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Free fatty acid receptors: structural models and elucidation of ligand binding interactions.

Tikhonova IG, Poerio E - BMC Struct. Biol. (2015)

Bottom Line: This binding mode can explain mutagenesis results for residues at positions 4.56 and 5.42.The novel structural models of FFAs provide information on specific modes of ligand binding at FFA subtypes and new suggestions for mutagenesis and ligand modification, guiding the development of novel orthosteric and allosteric chemical probes to validate the importance of FFAs in metabolic and inflammatory conditions.Using our FFA homology modelling experience, a strategy to model a GPCR, which is phylogenetically distant from GPCRs with the available crystal structures, is discussed.

View Article: PubMed Central - PubMed

Affiliation: Molecular Therapeutics, School of Pharmacy, Medical Biology Centre, Queen's University Belfast, Belfast, BT9 7BL, Northern Ireland, UK. i.tikhonova@qub.ac.uk.

ABSTRACT

Background: The free fatty acid receptors (FFAs), including FFA1 (orphan name: GPR40), FFA2 (GPR43) and FFA3 (GPR41) are G protein-coupled receptors (GPCRs) involved in energy and metabolic homeostasis. Understanding the structural basis of ligand binding at FFAs is an essential step toward designing potent and selective small molecule modulators.

Results: We analyse earlier homology models of FFAs in light of the newly published FFA1 crystal structure co-crystallized with TAK-875, an ago-allosteric ligand, focusing on the architecture of the extracellular binding cavity and agonist-receptor interactions. The previous low-resolution homology models of FFAs were helpful in highlighting the location of the ligand binding site and the key residues for ligand anchoring. However, homology models were not accurate in establishing the nature of all ligand-receptor contacts and the precise ligand-binding mode. From analysis of structural models and mutagenesis, it appears that the position of helices 3, 4 and 5 is crucial in ligand docking. The FFA1-based homology models of FFA2 and FFA3 were constructed and used to compare the FFA subtypes. From docking studies we propose an alternative binding mode for orthosteric agonists at FFA1 and FFA2, involving the interhelical space between helices 4 and 5. This binding mode can explain mutagenesis results for residues at positions 4.56 and 5.42. The novel FFAs structural models highlight higher aromaticity of the FFA2 binding cavity and higher hydrophilicity of the FFA3 binding cavity. The role of the residues at the second extracellular loop used in mutagenesis is reanalysed. The third positively-charged residue in the binding cavity of FFAs, located in helix 2, is identified and predicted to coordinate allosteric modulators.

Conclusions: The novel structural models of FFAs provide information on specific modes of ligand binding at FFA subtypes and new suggestions for mutagenesis and ligand modification, guiding the development of novel orthosteric and allosteric chemical probes to validate the importance of FFAs in metabolic and inflammatory conditions. Using our FFA homology modelling experience, a strategy to model a GPCR, which is phylogenetically distant from GPCRs with the available crystal structures, is discussed.

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The superimposition of the FFA1 crystal structure and homology models base on the backbone of the helices in the extracellular side. The crystal structure, rhodopsin, β2 adrenergic and PAR1-based homology models are in yellow, cyan, pink and grey colour, respectively. Residues predicted to be important for ligand coordination based on mutagenesis and residue K622.60, representing the possible anchoring point for allosteric ligands are shown in stick-like representation. The green arrows indicate the large movement of helices 3, 4 and 5 in the FFA1 crystal structure compared to the homology models
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Fig2: The superimposition of the FFA1 crystal structure and homology models base on the backbone of the helices in the extracellular side. The crystal structure, rhodopsin, β2 adrenergic and PAR1-based homology models are in yellow, cyan, pink and grey colour, respectively. Residues predicted to be important for ligand coordination based on mutagenesis and residue K622.60, representing the possible anchoring point for allosteric ligands are shown in stick-like representation. The green arrows indicate the large movement of helices 3, 4 and 5 in the FFA1 crystal structure compared to the homology models

Mentions: We evaluate the earlier homology models of FFA1 built by Tikhonova et al. [13, 15] and Sum, et al. [14, 17] based on rhodopsin and β2 adrenergic (β2) receptor crystal structures, templates with 16 % sequence identity to model the ligand binding site with the FFA1 crystal structure. In addition, the homology model based on the protease-activated receptor 1 (PAR1) crystal structure [18], the available template with the highest sequence identity, 26 % is constructed and included to the analysis. The backbone superimposition of helices of the crystal structure and homology models is shown in Fig. 2. The root-mean-square deviation (RMSD) value for the helix backbone is 2.8 Å for the rhodopsin-based and 1.9 Å for β2- and PAR1 -based FFA1 homology models. In the upper side of the helical bundle forming the ligand binding site a significant deviation is observed for helices 3, 4 and 5 with a backbone RMSD of 2–3.4 Å.Fig. 2


Free fatty acid receptors: structural models and elucidation of ligand binding interactions.

Tikhonova IG, Poerio E - BMC Struct. Biol. (2015)

The superimposition of the FFA1 crystal structure and homology models base on the backbone of the helices in the extracellular side. The crystal structure, rhodopsin, β2 adrenergic and PAR1-based homology models are in yellow, cyan, pink and grey colour, respectively. Residues predicted to be important for ligand coordination based on mutagenesis and residue K622.60, representing the possible anchoring point for allosteric ligands are shown in stick-like representation. The green arrows indicate the large movement of helices 3, 4 and 5 in the FFA1 crystal structure compared to the homology models
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4561419&req=5

Fig2: The superimposition of the FFA1 crystal structure and homology models base on the backbone of the helices in the extracellular side. The crystal structure, rhodopsin, β2 adrenergic and PAR1-based homology models are in yellow, cyan, pink and grey colour, respectively. Residues predicted to be important for ligand coordination based on mutagenesis and residue K622.60, representing the possible anchoring point for allosteric ligands are shown in stick-like representation. The green arrows indicate the large movement of helices 3, 4 and 5 in the FFA1 crystal structure compared to the homology models
Mentions: We evaluate the earlier homology models of FFA1 built by Tikhonova et al. [13, 15] and Sum, et al. [14, 17] based on rhodopsin and β2 adrenergic (β2) receptor crystal structures, templates with 16 % sequence identity to model the ligand binding site with the FFA1 crystal structure. In addition, the homology model based on the protease-activated receptor 1 (PAR1) crystal structure [18], the available template with the highest sequence identity, 26 % is constructed and included to the analysis. The backbone superimposition of helices of the crystal structure and homology models is shown in Fig. 2. The root-mean-square deviation (RMSD) value for the helix backbone is 2.8 Å for the rhodopsin-based and 1.9 Å for β2- and PAR1 -based FFA1 homology models. In the upper side of the helical bundle forming the ligand binding site a significant deviation is observed for helices 3, 4 and 5 with a backbone RMSD of 2–3.4 Å.Fig. 2

Bottom Line: This binding mode can explain mutagenesis results for residues at positions 4.56 and 5.42.The novel structural models of FFAs provide information on specific modes of ligand binding at FFA subtypes and new suggestions for mutagenesis and ligand modification, guiding the development of novel orthosteric and allosteric chemical probes to validate the importance of FFAs in metabolic and inflammatory conditions.Using our FFA homology modelling experience, a strategy to model a GPCR, which is phylogenetically distant from GPCRs with the available crystal structures, is discussed.

View Article: PubMed Central - PubMed

Affiliation: Molecular Therapeutics, School of Pharmacy, Medical Biology Centre, Queen's University Belfast, Belfast, BT9 7BL, Northern Ireland, UK. i.tikhonova@qub.ac.uk.

ABSTRACT

Background: The free fatty acid receptors (FFAs), including FFA1 (orphan name: GPR40), FFA2 (GPR43) and FFA3 (GPR41) are G protein-coupled receptors (GPCRs) involved in energy and metabolic homeostasis. Understanding the structural basis of ligand binding at FFAs is an essential step toward designing potent and selective small molecule modulators.

Results: We analyse earlier homology models of FFAs in light of the newly published FFA1 crystal structure co-crystallized with TAK-875, an ago-allosteric ligand, focusing on the architecture of the extracellular binding cavity and agonist-receptor interactions. The previous low-resolution homology models of FFAs were helpful in highlighting the location of the ligand binding site and the key residues for ligand anchoring. However, homology models were not accurate in establishing the nature of all ligand-receptor contacts and the precise ligand-binding mode. From analysis of structural models and mutagenesis, it appears that the position of helices 3, 4 and 5 is crucial in ligand docking. The FFA1-based homology models of FFA2 and FFA3 were constructed and used to compare the FFA subtypes. From docking studies we propose an alternative binding mode for orthosteric agonists at FFA1 and FFA2, involving the interhelical space between helices 4 and 5. This binding mode can explain mutagenesis results for residues at positions 4.56 and 5.42. The novel FFAs structural models highlight higher aromaticity of the FFA2 binding cavity and higher hydrophilicity of the FFA3 binding cavity. The role of the residues at the second extracellular loop used in mutagenesis is reanalysed. The third positively-charged residue in the binding cavity of FFAs, located in helix 2, is identified and predicted to coordinate allosteric modulators.

Conclusions: The novel structural models of FFAs provide information on specific modes of ligand binding at FFA subtypes and new suggestions for mutagenesis and ligand modification, guiding the development of novel orthosteric and allosteric chemical probes to validate the importance of FFAs in metabolic and inflammatory conditions. Using our FFA homology modelling experience, a strategy to model a GPCR, which is phylogenetically distant from GPCRs with the available crystal structures, is discussed.

Show MeSH
Related in: MedlinePlus