RESUMO
Genetic sensors with unique combinations of DNA recognition and allosteric response can be created by hybridizing DNA-binding modules (DBMs) and ligand-binding modules (LBMs) from distinct transcriptional repressors. This module swapping approach is limited by incompatibility between DBMs and LBMs from different proteins, due to the loss of critical module-module interactions after hybridization. We determine a design strategy for restoring key interactions between DBMs and LBMs by using a computational model informed by coevolutionary traits in the LacI family. This model predicts the influence of proposed mutations on protein structure and function, quantifying the feasibility of each mutation for rescuing hybrid repressors. We accurately predict which hybrid repressors can be rescued by mutating residues to reinstall relevant module-module interactions. Experimental results confirm that dynamic ranges of gene expression induction were improved significantly in these mutants. This approach enhances the molecular and mechanistic understanding of LacI family proteins, and advances the ability to design modular genetic parts.
Assuntos
Modelos Genéticos , Engenharia de Proteínas/métodos , Proteínas Repressoras/química , Proteínas Repressoras/genética , Regulação Alostérica , Sítios de Ligação , Mutação , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , Biologia SintéticaRESUMO
The development of synthetic biological systems requires modular biomolecular components to flexibly alter response pathways. In previous studies, we have established a module-swapping design principle to engineer allosteric response and DNA recognition properties among regulators in the LacI family, in which the engineered regulators served as effective components for implementing new cellular behavior. Here we introduced this protein engineering strategy to two regulators in the TetR family: TetR (UniProt Accession ID: P04483) and MphR (Q9EVJ6). The TetR DNA-binding module and the MphR ligand-binding module were used to create the TetR-MphR. This resulting hybrid regulator possesses DNA-binding properties of TetR and ligand response properties of MphR, which is able to control gene expression in response to a molecular signal in cells. Furthermore, we studied molecular interactions between the TetR DNA-binding module and MphR ligand-binding module by using mutant analysis. Together, we demonstrated that TetR family regulators contain discrete and functional modules that can be used to build biological components with novel properties. This work highlights the utility of rational design as a means of creating modular parts for cell engineering and introduces new possibilities in rewiring cellular response pathways.
Assuntos
DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Repressoras/química , Fatores de Transcrição/química , Regulação Alostérica , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Engineering allosteric transcriptional repressors containing an environmental sensing module (ESM) and a DNA recognition module (DRM) has the potential to unlock a combinatorial set of rationally designed biological responses. We demonstrated that constructing hybrid repressors by fusing distinct ESMs and DRMs provides a means to flexibly rewire genetic networks for complex signal processing. We have used coevolutionary traits among LacI homologs to develop a model for predicting compatibility between ESMs and DRMs. Our predictions accurately agree with the performance of 40 engineered repressors. We have harnessed this framework to develop a system of multiple toggle switches with a master OFF signal that produces a unique behavior: each engineered biological activity is switched to a stable ON state by different chemicals and returned to OFF in response to a common signal. One promising application of this design is to develop living diagnostics for monitoring multiple parameters in complex physiological environments and it represents one of many circuit topologies that can be explored with modular repressors designed with coevolutionary information.
