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Stabilization of G protein-coupled receptors by point mutations.

Heydenreich FM, Vuckovic Z, Matkovic M, Veprintsev DB - Front Pharmacol (2015)

Bottom Line: Their involvement in many physiological processes makes them interesting targets for drug development.Several approaches to stabilize the receptors in a particular conformation have led to breakthroughs in GPCR structure determination.We also discuss whether mutations alter the structure and pharmacological properties of GPCRs.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biomolecular Research, Paul Scherrer Institut Villigen, Switzerland ; Department of Biology, ETH Zürich Zürich, Switzerland.

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.

No MeSH data available.


Molecular basis of stabilization by mutation of the 3.41 position to tryptophan. The 3.41W mutation stabilizes 5-HT1B (green), 5-HT2B (light pink) and CXCR4 (blue) through its interaction with the proline in position 5.50 and the carbonyl of the amino acid in position 5.45.
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Figure 4: Molecular basis of stabilization by mutation of the 3.41 position to tryptophan. The 3.41W mutation stabilizes 5-HT1B (green), 5-HT2B (light pink) and CXCR4 (blue) through its interaction with the proline in position 5.50 and the carbonyl of the amino acid in position 5.45.

Mentions: One mutation, E1223.41W, identified in human β2-adrenergic receptor has been shown to be transferable to different GPCRs and increased thermostability as well as total expression level and expression at the cell surface (Roth et al., 2008). In rhodopsin, W1263.41 is the only residue in transmembrane (TM) domain 3 which contacts both TM4 and TM5. Since position 3.41 is not conserved, introduction of a mutation is less likely to disturb the overall fold of the receptor. The tryptophan at the TM3-TM4-TM5 interface is thought to stabilize the conformationally flexible TM5, and thereby increase thermostability and expression. The effect on β1-adrenergic receptor was similar to thermostabilization by addition of antagonist (Roth et al., 2008). Other mutations of the E1223.41 residue also led to higher thermostability and expression, especially E122Y and E122L. However, all mutations showed a loss in affinity for the ligand. The mutation which showed the highest increase in thermal stability has been successfully transferred to the serotonin receptors 5-HT1B (L138W) and 5-HT2B (M144W) as well as the CXCR4 chemokine receptor (L125W) (Wu et al., 2010; Wacker et al., 2013; Wang et al., 2013) and D3 dopamine receptor (L119W) (Chien et al., 2010). As proposed by molecular modeling of human β2-adrenergic receptor (hβ2AR), W3.41 stabilizes 5-HT1B, 5-HT2B and CXCR4 by its interaction with P5.50 and the carbonyl of 5.46 (Figure 4). Since conformational flexibility of TM5 is an inherent feature of class A GPCRs, transfer of this mutation is likely to be successful (Roth et al., 2008).


Stabilization of G protein-coupled receptors by point mutations.

Heydenreich FM, Vuckovic Z, Matkovic M, Veprintsev DB - Front Pharmacol (2015)

Molecular basis of stabilization by mutation of the 3.41 position to tryptophan. The 3.41W mutation stabilizes 5-HT1B (green), 5-HT2B (light pink) and CXCR4 (blue) through its interaction with the proline in position 5.50 and the carbonyl of the amino acid in position 5.45.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403299&req=5

Figure 4: Molecular basis of stabilization by mutation of the 3.41 position to tryptophan. The 3.41W mutation stabilizes 5-HT1B (green), 5-HT2B (light pink) and CXCR4 (blue) through its interaction with the proline in position 5.50 and the carbonyl of the amino acid in position 5.45.
Mentions: One mutation, E1223.41W, identified in human β2-adrenergic receptor has been shown to be transferable to different GPCRs and increased thermostability as well as total expression level and expression at the cell surface (Roth et al., 2008). In rhodopsin, W1263.41 is the only residue in transmembrane (TM) domain 3 which contacts both TM4 and TM5. Since position 3.41 is not conserved, introduction of a mutation is less likely to disturb the overall fold of the receptor. The tryptophan at the TM3-TM4-TM5 interface is thought to stabilize the conformationally flexible TM5, and thereby increase thermostability and expression. The effect on β1-adrenergic receptor was similar to thermostabilization by addition of antagonist (Roth et al., 2008). Other mutations of the E1223.41 residue also led to higher thermostability and expression, especially E122Y and E122L. However, all mutations showed a loss in affinity for the ligand. The mutation which showed the highest increase in thermal stability has been successfully transferred to the serotonin receptors 5-HT1B (L138W) and 5-HT2B (M144W) as well as the CXCR4 chemokine receptor (L125W) (Wu et al., 2010; Wacker et al., 2013; Wang et al., 2013) and D3 dopamine receptor (L119W) (Chien et al., 2010). As proposed by molecular modeling of human β2-adrenergic receptor (hβ2AR), W3.41 stabilizes 5-HT1B, 5-HT2B and CXCR4 by its interaction with P5.50 and the carbonyl of 5.46 (Figure 4). Since conformational flexibility of TM5 is an inherent feature of class A GPCRs, transfer of this mutation is likely to be successful (Roth et al., 2008).

Bottom Line: Their involvement in many physiological processes makes them interesting targets for drug development.Several approaches to stabilize the receptors in a particular conformation have led to breakthroughs in GPCR structure determination.We also discuss whether mutations alter the structure and pharmacological properties of GPCRs.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biomolecular Research, Paul Scherrer Institut Villigen, Switzerland ; Department of Biology, ETH Zürich Zürich, Switzerland.

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.

No MeSH data available.