Engineering allostery

Abstract

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.

Figure 1. Engineering novel allosteric proteins

(A) Designing novel ligand specificities: a known allosteric protein can be made to recognize different small molecule inducers by mutating the amino acids in the ligand binding domain. (B) Designing novel DNA specificities: allosteric transcription factors can be targeted to new DNA sequences by altering the DNA-binding domain. (C) Novel allosteric chimeras: allosteric protein domains that are not capable of binding DNA, such as periplasmic binding proteins, can be attached to a DNA-binding domain to create novel, chimeric transcription factors.

Figure 2. LacI protein architecture and long-range allosteric connections

Structure of a LacI dimer bound to DNA is shown on the left side, key regions highlighted in different colors. The dotted lines on the right side show long-range connection between four mutations (M42, A133, D149 and S151) that rescue allostery in Y282D.

Figure 3. Toggled selection scheme for high-throughput functional evaluation of LacI mutants

LacI mutants are shown in yellow; red circle represents a mutation. Positive selection in the absence of the inducer enriches for I- mutants (left arrow). Negative selection without inducer followed by positive selection with inducer enriches for WT-like mutants (center arrow). Negative selection with inducer enriches for Is mutants (right arrow).

Figure 4. Functional assays for other allosteric protein classes

(A) Direct transcriptional readout: applicable to allosteric transcription factors like nuclear receptors. Presence of the receptor ligand is necessary to activate expression of a selectable marker. (B) Indirect transcriptional readout: applicable to two-component systems. Allosteric activation of a histidine kinase causes phosphorylation of a response regulator, leading to transcription of a selectable marker. (C) Split reporter assay: applicable to GPCRs and heterodimeric receptor tyrosine kinases. Complementary halves of GFP are attached to the receptor and the factor recruited upon allosteric activation. A functional GFP is formed only when the activated allosteric protein binds its partner and brings the two GFP halves into sufficient proximity. (D) Environment-sensitive fluorophores: applicable to protein kinases. A fluorophore is attached to a peptide substrate of the kinase in close proximity to the phosphorylated residue, such that the fluorophore will fluoresce only when the phosphate group is present. (E) Domain-inserted reporter: applicable to cytosolic allosteric proteins. The allosteric protein is inserted into GFP, such that a functional GFP is formed only when the allosteric protein changes to a ligand-bound conformation.