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| author | McClune, Conor J. Alvarez-Buylla, Aurora Voigt, Christopher A. Laub, Michael T. |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology McClune, Conor J. Alvarez-Buylla, Aurora Voigt, Christopher A. Laub, Michael T. |
| author_sort | McClune, Conor J. |
| collection | MIT |
| description | Gene duplication is a common and powerful mechanism by which cells create new signalling pathways1,2, but recently duplicated proteins typically must become insulated from each other and from other paralogues to prevent unwanted crosstalk3. A similar challenge arises when new sensors or synthetic signalling pathways are engineered within cells or transferred between genomes. How easily new pathways can be introduced into cells depends on the density and distribution of paralogous pathways in the sequence space that is defined by their specificity-determining residues4,5. Here we directly investigate how crowded this sequence space is, by generating novel two-component signalling proteins in Escherichia coli using cell sorting coupled to deep sequencing to analyse large libraries designed on the basis of coevolutionary patterns. We produce 58 insulated pathways comprising functional kinase–substrate pairs that have different specificities than their parent proteins, and demonstrate that several of these new pairs are orthogonal to all 27 paralogous pathways in E. coli. Additionally, from the kinase–substrate pairs generated, we identify sets consisting of six pairs that are mutually orthogonal to each other, which considerably increases the two-component signalling capacity of E. coli. These results indicate that sequence space is not densely occupied. The relative sparsity of paralogues in sequence space suggests that new insulated pathways can arise easily during evolution, or be designed de novo. We demonstrate the latter by engineering a signalling pathway in E. coli that responds to a plant cytokinin, without crosstalk to extant pathways. Our work also demonstrates how coevolution-guided mutagenesis and the mapping of sequence space can be used to design large sets of orthogonal protein–protein interactions. |
| first_indexed | 2024-09-23T09:40:33Z |
| format | Article |
| id | mit-1721.1/125163 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T09:40:33Z |
| publishDate | 2020 |
| publisher | Springer Science and Business Media LLC |
| record_format | dspace |
| spelling | mit-1721.1/1251632022-09-30T16:06:55Z Engineering orthogonal signalling pathways reveals the sparse occupancy of sequence space McClune, Conor J. Alvarez-Buylla, Aurora Voigt, Christopher A. Laub, Michael T. Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Biological Engineering Gene duplication is a common and powerful mechanism by which cells create new signalling pathways1,2, but recently duplicated proteins typically must become insulated from each other and from other paralogues to prevent unwanted crosstalk3. A similar challenge arises when new sensors or synthetic signalling pathways are engineered within cells or transferred between genomes. How easily new pathways can be introduced into cells depends on the density and distribution of paralogous pathways in the sequence space that is defined by their specificity-determining residues4,5. Here we directly investigate how crowded this sequence space is, by generating novel two-component signalling proteins in Escherichia coli using cell sorting coupled to deep sequencing to analyse large libraries designed on the basis of coevolutionary patterns. We produce 58 insulated pathways comprising functional kinase–substrate pairs that have different specificities than their parent proteins, and demonstrate that several of these new pairs are orthogonal to all 27 paralogous pathways in E. coli. Additionally, from the kinase–substrate pairs generated, we identify sets consisting of six pairs that are mutually orthogonal to each other, which considerably increases the two-component signalling capacity of E. coli. These results indicate that sequence space is not densely occupied. The relative sparsity of paralogues in sequence space suggests that new insulated pathways can arise easily during evolution, or be designed de novo. We demonstrate the latter by engineering a signalling pathway in E. coli that responds to a plant cytokinin, without crosstalk to extant pathways. Our work also demonstrates how coevolution-guided mutagenesis and the mapping of sequence space can be used to design large sets of orthogonal protein–protein interactions. Office of Naval Research (N000141310074) NIH pre-doctoral training grant T32GM007287 2020-05-11T20:28:31Z 2020-05-11T20:28:31Z 2019-10 2018-10 2020-05-11T15:31:06Z Article http://purl.org/eprint/type/JournalArticle 0028-0836 1476-4687 https://hdl.handle.net/1721.1/125163 McClune, Conor J., et al. "Engineering orthogonal signalling pathways reveals the sparse occupancy of sequence space." Nature, 574, 7780 (October 2019): 702-706. en http://dx.doi.org/10.1038/s41586-019-1639-8 Nature Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf Springer Science and Business Media LLC PMC |
| spellingShingle | McClune, Conor J. Alvarez-Buylla, Aurora Voigt, Christopher A. Laub, Michael T. Engineering orthogonal signalling pathways reveals the sparse occupancy of sequence space |
| title | Engineering orthogonal signalling pathways reveals the sparse occupancy of sequence space |
| title_full | Engineering orthogonal signalling pathways reveals the sparse occupancy of sequence space |
| title_fullStr | Engineering orthogonal signalling pathways reveals the sparse occupancy of sequence space |
| title_full_unstemmed | Engineering orthogonal signalling pathways reveals the sparse occupancy of sequence space |
| title_short | Engineering orthogonal signalling pathways reveals the sparse occupancy of sequence space |
| title_sort | engineering orthogonal signalling pathways reveals the sparse occupancy of sequence space |
| url | https://hdl.handle.net/1721.1/125163 |
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