Sandbox Reserved 1170: Difference between revisions

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== Structure ==

[[Image:HGPR40bind2.png|~~250~~ px|right|thumb|Figure 1. Second proposed binding site of hGPR40 with surface shown. Substrate would bind in the deep pocket shown between TM3, 4, and 5.]]

[[Image:HGPR40bind2.png|200 px|right|thumb|Figure 1. Second proposed binding site of hGPR40 with surface shown. Substrate would bind in the deep pocket shown between TM3, 4, and 5.]]

Like most G-protein coupled receptors, hGPR40 contains <scene name='72/721541/Top_view_transmembrane_helices/2'>seven transmembrane helices</scene> (<scene name='72/721541/Top_view_transmembrane_helices/1'>top view of TM helices</scene>). To obtain a [https://en.wikipedia.org/wiki/Protein_crystallization crystallized structure] of the protein, four <scene name='72/721541/Stabilizing_mutations/4'>stabilizing mutations</scene> (<scene name='72/721541/L42a/3'>L42A</scene>, <scene name='72/721541/F88a/4'>F88A</scene>, <scene name='72/721541/G103a/3'>G103A</scene>, <scene name='72/721541/Y202f/3'>Y202F</scene>) were made to increase expression levels and thermal stability of the protein. These mutations did not significantly impact the enzyme's binding affinity with a known agonist, TAK-875.<ref name="Srivastava"/> A <scene name='72/721541/Lysozyme_crimson/2'>T4 Lysozyme</scene> (shown in crimson) was also added to intracellular loop 3 to aid in the formation of crystals. T4 Lysozyme also had little effect on TAK-875 binding.<ref name="Srivastava"/> For clarity, lysozyme is removed in all further renderings of hGPR40.

=== Binding Sites ===

[[Image:HGPR40bind3.png|~~250~~ px|right|thumb|Figure 2. Third proposed binding site of hGPR40 with surface shown. Substrate would bind in the pocket below TM7 and above and inbetween TM 1 and 2.]][http://metislabs.com/radioligand-binding-assays Radioligand binding studies] identified multiple [https://en.wikipedia.org/wiki/Binding_site binding sites] in hGPR40.<ref name="Srivastava"/> [https://en.wikipedia.org/wiki/Agonist Full agonists] and [https://en.wikipedia.org/wiki/Partial_agonist partial agonists] were shown to bind in separate sites with positive [http://www.britannica.com/science/cooperativity cooperativity].<ref name="Lin">PMID:22859723</ref> The <scene name='72/721541/Tak_binding_site/4'>binding site for the partial agonist TAK-875</scene> has been identified, but other binding sites were hypothesized. TAK-875 binds between transmembrane helices 3, 4, and 5 and underneath ECL2. By visual inspection, a second possible binding site was proposed between transmembrane helices 3, 4, and 5 on the intracellular side of the transmembrane helices (Figure 1). Also by visual inspection, a third possible binding site was proposed between transmembrane helices 1, 2, and 7 on the extracellular side of hGPR40, close to the TAK-875 binding site (Figure 2).<ref name="Srivastava"/> These binding sites could potentially serve as regulation points for hGPR40. Many proteins are regulated by the binding of inhibitors.

[[Image:HGPR40bind3.png|200 px|right|thumb|Figure 2. Third proposed binding site of hGPR40 with surface shown. Substrate would bind in the pocket below TM7 and above and inbetween TM 1 and 2.]][http://metislabs.com/radioligand-binding-assays Radioligand binding studies] identified multiple [https://en.wikipedia.org/wiki/Binding_site binding sites] in hGPR40.<ref name="Srivastava"/> [https://en.wikipedia.org/wiki/Agonist Full agonists] and [https://en.wikipedia.org/wiki/Partial_agonist partial agonists] were shown to bind in separate sites with positive [http://www.britannica.com/science/cooperativity cooperativity].<ref name="Lin">PMID:22859723</ref> The <scene name='72/721541/Tak_binding_site/4'>binding site for the partial agonist TAK-875</scene> has been identified, but other binding sites were hypothesized. TAK-875 binds between transmembrane helices 3, 4, and 5 and underneath ECL2. By visual inspection, a second possible binding site was proposed between transmembrane helices 3, 4, and 5 on the intracellular side of the transmembrane helices (Figure 1). Also by visual inspection, a third possible binding site was proposed between transmembrane helices 1, 2, and 7 on the extracellular side of hGPR40, close to the TAK-875 binding site (Figure 2).<ref name="Srivastava"/> These binding sites could potentially serve as regulation points for hGPR40. Many proteins are regulated by the binding of inhibitors.

=== Charge Network ===

[[Image:hydrogen bonding black.png|~~250~~ px|right|thumb|Figure 3. TAK-875 with key binding residues Tyr91, Arg183, Tyr240, and Arg258. These residues all hydrogen bond to the carboxylate moiety of TAK-875.]]hGPR40 has a distinct binding pocket that is established by <scene name='72/721541/All_binding_residues/1'>eight key residues</scene>: Tyr91, Glu172, Arg183, Ser187, Tyr240, Asn241, Asn244, and Arg258. The importance of these residues for agonist binding was determined by alanine [https://www.neb.com/applications/cloning-and-synthetic-biology/site-directed-mutagenesis mutagenesis] studies. Each of these residues have either a [http://www.proteinstructures.com/Structure/Structure/amino-acids.html charged or polar R-group] that allows them to develop a charge network. This network keeps the residues in a stable, unbound state until exposed to a substrate. When the substrate (an agonist) enters the binding pocket, four of the eight <scene name='72/721541/Hydrogen_binding_1/6'>key binding residues</scene> interact directly with the carboxylate moiety of the agonist. In 2007 and 2009, researchers showed the presence of Arg183 and Arg258 in the binding pocket.<ref name="Sum">PMID: 17699519</ref><ref name="Sum, C.">PMID:19068482</ref> Along with the two arginine residues, the charge network incorporates two tyrosine residues. These residues (Tyr91 and Tyr240) also stabilize the carboxylate group on the agonists. It was further determined that Tyr240 is especially important for binding. Mutation of Tyr240 caused a reduction in the binding affinity of TAK-875 by eight fold and had a significant effect on the [https://en.wikipedia.org/wiki/Dissociation_constant KD] of the protein.<ref name="Srivastava"/>

[[Image:hydrogen bonding black.png|200 px|right|thumb|Figure 3. TAK-875 with key binding residues Tyr91, Arg183, Tyr240, and Arg258. These residues all hydrogen bond to the carboxylate moiety of TAK-875.]]hGPR40 has a distinct binding pocket that is established by <scene name='72/721541/All_binding_residues/1'>eight key residues</scene>: Tyr91, Glu172, Arg183, Ser187, Tyr240, Asn241, Asn244, and Arg258. The importance of these residues for agonist binding was determined by alanine [https://www.neb.com/applications/cloning-and-synthetic-biology/site-directed-mutagenesis mutagenesis] studies. Each of these residues have either a [http://www.proteinstructures.com/Structure/Structure/amino-acids.html charged or polar R-group] that allows them to develop a charge network. This network keeps the residues in a stable, unbound state until exposed to a substrate. When the substrate (an agonist) enters the binding pocket, four of the eight <scene name='72/721541/Hydrogen_binding_1/6'>key binding residues</scene> interact directly with the carboxylate moiety of the agonist. In 2007 and 2009, researchers showed the presence of Arg183 and Arg258 in the binding pocket.<ref name="Sum">PMID: 17699519</ref><ref name="Sum, C.">PMID:19068482</ref> Along with the two arginine residues, the charge network incorporates two tyrosine residues. These residues (Tyr91 and Tyr240) also stabilize the carboxylate group on the agonists. It was further determined that Tyr240 is especially important for binding. Mutation of Tyr240 caused a reduction in the binding affinity of TAK-875 by eight fold and had a significant effect on the [https://en.wikipedia.org/wiki/Dissociation_constant KD] of the protein.<ref name="Srivastava"/>

=== ECL2 ===

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 == Structure ==
-[[Image:HGPR40bind2.png|250 px|right|thumb|Figure 1. Second proposed binding site of hGPR40 with surface shown. Substrate would bind in the deep pocket shown between TM3, 4, and 5.]]
+[[Image:HGPR40bind2.png|200 px|right|thumb|Figure 1. Second proposed binding site of hGPR40 with surface shown. Substrate would bind in the deep pocket shown between TM3, 4, and 5.]]
 Like most G-protein coupled receptors, hGPR40 contains <scene name='72/721541/Top_view_transmembrane_helices/2'>seven transmembrane helices</scene> (<scene name='72/721541/Top_view_transmembrane_helices/1'>top view of TM helices</scene>). To obtain a [https://en.wikipedia.org/wiki/Protein_crystallization crystallized structure] of the protein, four <scene name='72/721541/Stabilizing_mutations/4'>stabilizing mutations</scene> (<scene name='72/721541/L42a/3'>L42A</scene>, <scene name='72/721541/F88a/4'>F88A</scene>, <scene name='72/721541/G103a/3'>G103A</scene>, <scene name='72/721541/Y202f/3'>Y202F</scene>) were made to increase expression levels and thermal stability of the protein. These mutations did not significantly impact the enzyme's binding affinity with a known agonist, TAK-875.<ref name="Srivastava"/> A <scene name='72/721541/Lysozyme_crimson/2'>T4 Lysozyme</scene> (shown in <FONT COLOR="#DC143C">crimson</FONT>) was also added to intracellular loop 3 to aid in the formation of crystals. T4 Lysozyme also had little effect on TAK-875 binding.<ref name="Srivastava"/> For clarity, lysozyme is removed in all further renderings of hGPR40.
 === Binding Sites ===
-[[Image:HGPR40bind3.png|250 px|right|thumb|Figure 2. Third proposed binding site of hGPR40 with surface shown. Substrate would bind in the pocket below TM7 and above and inbetween TM 1 and 2.]][http://metislabs.com/radioligand-binding-assays Radioligand binding studies] identified multiple [https://en.wikipedia.org/wiki/Binding_site binding sites] in hGPR40.<ref name="Srivastava"/> [https://en.wikipedia.org/wiki/Agonist Full agonists] and [https://en.wikipedia.org/wiki/Partial_agonist partial agonists] were shown to bind in separate sites with positive [http://www.britannica.com/science/cooperativity cooperativity].<ref name="Lin">PMID:22859723</ref> The <scene name='72/721541/Tak_binding_site/4'>binding site for the partial agonist TAK-875</scene> has been identified, but other binding sites were hypothesized. TAK-875 binds between transmembrane helices 3, 4, and 5 and underneath ECL2. By visual inspection, a second possible binding site was proposed between transmembrane helices 3, 4, and 5 on the intracellular side of the transmembrane helices (Figure 1). Also by visual inspection, a third possible binding site was proposed between transmembrane helices 1, 2, and 7 on the extracellular side of hGPR40, close to the TAK-875 binding site (Figure 2).<ref name="Srivastava"/> These binding sites could potentially serve as regulation points for hGPR40. Many proteins are regulated by the binding of inhibitors.
+[[Image:HGPR40bind3.png|200 px|right|thumb|Figure 2. Third proposed binding site of hGPR40 with surface shown. Substrate would bind in the pocket below TM7 and above and inbetween TM 1 and 2.]][http://metislabs.com/radioligand-binding-assays Radioligand binding studies] identified multiple [https://en.wikipedia.org/wiki/Binding_site binding sites] in hGPR40.<ref name="Srivastava"/> [https://en.wikipedia.org/wiki/Agonist Full agonists] and [https://en.wikipedia.org/wiki/Partial_agonist partial agonists] were shown to bind in separate sites with positive [http://www.britannica.com/science/cooperativity cooperativity].<ref name="Lin">PMID:22859723</ref> The <scene name='72/721541/Tak_binding_site/4'>binding site for the partial agonist TAK-875</scene> has been identified, but other binding sites were hypothesized. TAK-875 binds between transmembrane helices 3, 4, and 5 and underneath ECL2. By visual inspection, a second possible binding site was proposed between transmembrane helices 3, 4, and 5 on the intracellular side of the transmembrane helices (Figure 1). Also by visual inspection, a third possible binding site was proposed between transmembrane helices 1, 2, and 7 on the extracellular side of hGPR40, close to the TAK-875 binding site (Figure 2).<ref name="Srivastava"/> These binding sites could potentially serve as regulation points for hGPR40. Many proteins are regulated by the binding of inhibitors.
 === Charge Network ===
-[[Image:hydrogen bonding black.png|250 px|right|thumb|Figure 3. TAK-875 with key binding residues Tyr91, Arg183, Tyr240, and Arg258. These residues all hydrogen bond to the carboxylate moiety of TAK-875.]]hGPR40 has a distinct binding pocket that is established by <scene name='72/721541/All_binding_residues/1'>eight key residues</scene>: Tyr91, Glu172, Arg183, Ser187, Tyr240, Asn241, Asn244, and Arg258. The importance of these residues for agonist binding was determined by alanine [https://www.neb.com/applications/cloning-and-synthetic-biology/site-directed-mutagenesis mutagenesis] studies. Each of these residues have either a [http://www.proteinstructures.com/Structure/Structure/amino-acids.html charged or polar R-group] that allows them to develop a charge network. This network keeps the residues in a stable, unbound state until exposed to a substrate. When the substrate (an agonist) enters the binding pocket, four of the eight <scene name='72/721541/Hydrogen_binding_1/6'>key binding residues</scene> interact directly with the carboxylate moiety of the agonist. In 2007 and 2009, researchers showed the presence of Arg183 and Arg258 in the binding pocket.<ref name="Sum">PMID: 17699519</ref><ref name="Sum, C.">PMID:19068482</ref> Along with the two arginine residues, the charge network incorporates two tyrosine residues. These residues (Tyr91 and Tyr240) also stabilize the carboxylate group on the agonists. It was further determined that Tyr240 is especially important for binding. Mutation of Tyr240 caused a reduction in the binding affinity of TAK-875 by eight fold and had a significant effect on the [https://en.wikipedia.org/wiki/Dissociation_constant K<sub>D</sub>] of the protein.<ref name="Srivastava"/>
+[[Image:hydrogen bonding black.png|200 px|right|thumb|Figure 3. TAK-875 with key binding residues Tyr91, Arg183, Tyr240, and Arg258. These residues all hydrogen bond to the carboxylate moiety of TAK-875.]]hGPR40 has a distinct binding pocket that is established by <scene name='72/721541/All_binding_residues/1'>eight key residues</scene>: Tyr91, Glu172, Arg183, Ser187, Tyr240, Asn241, Asn244, and Arg258. The importance of these residues for agonist binding was determined by alanine [https://www.neb.com/applications/cloning-and-synthetic-biology/site-directed-mutagenesis mutagenesis] studies. Each of these residues have either a [http://www.proteinstructures.com/Structure/Structure/amino-acids.html charged or polar R-group] that allows them to develop a charge network. This network keeps the residues in a stable, unbound state until exposed to a substrate. When the substrate (an agonist) enters the binding pocket, four of the eight <scene name='72/721541/Hydrogen_binding_1/6'>key binding residues</scene> interact directly with the carboxylate moiety of the agonist. In 2007 and 2009, researchers showed the presence of Arg183 and Arg258 in the binding pocket.<ref name="Sum">PMID: 17699519</ref><ref name="Sum, C.">PMID:19068482</ref> Along with the two arginine residues, the charge network incorporates two tyrosine residues. These residues (Tyr91 and Tyr240) also stabilize the carboxylate group on the agonists. It was further determined that Tyr240 is especially important for binding. Mutation of Tyr240 caused a reduction in the binding affinity of TAK-875 by eight fold and had a significant effect on the [https://en.wikipedia.org/wiki/Dissociation_constant K<sub>D</sub>] of the protein.<ref name="Srivastava"/>
 === ECL2 ===