User:Alexander Rudecki/Sandbox 1: Difference between revisions

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The <scene name='58/580851/Composition/1'>composition</scene> of DromeQC consists of either {{Template:ColorKey Composition Protein}}, {{Template:ColorKey Composition Ligand}}, or the {{Template:ColorKey Composition Solvent}} in which it was crystallized (water). When DromeQC <scene name='58/580851/Secondary/1'>secondary structure</scene> is colour coated ({{Template:ColorKey_Helix}}, {{Template:ColorKey_Strand}}) the global fold may be visualized nicely. This fold is driven by <scene name='58/580851/Polar/1'>hydrophobic/polar</scene> residues. The majority of {{Template:ColorKey_Hydrophobic}} residues lie within the enzyme's interior, consistent with the hydrophobic collapse folding theory. In contrast, most {{Template:ColorKey_Polar}} residues reside on the exterior where they contact the polar solvent. Similarly, <scene name='58/580851/Charge/1'>charged residues</scene>, either {{Template:ColorKey_Charge_Anionic}} or {{Template:ColorKey_Charge_Cationic}}, appear to cluster on the outside of the protein. From analysis of charged residues, it can be seen that a salt bridge may connect the two monomers (Chain ~~A->Chain~~ B: ~~ARG35->GLU64~~).

The <scene name='58/580851/Composition/1'>composition</scene> of DromeQC consists of either {{Template:ColorKey Composition Protein}}, {{Template:ColorKey Composition Ligand}}, or the {{Template:ColorKey Composition Solvent}} in which it was crystallized (water). When DromeQC <scene name='58/580851/Secondary/1'>secondary structure</scene> is colour coated ({{Template:ColorKey_Helix}}, {{Template:ColorKey_Strand}}) the global fold may be visualized nicely. This fold is driven by <scene name='58/580851/Polar/1'>hydrophobic/polar</scene> residues. The majority of {{Template:ColorKey_Hydrophobic}} residues lie within the enzyme's interior, consistent with the hydrophobic collapse folding theory. In contrast, most {{Template:ColorKey_Polar}} residues reside on the exterior where they contact the polar solvent. Similarly, <scene name='58/580851/Charge/1'>charged residues</scene>, either {{Template:ColorKey_Charge_Anionic}} or {{Template:ColorKey_Charge_Cationic}}, appear to cluster on the outside of the protein. From analysis of charged residues, it can be seen that a salt bridge may connect the two monomers (Chain A→Chain B: ARG35→GLU64).

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[[Image:Cropped_dimer.jpg|thumb|400px|right|Figure 2. A 3D graphical representation displaying the homodimer glutaminyl cyclase from ''Drosophila melanogaster'' (PDB: 4F9U). Secondary structure is depicted by red (α-helix) and yellow (β-strand) ribbons, glycosyl groups are coloured ~~pinks~~, while hydrogen bonds between the two monomers are shown by dotted green lines. The active site of QC contains a chelated zinc ion represented by a gray sphere. Also bound to the active site of this crystal structure and depicted as blue is the inhibitor 1-(3,4-dimethoxyphenyl)-3-[3-(1H-imidazol-1-yl)propyl]thiourea.]][[Image:DromeQCActiveSite.png|thumb|300px|left|Figure 4. A comparison between the active sites of ~~Drosophila melanogaster~~ DromeQC crystalized either with a PBD150 inhibitor (right, 4F90) or without (left, 4FWU). Protein loops surrounding the active site are denoted in blue, ~~and~~ a ~~key~~ catalytic Zn2+ ~~is shown as a grey sphere,~~ chelated by three residues ~~shown in~~ light blue. The PBD150 inhibitor (red) involve interactions with W296 (yellow), F292 (green), W176 (beige) and D271 (pink).]]

[[Image:Cropped_dimer.jpg|thumb|400px|right|Figure 2. A 3D graphical representation displaying the homodimer glutaminyl cyclase from ''Drosophila melanogaster'' (PDB: 4F9U). Secondary structure is depicted by red (α-helix) and yellow (β-strand) ribbons, glycosyl groups are coloured pink, while hydrogen bonds between the two monomers are shown by dotted green lines. The active site of QC contains a chelated zinc ion represented by a gray sphere. Also bound to the active site of this crystal structure and depicted as blue is the inhibitor 1-(3,4-dimethoxyphenyl)-3-[3-(1H-imidazol-1-yl)propyl]thiourea.]][[Image:DromeQCActiveSite.png|thumb|300px|left|Figure 4. A comparison between the active sites of DromeQC crystalized either with a PBD150 inhibitor (right, PDB: 4F90) or without (left, PDB: 4FWU). Protein loops surrounding the active site are denoted in dark blue, providing a scaffold for a catalytic Zn2+ (gray) to be chelated by three residues (light blue).The PBD150 inhibitor (red) involve interactions with W296 (yellow), F292 (green), W176 (beige) and D271 (pink).]]

===Topology and Overall Structure<ref name="main"/>===

As shown above, DromeQC is made up of 2 identical, independent monomers that come together to form an asymmetric homodimer (Figure 2). The subunits are connected via 4 hydrogen bonds (Chain ~~1→Chain 2~~: ARG35 NH2→GLU64 OE2, ARG43 NH2→ASN71 O, ARG43 NH2→PHE75 O, ARG43 NH1→PHE75 O) and surface complementarity. The subunits exhibit a globular α/β-hydrolase fold, characterized by a central twisted β-sheet motif consisting of 5 parallel strands (β1 and β3-β6) and an antiparallel β2 strand (~~Figure~~ 3). This β-center is flanked by 9 surrounding α-helices; 2 fill the concave face (α5, α7), 7 fill the convex face (α1- α5, α8, α9) with one helix at the edge (α6) of each monomer. DromeQC is glycosylated (with up to 7 carbohydrate moieties) at the N42 position. These polysaccharide tags ~~increased~~ solubility of DromeQC, and appear to have no affect of protein activity<ref name="main"/>.

As shown above, DromeQC is made up of 2 identical, independent monomers that come together to form an asymmetric <scene name='58/580851/Two_chains/2'>homodimer</scene> (Figure 1). The subunits are connected via 4 hydrogen bonds (Chain A→Chain B: ARG35 NH2→GLU64 OE2, ARG43 NH2→ASN71 O, ARG43 NH2→PHE75 O, ARG43 NH1→PHE75 O) and surface complementarity (Figure 2). The subunits exhibit a globular α/β-hydrolase fold, characterized by a central twisted β-sheet motif consisting of 5 parallel strands (β1 and β3-β6) and an antiparallel β2 strand (Figures 2&3). This β-center is flanked by 9 surrounding α-helices; 2 fill the concave face (α5, α7), 7 fill the convex face (α1- α5, α8, α9) with one helix at the edge (α6) of each monomer. DromeQC is glycosylated (with up to 7 carbohydrate moieties) at the N42 position. These polysaccharide tags increase the solubility of DromeQC, and appear to have no affect of protein activity<ref name="main"/>.

Within each subunit is 1 ~~cysteine~~ bond (C113→C136) linking the β3 strand to the α3 helix. These cysteine residues are situated close to the active site, and are conserved in the human orthologue suggesting a pivotal role in catalysis<ref name="schilling"/>. However, when cysteines were replaced with alanines via site-directed mutagenesis, no kinetic differences were observed<ref name="main"/>. In contrast, ~~this mutation~~ did affect structural differences as determined by thermal unfolding experiments<ref name="main"/>. These results correspond to the structural stabilization of this disulfide bond in hQC, ~~and lack of its~~ effect on kinetic activity<ref>Ruiz-Carrillo, D., Koch, B., Parthier, C., Wermann, M., Dambe, T., Buchholz, M., Ludwig, H., Heiser, U., Rahfeld, J., Stubbs, M. T., Schilling, S., and H. Demuth. (2011) Structures of glycosylated mammalian glutaminyl cyclases reveal conformational variability near the active center. Biochemistry. 50: 6280-6288. [http://dx.doi.org/10.1021/bi200249h DOI: 10.1021/bi200249h]</ref>.

Within each subunit is 1 disulfide bond (C113→C136) linking the β3 strand to the α3 helix. These cysteine residues are situated close to the active site and are conserved in the human orthologue, suggesting a pivotal role in catalysis<ref name="schilling"/>. However, this hypothesis was ruled out when cysteines were replaced with alanines via site-directed mutagenesis; in these experiments no kinetic differences were observed in the DromeQC mutant<ref name="main"/>. In contrast, these mutations did affect structural differences of DromeQC, as determined by thermal unfolding experiments<ref name="main"/>. These results correspond to the structural stabilization of this disulfide bond in hQC, with no apparent effect on kinetic activity<ref>Ruiz-Carrillo, D., Koch, B., Parthier, C., Wermann, M., Dambe, T., Buchholz, M., Ludwig, H., Heiser, U., Rahfeld, J., Stubbs, M. T., Schilling, S., and H. Demuth. (2011) Structures of glycosylated mammalian glutaminyl cyclases reveal conformational variability near the active center. Biochemistry. 50: 6280-6288. [http://dx.doi.org/10.1021/bi200249h DOI: 10.1021/bi200249h]</ref>.

===Active Site===

The active site of DromeQC is located on four loops that lack secondary structure (Figure 4). Using these loops as a scaffold, a catalytic zinc ion is chelated via D131 OD2, E171 OE2, and H297 NE2. Thus, under the absence of substrate or inhibitor, Zn2+ exhibits trivalency (Figure 4). However, when DromeQC was crystalized in presence of a PBD150 inhibitor, Zn2+ was additionally chelated by the PBD150 imidazole moiety<ref name="main"/>. It is plausible that the ~~amide~~ oxygen of glutamine of peptide substrates ~~chelate the zinc ion in a similar fashion, leading~~ to position, polarization, and stabilization for cyclization.

The active site of DromeQC is located on four loops that lack secondary structure (Figure 4). Using these loops as a scaffold, a catalytic zinc ion is chelated via D131 OD2, E171 OE2, and H297 NE2. Thus, under the absence of substrate or inhibitor, Zn2+ exhibits trivalency (Figure 4). However, when DromeQC was crystalized in presence of a PBD150 inhibitor, Zn2+ was additionally chelated by the PBD150 imidazole moiety<ref name="main"/>. In a similar fashion, it is plausible that the γ oxygen of glutamine in peptide substrates chelates Zn2+. This would lead to proper position, polarization, and stabilization of substrate for cyclization.

===Inhibitor binding<ref name="main"/>===

Binding of PBD150 to DromeQC utilizes π-π, arene-H, and hydrogen bonding interactions (Figure 4). The dimethoxyphenyl phenyl group of PBD150 is stabilized by π-π interactions with F292. This phenyl group is highly flexibily, however, as only weak electron density was observed during crystal structure analysis<ref name=“main”/>. Such flexibility could be essential for substrates to cyclize. Also stabilizing the inhibitor is an arene-H interaction made between the imidazole moiety of PBD150 and W296. The first carbon upstream of this imidazole ring forms an additional arene-H contact with W176. Finally, the sulfur contained in the thiourea group makes a hydrogen bond with D271. This sulfur could mimic a carbonyl oxygen in the backbone of a peptide, suggesting a possible substrate binding mechanism.

==Function==

@@ Line 21: / Line 21: @@
-The <scene name='58/580851/Composition/1'>composition</scene> of DromeQC consists of either <span style="font-size:150%">{{Template:ColorKey Composition Protein}}, {{Template:ColorKey Composition Ligand}}</span>, or the <span style="font-size:150%">{{Template:ColorKey Composition Solvent}}</span> in which it was crystallized (water). When DromeQC <scene name='58/580851/Secondary/1'>secondary structure</scene> is colour coated <span style="font-size:150%">({{Template:ColorKey_Helix}}, {{Template:ColorKey_Strand}})</span> the global fold may be visualized nicely. This fold is driven by <scene name='58/580851/Polar/1'>hydrophobic/polar</scene> residues. The majority of <span style="font-size:150%">{{Template:ColorKey_Hydrophobic}}</span> residues lie within the enzyme's interior, consistent with the hydrophobic collapse folding theory. In contrast, most <span style="font-size:150%">{{Template:ColorKey_Polar}}</span> residues reside on the exterior where they contact the polar solvent. Similarly, <scene name='58/580851/Charge/1'>charged residues</scene>, either <span style="font-size:150%">{{Template:ColorKey_Charge_Anionic}}</span> or <span style="font-size:150%">{{Template:ColorKey_Charge_Cationic}}</span>, appear to cluster on the outside of the protein. From analysis of charged residues, it can be seen that a salt bridge may connect the two monomers (Chain A->Chain B: ARG35->GLU64).
+The <scene name='58/580851/Composition/1'>composition</scene> of DromeQC consists of either <span style="font-size:150%">{{Template:ColorKey Composition Protein}}, {{Template:ColorKey Composition Ligand}}</span>, or the <span style="font-size:150%">{{Template:ColorKey Composition Solvent}}</span> in which it was crystallized (water). When DromeQC <scene name='58/580851/Secondary/1'>secondary structure</scene> is colour coated <span style="font-size:150%">({{Template:ColorKey_Helix}}, {{Template:ColorKey_Strand}})</span> the global fold may be visualized nicely. This fold is driven by <scene name='58/580851/Polar/1'>hydrophobic/polar</scene> residues. The majority of <span style="font-size:150%">{{Template:ColorKey_Hydrophobic}}</span> residues lie within the enzyme's interior, consistent with the hydrophobic collapse folding theory. In contrast, most <span style="font-size:150%">{{Template:ColorKey_Polar}}</span> residues reside on the exterior where they contact the polar solvent. Similarly, <scene name='58/580851/Charge/1'>charged residues</scene>, either <span style="font-size:150%">{{Template:ColorKey_Charge_Anionic}}</span> or <span style="font-size:150%">{{Template:ColorKey_Charge_Cationic}}</span>, appear to cluster on the outside of the protein. From analysis of charged residues, it can be seen that a salt bridge may connect the two monomers (Chain A→Chain B: ARG35→GLU64).
@@ Line 30: / Line 30: @@
-[[Image:Cropped_dimer.jpg|thumb|400px|right|Figure 2. A 3D graphical representation displaying the homodimer glutaminyl cyclase from ''Drosophila melanogaster'' (PDB: 4F9U). Secondary structure is depicted by red (α-helix) and yellow (β-strand) ribbons, glycosyl groups are coloured pinks, while hydrogen bonds between the two monomers are shown by dotted green lines. The active site of QC contains a chelated zinc ion represented by a gray sphere. Also bound to the active site of this crystal structure and depicted as blue is the inhibitor 1-(3,4-dimethoxyphenyl)-3-[3-(1H-imidazol-1-yl)propyl]thiourea.]][[Image:DromeQCActiveSite.png|thumb|300px|left|Figure 4. A comparison between the active sites of Drosophila melanogaster DromeQC crystalized either with a PBD150 inhibitor (right, 4F90) or without (left, 4FWU). Protein loops surrounding the active site are denoted in blue, and a key catalytic Zn<sup>2+</sup> is shown as a grey sphere, chelated by three residues shown in light blue. The PBD150 inhibitor (red) involve interactions with W296 (yellow), F292 (green), W176 (beige) and D271 (pink).]]
+[[Image:Cropped_dimer.jpg|thumb|400px|right|Figure 2. A 3D graphical representation displaying the homodimer glutaminyl cyclase from ''Drosophila melanogaster'' (PDB: 4F9U). Secondary structure is depicted by red (α-helix) and yellow (β-strand) ribbons, glycosyl groups are coloured pink, while hydrogen bonds between the two monomers are shown by dotted green lines. The active site of QC contains a chelated zinc ion represented by a gray sphere. Also bound to the active site of this crystal structure and depicted as blue is the inhibitor 1-(3,4-dimethoxyphenyl)-3-[3-(1H-imidazol-1-yl)propyl]thiourea.]][[Image:DromeQCActiveSite.png|thumb|300px|left|Figure 4. A comparison between the active sites of DromeQC crystalized either with a PBD150 inhibitor (right, PDB: 4F90) or without (left, PDB: 4FWU). Protein loops surrounding the active site are denoted in dark blue, providing a scaffold for a catalytic Zn<sup>2+</sup> (gray) to be chelated by three residues (light blue).The PBD150 inhibitor (red) involve interactions with W296 (yellow), F292 (green), W176 (beige) and D271 (pink).]]
 ===Topology and Overall Structure<ref name="main"/>===
-As shown above, DromeQC is made up of 2 identical, independent monomers that come together to form an asymmetric homodimer (Figure 2). The subunits are connected via 4 hydrogen bonds (Chain 1→Chain 2: ARG35 NH2→GLU64 OE2, ARG43 NH2→ASN71 O, ARG43 NH2→PHE75 O, ARG43 NH1→PHE75 O) and surface complementarity. The subunits exhibit a globular α/β-hydrolase fold, characterized by a central twisted β-sheet motif consisting of 5 parallel strands (β1 and β3-β6) and an antiparallel β2 strand (Figure 3). This β-center is flanked by 9 surrounding α-helices; 2 fill the concave face (α5, α7), 7 fill the convex face (α1- α5, α8, α9) with one helix at the edge (α6) of each monomer. DromeQC is glycosylated (with up to 7 carbohydrate moieties) at the N42 position. These polysaccharide tags increased solubility of DromeQC, and appear to have no affect of protein activity<ref name="main"/>.
+As shown above, DromeQC is made up of 2 identical, independent monomers that come together to form an asymmetric <scene name='58/580851/Two_chains/2'>homodimer</scene> (Figure 1). The subunits are connected via 4 hydrogen bonds (Chain A→Chain B: ARG35 NH2→GLU64 OE2, ARG43 NH2→ASN71 O, ARG43 NH2→PHE75 O, ARG43 NH1→PHE75 O) and surface complementarity (Figure 2). The subunits exhibit a globular α/β-hydrolase fold, characterized by a central twisted β-sheet motif consisting of 5 parallel strands (β1 and β3-β6) and an antiparallel β2 strand (Figures 2&3). This β-center is flanked by 9 surrounding α-helices; 2 fill the concave face (α5, α7), 7 fill the convex face (α1- α5, α8, α9) with one helix at the edge (α6) of each monomer. DromeQC is glycosylated (with up to 7 carbohydrate moieties) at the N42 position. These polysaccharide tags increase the solubility of DromeQC, and appear to have no affect of protein activity<ref name="main"/>.
-Within each subunit is 1 cysteine bond (C113→C136) linking the β3 strand to the α3 helix. These cysteine residues are situated close to the active site, and are conserved in the human orthologue suggesting a pivotal role in catalysis<ref name="schilling"/>. However, when cysteines were replaced with alanines via site-directed mutagenesis, no kinetic differences were observed<ref name="main"/>. In contrast, this mutation did affect structural differences as determined by thermal unfolding experiments<ref name="main"/>. These results correspond to the structural stabilization of this disulfide bond in hQC, and lack of its effect on kinetic activity<ref>Ruiz-Carrillo, D., Koch, B., Parthier, C., Wermann, M., Dambe, T., Buchholz, M., Ludwig, H., Heiser, U., Rahfeld, J., Stubbs, M. T., Schilling, S., and H. Demuth. (2011) Structures of glycosylated mammalian glutaminyl cyclases reveal conformational variability near the active center. Biochemistry. 50: 6280-6288. [http://dx.doi.org/10.1021/bi200249h DOI: 10.1021/bi200249h]</ref>.
+Within each subunit is 1 disulfide bond (C113→C136) linking the β3 strand to the α3 helix. These cysteine residues are situated close to the active site and are conserved in the human orthologue, suggesting a pivotal role in catalysis<ref name="schilling"/>. However, this hypothesis was ruled out when cysteines were replaced with alanines via site-directed mutagenesis; in these experiments no kinetic differences were observed in the DromeQC mutant<ref name="main"/>. In contrast, these mutations did affect structural differences of DromeQC, as determined by thermal unfolding experiments<ref name="main"/>. These results correspond to the structural stabilization of this disulfide bond in hQC, with no apparent effect on kinetic activity<ref>Ruiz-Carrillo, D., Koch, B., Parthier, C., Wermann, M., Dambe, T., Buchholz, M., Ludwig, H., Heiser, U., Rahfeld, J., Stubbs, M. T., Schilling, S., and H. Demuth. (2011) Structures of glycosylated mammalian glutaminyl cyclases reveal conformational variability near the active center. Biochemistry. 50: 6280-6288. [http://dx.doi.org/10.1021/bi200249h DOI: 10.1021/bi200249h]</ref>.
 ===Active Site===
-The active site of DromeQC is located on four loops that lack secondary structure (Figure 4). Using these loops as a scaffold, a catalytic zinc ion is chelated via D131 OD2, E171 OE2, and H297 NE2. Thus, under the absence of substrate or inhibitor, Zn<sup>2+</sup> exhibits trivalency (Figure 4). However, when DromeQC was crystalized in presence of a PBD150 inhibitor, Zn<sup>2+</sup> was additionally chelated by the PBD150 imidazole moiety<ref name="main"/>. It is plausible that the amide oxygen of glutamine of peptide substrates chelate the zinc ion in a similar fashion, leading to position, polarization, and stabilization for cyclization.
+The active site of DromeQC is located on four loops that lack secondary structure (Figure 4). Using these loops as a scaffold, a catalytic zinc ion is chelated via D131 OD2, E171 OE2, and H297 NE2. Thus, under the absence of substrate or inhibitor, Zn<sup>2+</sup> exhibits trivalency (Figure 4). However, when DromeQC was crystalized in presence of a PBD150 inhibitor, Zn<sup>2+</sup> was additionally chelated by the PBD150 imidazole moiety<ref name="main"/>. In a similar fashion, it is plausible that the γ oxygen of glutamine in peptide substrates chelates Zn<sup>2+</sup>. This would lead to proper position, polarization, and stabilization of substrate for cyclization.
+===Inhibitor binding<ref name="main"/>===
+Binding of PBD150 to DromeQC utilizes π-π, arene-H, and hydrogen bonding interactions (Figure 4). The dimethoxyphenyl phenyl group of PBD150 is stabilized by π-π interactions with F292. This phenyl group is highly flexibily, however, as only weak electron density was observed during crystal structure analysis<ref name=“main”/>. Such flexibility could be essential for substrates to cyclize. Also stabilizing the inhibitor is an arene-H interaction made between the imidazole moiety of PBD150 and W296. The first carbon upstream of this imidazole ring forms an additional arene-H contact with W176. Finally, the sulfur contained in the thiourea group makes a hydrogen bond with D271. This sulfur could mimic a carbonyl oxygen in the backbone of a peptide, suggesting a possible substrate binding mechanism.
 ==Function==