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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.


Comparison of GPCRs containing either a fusion protein or thermostabilizing mutations. Structures of neurotensin receptor 1 (A) and adenosine A2A receptor in the inactive (B) and active (C) state solved using fusion-protein or point-mutagenesis approaches show high similarity [overall RMSD = 0.95 Å (A) and 0.5 Å (B,C)]. The differences in helices five and six in the two neurotensin receptor structures may be due to mutation of an amino acid involved in activation, R1673.50L, in one receptor and the T4 lysozyme fusion in the other receptor. PDB IDs: 3ZEV (NTR1, directed evolution), 4GRV (NTR1, fusion with T4 lysozyme), 3PWH (A2A, antagonist-bound, with thermostabilizing mutations), 3EML (A2A, antagonist-bound, T4 lysozyme fusion), 3QAK (A2A, with agonist UK-432097 and T4 lysozyme) and 2YDO (with agonist adenosine and thermostabilizing mutations).
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Figure 5: Comparison of GPCRs containing either a fusion protein or thermostabilizing mutations. Structures of neurotensin receptor 1 (A) and adenosine A2A receptor in the inactive (B) and active (C) state solved using fusion-protein or point-mutagenesis approaches show high similarity [overall RMSD = 0.95 Å (A) and 0.5 Å (B,C)]. The differences in helices five and six in the two neurotensin receptor structures may be due to mutation of an amino acid involved in activation, R1673.50L, in one receptor and the T4 lysozyme fusion in the other receptor. PDB IDs: 3ZEV (NTR1, directed evolution), 4GRV (NTR1, fusion with T4 lysozyme), 3PWH (A2A, antagonist-bound, with thermostabilizing mutations), 3EML (A2A, antagonist-bound, T4 lysozyme fusion), 3QAK (A2A, with agonist UK-432097 and T4 lysozyme) and 2YDO (with agonist adenosine and thermostabilizing mutations).

Mentions: Two rat neurotensin receptor 1 variants selected by CHESS and a variant harboring 11 mutations uncovered by recombination and evolution of position-specific libraries have been crystallized (Schlinkmann et al., 2012a,b; Egloff et al., 2014). All three structures are almost identical even though the sets of mutations are different (overall RMSD = 0.4Å). In addition, one variant was competent of G-protein activation, though at a reduced level, ligand binding with native-like affinities and desensitization. The structure looks like an inactive agonist-bound conformation since TM6 did not move outwards, which agrees well with the reduced G-protein activation observed and the fact that R1673.50, a residue involved in the activation mechanism has been mutated to leucine. The T4L structure of NTR1 (White et al., 2012, PDB ID 4GRV), supposedly in a semi-active conformation, is very similar to the structures obtained by directed evolution. They only differ at the intracellular ends of TM5 and 6 which might be due to different degrees of activation and the fusion with T4L (Figure 5A).


Stabilization of G protein-coupled receptors by point mutations.

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

Comparison of GPCRs containing either a fusion protein or thermostabilizing mutations. Structures of neurotensin receptor 1 (A) and adenosine A2A receptor in the inactive (B) and active (C) state solved using fusion-protein or point-mutagenesis approaches show high similarity [overall RMSD = 0.95 Å (A) and 0.5 Å (B,C)]. The differences in helices five and six in the two neurotensin receptor structures may be due to mutation of an amino acid involved in activation, R1673.50L, in one receptor and the T4 lysozyme fusion in the other receptor. PDB IDs: 3ZEV (NTR1, directed evolution), 4GRV (NTR1, fusion with T4 lysozyme), 3PWH (A2A, antagonist-bound, with thermostabilizing mutations), 3EML (A2A, antagonist-bound, T4 lysozyme fusion), 3QAK (A2A, with agonist UK-432097 and T4 lysozyme) and 2YDO (with agonist adenosine and thermostabilizing mutations).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Comparison of GPCRs containing either a fusion protein or thermostabilizing mutations. Structures of neurotensin receptor 1 (A) and adenosine A2A receptor in the inactive (B) and active (C) state solved using fusion-protein or point-mutagenesis approaches show high similarity [overall RMSD = 0.95 Å (A) and 0.5 Å (B,C)]. The differences in helices five and six in the two neurotensin receptor structures may be due to mutation of an amino acid involved in activation, R1673.50L, in one receptor and the T4 lysozyme fusion in the other receptor. PDB IDs: 3ZEV (NTR1, directed evolution), 4GRV (NTR1, fusion with T4 lysozyme), 3PWH (A2A, antagonist-bound, with thermostabilizing mutations), 3EML (A2A, antagonist-bound, T4 lysozyme fusion), 3QAK (A2A, with agonist UK-432097 and T4 lysozyme) and 2YDO (with agonist adenosine and thermostabilizing mutations).
Mentions: Two rat neurotensin receptor 1 variants selected by CHESS and a variant harboring 11 mutations uncovered by recombination and evolution of position-specific libraries have been crystallized (Schlinkmann et al., 2012a,b; Egloff et al., 2014). All three structures are almost identical even though the sets of mutations are different (overall RMSD = 0.4Å). In addition, one variant was competent of G-protein activation, though at a reduced level, ligand binding with native-like affinities and desensitization. The structure looks like an inactive agonist-bound conformation since TM6 did not move outwards, which agrees well with the reduced G-protein activation observed and the fact that R1673.50, a residue involved in the activation mechanism has been mutated to leucine. The T4L structure of NTR1 (White et al., 2012, PDB ID 4GRV), supposedly in a semi-active conformation, is very similar to the structures obtained by directed evolution. They only differ at the intracellular ends of TM5 and 6 which might be due to different degrees of activation and the fusion with T4L (Figure 5A).

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.