Trends in Genetics

Trends in Genetics

Volume 30, Issue 12, December 2014, Pages 521-528
Journal home page for Trends in Genetics

Opinion
Engineering allostery

https://doi.org/10.1016/j.tig.2014.09.004Get rights and content

Allosteric proteins have great potential in synthetic biology, but our limited understanding of the molecular underpinnings of allostery has hindered the development of designer molecules, including transcription factors with new DNA-binding or ligand-binding specificities that respond appropriately to inducers. Such allosteric proteins could function as novel switches in complex circuits, metabolite sensors, or as orthogonal regulators for independent, inducible control of multiple genes. Advances in DNA synthesis and next-generation sequencing technologies have enabled the assessment of millions of mutants in a single experiment, providing new opportunities to study allostery. Using the classic LacI protein as an example, we describe a genetic selection system using a bidirectional reporter to capture mutants in both allosteric states, allowing the positions most crucial for allostery to be identified. This approach is not limited to bacterial transcription factors, and could reveal new mechanistic insights and facilitate engineering of other major classes of allosteric proteins such as nuclear receptors, two-component systems, G protein-coupled receptors, and protein kinases.

Section snippets

Unlocking the power of allostery in synthetic biology

Allosteric regulation mediates virtually every biological process, including transcription, signal transduction, and enzyme activity and transport. Allostery can be broadly defined as activity at one site in a protein regulating function at a spatially distant site. Allosteric regulation occurs through an allosteric effector, generally a small molecule, which binds at one active site and triggers a conformational change that affects function at a distant site. Because of their ability to

Lessons from LacI

Allosteric transcription factors in bacteria, one of the largest annotated families of proteins, regulate adaptive responses to environmental cues. The best- and longest-studied allosteric protein is the E. coli repressor LacI which regulates the lac (lactose-utilization) operon 2, 12. LacI is composed of ligand-binding and DNA-binding domains. In the absence of the ligand, LacI has high affinity for DNA; when bound to inducer, the protein undergoes a conformational change that causes the

Deep mutational scanning using a toggled selection system for allostery

Biochemical and evolutionary studies of LacI suggest that amino acids involved in allostery are intricately coupled with amino acids recognizing ligand and DNA. Thus, a first step toward unraveling this allosteric network would be to determine the role of all the amino acids in LacI function, classifying each as participating in one of the following categories: binds to DNA or to ligand, is required for structural stability, is involved in allostery, or none of the above. As with conventional

New ligand and DNA specificities engineered into existing proteins

To engineer an orthogonal transcription factor, we can begin by redesigning the ligand- and DNA-binding specificities of natural transcription factors. Once the allosteric connections have been identified, and the residues responsible for allostery have been distinguished from those involved in binding ligand or DNA, we can incorporate this information into the design protocol. For example, in the case of LacI, mutating residues that cause an Is phenotype is more likely to result in altered

Application to the wider world of allostery

As DNA synthesis and sequencing become increasingly cheaper and higher in throughput, the rate-limiting step for the analysis of other classes of allosteric proteins is the development of functional assays. The bacterial transcription factor family allows one of the easiest functional assays because allostery is directly coupled to transcription. In this section we briefly describe functional assays for other major classes of allosteric proteins.

Concluding remarks

The ability to engineer the above allosteric protein classes paves the way for new synthetic biology applications: designer GPCRs that can respond to a drug overdose, two-component system proteins that enable bacterial chemotaxis toward a specific molecule, dynamic rewiring of kinase signaling, and controlling the composition and function of engineered microbiota with quorum-sensing switches.

The approach outlined here shows how the power of deep sequencing can be harnessed to address a

Acknowledgments

We thank Lea Starita and James Carothers for comments on the manuscript. This work has been supported by the Wyss Technology Development Fellowship to S.R, the US Department of Energy (DE-FG02-02ER63445 to G.M.C.), and the US National Institutes of Health (P41 GM103533 to S.F.). S.F. is an investigator of the Howard Hughes Medical Institute.

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    These authors contributed equally to this work.

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