Abstract
Membrane proteins such as G protein-coupled receptors (GPCRs) exert fundamental biological functions and are involved in a multitude of physiological responses, making these receptors ideal drug targets. Drug discovery programs targeting GPCRs have been greatly facilitated by the emergence of high-resolution structures and the resulting opportunities to identify new chemical entities through structure-based drug design. To enable the determination of high-resolution structures of GPCRs, most receptors have to be engineered to overcome intrinsic hurdles such as their poor stability and low expression levels. In recent years, multiple engineering approaches have been developed to specifically address the technical difficulties of working with GPCRs, which are now beginning to make more challenging receptors accessible to detailed studies. Importantly, successfully engineered GPCRs are not only valuable in X-ray crystallography, but further enable biophysical studies with nuclear magnetic resonance spectroscopy, surface plasmon resonance, native mass spectrometry, and fluorescence anisotropy measurements, all of which are important for the detailed mechanistic understanding, which is the prerequisite for successful drug design. Here, we summarize engineering strategies based on directed evolution to reduce workload and enable biophysical experiments of particularly challenging GPCRs.
Highlights
Membrane proteins such as G protein-coupled receptors (GPCRs) exert fundamental biological functions and are involved in a multitude of physiological responses, making these receptors ideal drug targets
The structures of neurotensin 1 receptor (NTS1 R):NT8-13 revealed the intermediate state in which the receptor had been stabilized, showing the agonist bound in the orthosteric pocket on the extracellular receptor portion, but with the intracellular receptor portion still found in an inactive conformation in the absence of a G protein (Figure 5)
The influence of lipids on G protein coupling selectivity was demonstrated for three different thermostabilized GPCRs using native mass spectrometry (nMS) [77], whereas different conformational states of a stabilized β1 adrenergic receptor in complex with G protein mimics were assessed by nMS, suggesting that this method is suitable for drug screening purposes [78]
Summary
GPCRs have evolved as intrinsically flexible proteins that sample a multitude of different conformations They have to convey an extracellular signal, triggered by the binding of an agonist molecule, into the cytoplasm. In extensive site-directed mutagenesis approaches such as alanine scanning [29], every receptor amino acid is individually mutated, and the corresponding mutant is expressed and evaluated. When combining mutations, their effects can be additive, cancel out, or even be deleterious, requiring again an experimental screening of all combinatorial possibilities. Cells displaying an increased functional receptor expression are detected and enriched by assessing the fluorescence brightness emitted by either a specially constructed receptor-fused fluorescent protein that reports on correct membrane insertion [36] or by probing functional receptor expression directly with a fluorescently labelled ligand, which can be an agonist or antagonist [33,37]
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