User:Alexander Rudecki/Sandbox 1

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IntroductionIntroduction

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[1]. It has been shown that DromeQC has a similar overall fold to hQC, as well as a conserved active site[2]. Thus DromeQC is an attractive candidate for transgenic models and mechanistic studies.

DNA-->RNA-->ProteinDNA-->RNA-->Protein

DromeQC is encoded by chromosome 3L, locus 64F4-64F5 in the D. melanogaster genome[3]. It is transcribed into a 1622 nucleotide transcript, containing 5' (36 nucleotides) and 3' (83 nucleotides) untranslated regions [3]. The translated protein contains 340 residues corresponding to a M=38,028 Da[4]. It contains a 27 residue signal sequence, suggesting its involvement in the secretory pathway [1].

StructureStructure

A 3D ribbon model of DromeQC as determined by X-ray crystallography

Drag the structure with the mouse to rotate

DromeQC is a consisting of an A Chain and a B Chain. This homodimer can also be viewed in and representations. The backbone of DromeQC can be easily seen as an . Here the polypeptide progression is depicted by the following template:

 Amino Terminus                 Carboxy Terminus 

The of DromeQC consists of either Protein, Ligand, or Solvent in which it was crystallized (water). When DromeQC is colour coated (Alpha Helices,  Beta Strands ) the global fold may be visualized nicely. This fold is driven by residues. The majority of Hydrophobic residues lie in the interior, consistent with the hydrophobic collapse folding theory. Likewise, most Polar residues reside on the exterior where they contact polar solvent molecules. Similarly, , either Anionic (-) or Cationic (+) appear to cluster on the outside of the protein. From these analyses, it can be seen that a salt bridge connects the two monomers.





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.
Figure 2. Topology of DromeQC[5]

Topology and Overall StructureTopology 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. [2].


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[1]. However, when cysteines were replaced with alanines via site-directed mutagenesis, no kinetic differences were observed[2]. In contrast, this mutation did affect structural differences as determined by thermal unfolding experiments[2]. These results correspond to the structural stabilization of this disulfide bond in hQC, and lack of its effect on kinetic activity[6].

Active SiteActive Site

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 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).

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, Zn2+ exhibits trivalency (Figure 3). However, when DromeQC was crystalized in presence of a PBD150 inhibitor, Zn2+ was additionally chelated by the PBD150 imidazole moiety[2]. 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 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).]]

FunctionFunction

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[7]. 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[1]. 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,[8] N terminal pGlu formation must be post translational due to an essential methionine that initiates translation in all organisms.

Catalytic MechanismCatalytic 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[9]. 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 Zn2+. 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.












ReferencesReferences

  1. ↑ 1.0 1.1 1.2 1.3 Schilling S, Zeitschel U, Hoffmann T, Heiser U, Francke M, Kehlen A, Holzer M, Hutter-Paier B, Prokesch M, Windisch M, Jagla W, Schlenzig D, Lindner C, Rudolph T, Reuter G, Cynis H, Montag D, Demuth HU, Rossner S. Glutaminyl cyclase inhibition attenuates pyroglutamate Abeta and Alzheimer's disease-like pathology. Nat Med. 2008 Oct;14(10):1106-11. doi: 10.1038/nm.1872. Epub 2008 Sep 28. PMID:18836460 doi:http://dx.doi.org/10.1038/nm.1872
  2. ↑ 2.0 2.1 2.2 2.3 2.4 Koch B, Kolenko P, Buchholz M, Ruiz Carrillo D, Parthier C, Wermann M, Rahfeld JU, Reuter G, Schilling S, Stubbs MT, Demuth HU. Crystal Structures of Glutaminyl Cyclases from Drosophila melanogaster Reveal Active Site Conservation between Insect and Mammalian QCs. Biochemistry. 2012 Aug 16. PMID:22897232 doi:10.1021/bi300687g
  3. ↑ 3.0 3.1 DromeQC Gene Card. NCBI. [1]
  4. ↑ DromeQC. UniProt. [2]
  5. ↑ PDB Sum Entry 4F9U. EMBL-EBI. [3]
  6. ↑ 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. DOI: 10.1021/bi200249h
  7. ↑ Goren HJ, Bauce LG, Vale W. Forces and structural limitations of binding of thyrotrophin-releasing factor to the thyrotrophin-releasing receptor: the pyroglutamic acid moiety. Mol Pharmacol. 1977 Jul;13(4):606-14. PMID:196172
  8. ↑ Bernfield MR, Nestor L. The enzymatic conversion of glutaminyl-tRNA to pyrrolidone carboxylate-tRNA. Biochem Biophys Res Commun. 1968 Dec 9;33(5):843-9. PMID:4881333
  9. ↑ Calvaresi M, Garavelli M, Bottoni A. Computational evidence for the catalytic mechanism of glutaminyl cyclase. A DFT investigation. Proteins. 2008 Nov 15;73(3):527-38. doi: 10.1002/prot.22061. PMID:18470930 doi:http://dx.doi.org/10.1002/prot.22061
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