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Abstract
G protein-coupled receptors (GPCRs) represent a large family of different proteins, which are involved in physiological processes throughout the entire body. Furthermore, they represent important drug targets. For rational drug design, it is important to get further insights into the binding mode of endogenous ligands as well as of therapeutic agents at the respective target receptors. However, structural investigations usually require homogenous, solubilized and functional receptors, which is still challenging. Cell-free expression methods have emerged in the last years and many different proteins are successfully expressed, including hydrophobic membrane proteins like GPCRs. In this work, an Escherichia coli based cell-free expression system was used to express the neuropeptide Y2 receptor (Y2R) for structural investigations. This GPCR was expressed in two different variants, a C-terminal enhanced green fluorescent fusion protein and a cysteine deficient variant. In order to obtain soluble receptors, the expression was performed in the presence of mild detergents, either Brij-35 or Brij-58, which led to high amounts of soluble receptor. Furthermore, the influence of temperature, pH value and additives on protein expression and solubilization was tested. For functional and structural investigations, the receptors were expressed at 37°C, pH 7.4 in the presence of 1 mM oxidized and 5 mM reduced glutathione. The expressed receptors were purified by ligand affinity chromatography and functionality of Y2R_cysteine_deficient was verified by a homogenous binding assay. Finally, photo-crosslinking studies were performed between cell-free expressed Y2R_cysteine_deficient and a neuropeptide Y (NPY) analog bearing the photoactive, unnatural amino acid p-benzoyl-phenylalanine at position 27 and biotin at position 22 for purification. After enzymatic digestion, fragments of crosslinked receptor were identified by mass spectrometry. Our findings demonstrate that, in contrast to Y1R, NPY position 27 remains flexible when bound to Y2R. These results are in agreement with the suggested binding mode of NPY at Y2R.
Highlights
G protein-coupled receptors (GPCR) are involved in the regulation of processes throughout the whole body and are potential pharmaceutical targets
In order to identify new drugs, new GPCR targets can be approached. Another concept is to find compounds for the fine-tuning of the GPCR signaling, which provides the opportunity to add other dimensions of regulation besides “on” or “off.” The rational design of novel drug compounds requires detailed knowledge of the binding pocket as well as the dynamic processes GPCRs undergo during activation
As an alternative the polyoxyethylene alkyl-ether Brij-35 and Brij-58, both with a critical micellar concentration below 0.1 mM, were added in final concentrations of 0.01–0.5% (w/v). These detergents have been used for soluble expression of a number of GPCRs by cell-free systems (Klammt et al, 2005; Schwarz et al, 2007; Corin et al, 2011a; Junge et al, 2011; Isaksson et al, 2012; Orban et al, 2015)
Summary
G protein-coupled receptors (GPCR) are involved in the regulation of processes throughout the whole body and are potential pharmaceutical targets. In 2017, about 35% of all approved drugs used GPCRs as targets and still, there is huge potential left. With about 800 members, these proteins are the largest family of integral membrane proteins, and only 134 of them are targeted by pharmaceuticals (Sriram and Insel, 2018). In order to identify new drugs, new GPCR targets can be approached. Another concept is to find compounds for the fine-tuning of the GPCR signaling, which provides the opportunity to add other dimensions of regulation besides “on” or “off.” The rational design of novel drug compounds requires detailed knowledge of the binding pocket as well as the dynamic processes GPCRs undergo during activation. Direct structural investigations of GPCRs remain challenging owing to their hydrophobicity and structural flexibility, since GPCRs adapt many distinct conformations in the basal, as well in the active state (Katritch et al, 2013; Venkatakrishnan et al, 2013; Manglik et al, 2015; Latorraca et al, 2017)
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