A GAL4-based yeast three-hybrid system for the identification of small molecule–target protein interactions
Introduction
Interactions between small molecules and their receptor proteins modulate many fundamental cellular processes. Synthetic small molecules are used widely in basic biological research and form the basis for disease-modification through pharmacological agents. Despite their widespread use, the efficient identification of target proteins for small molecular weight ligands is a time consuming and technically challenging process. Current biochemical strategies involve sequencing of the interacting protein only after it has been purified from a highly complex cell extract via radiolabelled ligand-binding, photocrosslinking and affinity chromatography [1], [2].
An alternative route for identifying proteins that interact with a small molecule of interest uses an approach that exploits the genetic and molecular tools developed in the yeast two-hybrid system for studying protein–protein interactions [3], [4]. The two-hybrid system uses a transcription factor that can be separated into two domains, a DNA binding domain (BD) and an activation domain (AD). When co-expressed within the same cell, transcriptional activation can only occur if these composite domains are brought into close proximity. In the yeast two-hybrid system, functional assembly of the transcription factor can be made to occur in the nucleus of living yeast cells by fusing the domains with two proteins that interact. A yeast ‘three-hybrid’ approach has been developed through a modification of the two-hybrid system to incorporate a small molecule heterodimer [5]. Functional assembly of the transcription factor is driven by two independent small molecule–protein interactions (Fig. 1). As a result, interaction of the test compound with its binding protein drives activation of measurable reporter gene expression.
A yeast three-hybrid system has been described in which a glucocorticoid receptor hormone binding domain (GRhbd)-fusion and an FKBP12-fusion can be dimerised by a Dex–FK506 heterodimer [5]. The heterodimeric molecule was chosen because Dex and FK506 possess the well characterised binding partners GRhbd and FKBP12, respectively, they are both permeable to yeast cells and they both possess chemical functionality that can be modified without disrupting receptor binding. By similar reasoning, another yeast three-hybrid system has been reported more recently, in which a GRhbd-fusion and a DHFR-fusion can be dimerised by a Dex–MTX heterodimer [6]. The success of the two-hybrid system in identifying novel protein interactions with a protein of interest suggests that the three-hybrid system may have similar potential for identifying novel protein interactions with a compound of interest. Establishing the true potential of this approach requires the construction of an appropriate yeast strain and an assessment of practicalities such as signal to noise ratio, sensitivity, ease of eliminating false positives and breadth of application.
We present modifications of the published system that for the first time use the GAL4 transcription factor as the basis for the yeast three-hybrid strain. The large number of GAL4 activation domain libraries available suggests screens should be feasible for proteins from many different organisms or specific mammalian tissues. We demonstrate the sensitivity of this system, its efficacy for multiple target compounds and its utility for cDNA library screening. In addition to identifying proteins that interact with a compound of interest, we also highlight the ease with which false positives can be eliminated and describe the nature of these false positives. Our results demonstrate that the identification of novel proteins that interact with small molecules is now technically feasible and comparatively easy for any laboratory with rudimentary molecular biology capability. The subsequent identification of such proteins has potential implications relating to understanding both compound mode of action and side effect profiles.
Section snippets
Dexamethasone–methotrexate synthesis
The Dex–MTX heterodimer was synthesised as shown in Fig. 2. The two molecules were covalently linked with a 10-carbon (n=8) tether through positions that when modified did not abrogate target protein binding. The Dex–FK506 heterodimer was obtained from Liu and co-worker [5].
Construction of expression vectors
All oligonucleotides used in this study are listed in Table 1. Full-length rat glucocorticoid receptor was amplified by PCR using p6RGR [5] as a template and the primer pair 5′RGRdch/3′RGRii. This was subcloned into pYX032
Generation of functional yeast three-hybrid strains
To determine whether a GAL4-based three-hybrid system could be developed to test for compound–protein interactions, a number of yeast strains were constructed (Fig. 3, Table 2). Yeast strain DCH1 expresses fusion proteins of the hormone binding domain of the rat glucocorticoid receptor fused to the GAL4 DNA-binding domain and of FKBP12 fused to the GAL4 transcriptional activation domain. An efficient three-hybrid strain requires transcription to be stimulated only in the presence of compound
Discussion
In this report we describe a yeast three-hybrid strain that for the first time uses the functional reconstitution of a GAL4 transcription factor to activate reporter gene expression. We have shown the strain is functional using known compound–protein interactions between FK506–FKBP12 and MTX–DHFR to activate lacZ expression. These interactions can be competed by the presence of excess FK506 or MTX, respectively, resulting in loss of reporter gene expression. In addition, a strain expressing a
Acknowledgements
We thank Jun Liu (Centre for Cancer Research, Massachusetts Institute of Technology) for the Dex–FK506 compound and Carla Smith at GlaxoSmithKline for synthesis of the Dex–MTX compound. We would also like to thank colleagues at GlaxoSmithKline; Stuart Farrow for useful discussions and critical review of the manuscript as well as Robin Brown, Peter Lowe and Trevor Chapman for helpful advice.
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