Abstract
G protein-coupled receptors (GPCRs) are flexible integral membrane proteins involved in transmembrane signaling. Their involvement in many physiological processes makes them interesting targets for drug development. Determination of the structure of these receptors will help to design more specific drugs, however, their structural characterization has so far been hampered by the low expression and their inherent instability in detergents which made protein engineering indispensable for structural and biophysical characterization. Several approaches to stabilize the receptors in a particular conformation have led to breakthroughs in GPCR structure determination. These include truncations of the flexible regions, stabilization by antibodies and nanobodies, fusion partners, high affinity and covalently bound ligands as well as conformational stabilization by mutagenesis. In this review we focus on stabilization of GPCRs by insertion of point mutations, which lead to increased conformational and thermal stability as well as improved expression levels. We summarize existing mutagenesis strategies with different coverage of GPCR sequence space and depth of information, design and transferability of mutations and the molecular basis for stabilization. We also discuss whether mutations alter the structure and pharmacological properties of GPCRs.
Highlights
Gprotein-coupled receptors (GPCRs) are integral membrane proteins that play a central role in signaling pathways being key intermediaries between external stimuli and the intracellular signaling cascades
Conformational thermostabilization of many different receptors using alanine/leucine scanning or directed evolution has shown that point mutations are a valuable tool for GPCR structural biology and biophysics
A relatively large fraction (5–12%) of alanine mutations in scanning mutagenesis led to the stabilization of the receptors, suggesting that it is likely that many receptors could be stabilized by this approach
Summary
Gprotein-coupled receptors (GPCRs) are integral membrane proteins that play a central role in signaling pathways being key intermediaries between external stimuli and the intracellular signaling cascades. Alanine scanning combined with radio-ligand binding does identify stabilizing mutations but leads to a conformationally and thermally stabilized construct which can be used for crystallization and subsequent structure determination. Combined approaches with initial improvement of rat neurotensin receptor 1 by error-prone PCR-based evolution led to a variant with a 12-fold higher expression level (Dodevski and Plückthun, 2011). This variant was improved to 50fold increased expression compared to wild type using all-vs.-all mutations (Schlinkmann et al, 2012b). As our understanding of the role of the individual amino acids in GPCR structure and stability improves, and more experimental data become available, the computational approaches to receptor design will likely become more powerful and widely used in the future
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