Directed evolution of specific receptor–ligand pairs for use in the creation of gene switches

Abstract

Despite their versatility and power in controlling gene regulation in nature, nuclear hormone receptors (NHRs) have largely eluded utility in heterologous gene regulation applications such as gene therapy and metabolic engineering. The main reason for this void is the pleiotropic interference of the receptor-ligand combination with regulatory networks in the host organism. In recent years, numerous strategies have been developed to engineer ligandreceptor pairs that do not cross-interact with host regulatory pathways. However, these strategies have either met with limited success or cannot be readily extended to other ligand-receptor pairs. Here, we present a simple, effective, and readily generalizable strategy for reengineering NHRs to respond specifically to a selected synthetic ligand. The method involves generation of genetic diversity by stepwise individual site saturation mutagenesis of a fixed set of ligand-contacting residues and random point mutagenesis, followed by phenotypic screening based on a yeast two-hybrid system. As a test case, this method was used to alter the specificity of the NHR human estrogen receptor ␣ in favor of the synthetic ligand 4,4 -dihydroxybenzil, relative to the natural ligand 17␤-estradiol, by >10 7 -fold. The resulting ligand-receptor pair is highly sensitive to the synthetic ligand in human endometrial cancer cells and is essentially fully orthogonal to the wild-type receptor-natural ligand pair. This method should provide a powerful, broadly applicable tool for engineering receptors͞enzymes with improved or novel ligand͞substrate specificity.gene therapy ͉ nuclear hormone receptor ͉ protein engineering ͉ orthogonal ligand-receptor pairs T he ability to manipulate naturally occurring proteins to bind and respond to synthetic ligands in a manner independent, or orthogonal, from the influence of natural proteins and ligands, constitutes a significant challenge in protein engineering (1). Such a tool has important utility in the creation of gene switches for the control of heterologous gene expression in applications such as gene therapy and metabolic engineering (2, 3), as well as in the selective regulation of cellular processes such as apoptosis, genetic recombination, signal transduction, and motor protein function (4).To date, numerous synthetic ligand-mutant receptor pairs have been created that are orthogonal to the analogous natural interaction to varying degrees. Among the proteins used for this work, nuclear hormone receptors (5), naturally occurring transcription factors, have served as a prime target, owing largely to their ''gene switch-like'' attributes: rapid induction kinetics (6-8), dose-dependent ligand response, and readily interchangeable functional modules (9, 10). Despite the extensive work carried out to engineer new specific ligand-receptor pairs from nuclear hormone receptors, the absence of a conceptually simple, generally applicable engineering approach remains a concern. Rational design of ligands to rematch interaction with a given mutant of human...

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“…For example, most modifications in enantioselectivity or substrate specificity are located in the vicinity of the active site or in the access/exit of reactants/products [58,60,61]. Stability and activity however can be affected by mutations in any part of the protein, close or far from the active site [62], increasing significantly the number of possible mutations. To avoid screening massive number of mutations, one can reduce the region to explore by using functional information (from point mutations, random mutagenesis or deduction from sequence alignments) or when structural information exists (by visual inspection, analysis, etc.…”

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