View source for User:Alexander Rudecki/Sandbox 1

==Introduction==

''Drosophila melanogaster'' glutaminyl cyclase (DromeQC but also known as CG10487 CG32412 or Dmel\CG32412) is a globular protein part of the α/β-hydrolase superfamily. DromeQC is an aminoacyltransferase (EC 2.3.2.5) that acts on N-terminal glutamine or glutamate residues, producing a stable cap resistant to protease degradation. The human orthologue of DromeQC (hQC) has been implicated in stabilizing amyloid Aβ peptides involved in neurodegenerative disorders such as Alzheimers<ref name="schilling">PMID: 18836460</ref>. It has been shown that DromeQC has a similar overall fold to hQC, as well as a conserved active site<ref name="main">PMID: 22897232</ref>. Thus DromeQC is an attractive candidate for transgenic models and mechanistic studies.

===DNA-->RNA-->Protein===

DromeQC is encoded by chromosome 3L, locus 64F4-64F5 in the ''D. melanogaster'' genome<ref name="genecard">DromeQC Gene Card. NCBI. [http://www.ncbi.nlm.nih.gov/gene?cmd=Retrieve&dopt=Graphics&list_uids=38663]</ref>. It is transcribed into a 1622 nucleotide transcript, containing 5' (36 nucleotides) and 3' (83 nucleotides) untranslated regions <ref name="genecard"/>. The translated protein contains 340 residues corresponding to a M=38,028 Da<ref>DromeQC. UniProt. [http://www.uniprot.org/uniprot/Q9VRQ9]</ref>. It contains a 27 residue signal sequence, suggesting its involvement in the secretory pathway <ref name="schilling"/>.

==Structure==
<Structure load='4F9U' size='350' frame='true' align='right' caption='A 3D ribbon model of DromeQC as determined by X-ray crystallography' scene='Insert optional scene name here'/>
DromeQC is a <scene name='58/580851/Two_chains/2'>homodimer</scene> consisting of an <span style="font-size:150%"><font color='blue'>A Chain</font></span> and a <span style="font-size:150%"><font color='limegreen'>B Chain</font></span>. This homodimer can also be viewed in <scene name='58/580851/Trace/1'>trace</scene> and <scene name='58/580851/Space_fill/1'>space filling</scene> representations. The backbone of DromeQC can be easily seen as an <scene name='58/580851/N_to_c_rainbow/1'>N to C rainbow</scene>. Here the polypeptide progression is depicted by the following template:
{{Template:ColorKey_Amino2CarboxyRainbow}}
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 <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 in the interior, consistent with the hydrophobic collapse folding theory. Likewise, most <span style="font-size:150%">{{Template:ColorKey_Polar}}</span> residues reside on the exterior where they contact polar solvent molecules. 








[[Image:Cropped_dimer.jpg|thumb|400px|right|Figure 1. 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:QCtopology.gif|thumb|200px|left|Figure 2. Topology of DromeQC<ref>PDB Sum Entry 4F9U. EMBL-EBI. [https://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=4f9u&template=protein.html&r=wiring&l=1&chain=A]</ref>]]

===Topology and Overall Structure===

DromeQC is made up of 2 identical, independent monomers that come together to form an asymmetric homodimer (Figure 1). 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 2). 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"/>.


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>.

===Active Site===
[[Image:DromeQCActiveSite.png|thumb|300px|left|Figure 3. 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).]]
The active site of DromeQC is located on four loops that lack secondary structure (Figure 3). 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 3). 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. 

Binding ofomeQCActiveSite.png|thumb|700px|center|Figure 3. 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).]]

==Function==
[[Image:Cyclase_reaction.png|thumb|500px|right|Figure 4. Cyclization of a terminal glutamine residue via DromeQC.]]
The N-terminus of many proteins (ie gonadotropin releasing hormone and thyrotropin-releasing hormone) contain a pyroglutamic acid (pGlu) residue<ref>PMID: 196172</ref>. A pGlu ‘cap’ protects these proteins against degradation by aminopeptidases, and influences the conformation of the hormone or its associated receptor, leading to their activation<ref name="schilling"/>. Cyclization also leads to decreased basicity in the peptide. Though cyclization of Gln-tRNA to pGlu-tRNA has been shown to occur in papaya latex,<ref>PMID: 4881333</ref> N terminal pGlu formation must be post translational due to an essential methionine that initiates translation in all organisms.    

===Catalytic Mechanism===

DromeQC is the enzyme responsible for this post-translational processing of polypeptides. DromeQC catalyzes the cyclization of N-terminal glutamine, and to a lesser extent glutamate, into pyroglutamic acid (5-oxo-L-proline, or <Glu) (Figure 4). Cyclization occurs via a nucleophilic attack of the α-amine on the γ carbon in the glutamine side chain. The enzymatic mechanism for DromeQC is still undetermined, but it seems plausible that it follows that of its human orthologue. In hQC, the N-terminus of the peptide substrate is inserted into the active site pocket, where the γ amide oxygen chelates the catalytic zinc ion<ref name="mechanism">PMID: 18470930</ref>. This ion-dipole interaction causes carbonyl polarization, making it a better electrophile. To facilitate the reaction, a conserved glutamate (Glu201) acts as both a general base and acid. Glu201 abstracts a proton from the α-amino group, causing it to nucleophilically attack the γ amide oxygen. This produces a tetrahedral intermediate with a charged oxygen that is stabilized by Zn<sup>2+</sup>. Glu201 then protonates the γ amide nitrogen, and an amine group is expelled as the carbonyl reforms. Also essential to this mechanism is a conserved aspartate (Asp248) that coordinates/stabilizes the leaving amine group.  






















==References==
<references/>