Phage display selection of small molecule switchable transcription factors

The genetic information of organisms within DNA molecules instructs cells how to construct proteins which are key the structural building blocks and molecular machines of life. Many different proteins are present in a cell, although their amounts vary according to the stage of the lifecycle of the cell and in response to signals from its surroundings. Unravelling the roles of proteins in the processes that convert general purpose stem cells into specialized tissues allowing the development of multicellular organisms is an example of the type of problem that would benefit enormously from an ability to control the quantities of specific proteins present at a particular moment in time. Here we propose a means for controlling production of a protein by addition of a small molecule of our choice. Protein synthesis begins with the binding of specialized proteins known as transcription factors to regions of DNA adjacent to locations coding for proteins themselves. Blocking the transcription factor from binding DNA prevents the synthesis of the protein(s) coded by the genes that are targeted by that transcription factor. Our hypothesis is that by designing transcription factors that change shape when they bind a specific small molecule, we can flip them from an inactive form that is incapable of binding DNA to an active form that binds DNA and switches on protein synthesis. We will address the challenge of designing a switching transcription factor that responds to a small molecule chosen to display favourable properties in animals including lack of toxicity and good oral uptake. For this purpose we have chosen an antihistamine drug that interacts specifically with a protein involved in the inflammatory response and so exhibits few side effects. As it is difficult to design proteins from scratch, we will create genes encoding a large number of variants of a natural transcription factor that have been chosen to create a cavity in the transcription factor into which our small molecule could bind. Proteins rely on their interiors to maintain a precise shape, and in the absence of the small molecule we predict that the cavity will destabilize the shape of the transcription factor so that it no longer binds DNA. We will place our transcription factor genes into a virus that infects bacteria, so that bacteria produce virus particles which display the transcription factor on their exterior. Using DNA as bait, we will 'fish out' virus particles that bind to the DNA only when the small molecule is present, discarding the remaining virus. Although we may isolate only a very small number of virus particles, these can be amplified by replication in infected bacteria. By repeating the process of selection and amplification a number of times the majority of virus will display transcription factors that are able to switch their DNA binding in response to the small molecule. We will analyse how tightly these transcription factors bind DNA in the presence and absence of the small molecule, and determine information about their three dimensional structures to verify our hypothesis on their mode of action. Our final objective will be to place a new transcription factor into mammalian cells to test its ability to switch on a gene of interest in response to the small molecule. This work will be of interest to researchers involved in the structure and design of proteins, and how proteins interact with small molecules and DNA. There will be direct applications for the study of the roles of genes, by enabling researchers to selectively switch on proteins in cells and then monitor the outcome. This will yield insight into processes such as growth, cell differentiation, ageing and disease progression. Better understanding of these processes, the ways in which they can malfunction, and how to control them, will lead to an increase in the quality of life through enhanced avoidance of ill health in humans and improvements to agriculture and food production.

Our goal is to design a transcription factor that binds its DNA target in response to a small molecule effector of our choice. We propose construction of a library of zinc finger proteins, derived from the three-finger transcription factor Zif268, in which we introduce mutations replacing the zinc binding site of the middle of the three fingers with the aim of creating an allosteric small molecule binding site. Our hypothesis is that creation of a cavity within the middle finger will disrupt folding and inhibit DNA binding, but that folding and DNA binding can be restored by binding of a small molecule complementary to the cavity. The protein library will be fused to the M13 phage pIII coat protein, encoded on a phagmemid. Infection with helper phage will afford phage particles displaying zinc fingers for panning against the target DNA duplex to select those that bind in the presence of small molecule effector, but not in its absence. The small molecule effector that we have chosen for these experiments is levocetirizine, a selective histamine H1 receptor antagonist and commercially available drug with few side effects. Phage display selected proteins will be expressed as soluble GST fusions, and we will determine the cooperativity between small molecule and DNA binding by measurement of their affinities and specificities as a function of the concentration of small molecule effector using gel mobility shift assays, surface plasmon resonance and ELISA. We will attempt to obtain crystals of protein-DNA-small molecule complexes for crystallographic analysis. We will explore the effect of the position of the switch domain within a multi-finger protein on cooperativity, and study structure-affinity relationships of the small molecule. Using a luciferase reporter assay in HeLa cells we will test the hypothesis that our allosteric DNA binding domain fused to a VP16 activation domain will behave as a small molecule switchable transcription factor.