Consequently, a general method that is able to provide milligrams of soluble hQC is of great practical importance (Baneyx, 1999 ?). as a target for large-scale NMR and X-ray screening campaigns in the search for new inhibitors of hQC, the X-ray crystal structures of the hQC Y115ECY117E variant and of its adduct with the inhibitor PBD-150 were determined. insect cells (Booth (Ruiz-Carrillo as a thioredoxin-fusion protein (Huang, Liu & Wang, 2005 ?). These expression systems have drawbacks in terms of cost, ease of production or low yield. Consequently, a general Rabbit polyclonal to HMBOX1 method that is able to provide milligrams of soluble hQC is of great practical importance (Baneyx, 1999 ?). A soluble variant of hQC has been designed in order to overcome the tendency of recombinant bacterial hQC to be expressed as inclusion bodies (Castaldo strain BL21 (DE3) by thermal shock (see Table 1 ?). Table 1 Human glutaminyl cyclase hQC-2X production information Source organism BL21 (DE3)Complete amino-acid sequence of the construct produced ASAWPEEKNYHQPAILNSSALRQIAEGTSISEMWQNDLQPLLIERYPGSPGSYAARQHIMQRIQRLQADWVLEIDTFLSQTPEGERSFSNIISTLNPTAKRHLVLACHYDSKYFSHWNNRVFVGATDSAVPCAMMLELARALDKKLLSLKTVSDSKPDLSLQLIFFDGEEAFLHWSPQDSLYGSRHLAAKMASTPHPPGARGTSQLHGMDLLVLLDLIGAPNPTFPNFFPNSARWFERLQAIEHELHELGLLKDHSLEGRYFQNYSYGGVIQDDHIPFLRRGVPVLHLIPSPFPEVWHTMDDNEENLDESTIDNLNKILQVFVLEYLHL Open in a separate window The bacterial culture was grown at 37C in SB medium supplemented with 100?mg?l?1 ampicillin. Protein overexpression was induced with 0.1?mIPTG when the cell density reached an OD600 of 0.6C0.8 and the culture was incubated at 24C for 48?h. After 2?d, the cells were harvested by centrifugation (4000?rev?min?1 for 15?min), resuspended in lysis buffer (50?mTrisCHCl pH 8.5, 150?mNaCl, 20?mimidazole) and disrupted by sonication. The supernatant of the resulting crude extract was collected by centrifugation and further purified by nickel-affinity chromatography similar to the previously described method (Castaldo MES buffer pH 6.5, 1.6?ammonium sulfate as the precipitant solution (Huang, Liu & Wang, 2005 ?). Attempts to crystallize the hQC-2X variant in conditions similar to those for the native enzyme were unsuccessful. Crystals of hQC-2X were obtained using the sitting-drop vapour-diffusion technique (Benvenuti & Mangani, 2007 ?). Drops were prepared by mixing equal volumes (3?l) of 8?mg?ml?1 hQC-2X in 0.1?TrisCHCl pH 7.5, 0.15?NaCl with a precipitant solution composed of 0.2C0.4?ammonium sulfate, 0.1?MES 6 pH.5 (Table 2 ?). The different crystallization condition of hQC-2X compared with those for the native enzyme can possibly be attributed to the changed surface properties of our variant, which cause a different quaternary assembly of the molecules as indicated by the different space group and packing (see 3). Table 2 Crystallization MethodSitting-drop vapour diffusionPlate type24-well platesTemperature (K)277.15Protein concentration (mgml1)8Buffer composition of protein solution100mTrisHCl pH 7.5, 0.15NaClComposition of reservoir solution0.1MES buffer pH 6.5, 0.20.4ammonium ratio and Azatadine dimaleate sulfateVolume of drop6l, 1:1 ratioVolume of reservoir (l)600 Open in a separate window Drops were allowed to Azatadine dimaleate equilibrate at 277.15?K over wells containing 600?l precipitant solution. Crystals suitable for diffraction appeared within one week. Crystals of hQC-2X in complex with the PBD-150 inhibitor ({1-(3,4-dimethoxyphenyl)-3[3-(1PBD-150 dissolved in 1,4-dioxane. Open in a separate window Figure 1 Chemical structure of 1-(3,4-dimethoxyphenyl)-3[3-(1v.7.0.4 (Leslie, 2006 ?) and scaled with (Evans, 2006 ?) from the ()86.38, 149.63, 96.3086.43, 149.54, 96.21 ()96.796.82Mosaicity ()0.520.70Resolution range ()33.02.10 (2.212.10)33.01.95 (2.061.95)Total No. of reflections154214 (22314)257040 (36754)No. of unique reflections66975 (9807)87147 (12723)Completeness (%)95.0 (95.3)99.0 (98.9)Multiplicity2.3 (2.3)2.9 (2.9) factor from Wilson plot (2)9.689.41 Open in a separate window Structures were solved by molecular replacement using (Vagin & Teplyakov, 2010 ?) with a subunit of human glutaminyl cyclase (PDB entry 2afm; Huang, Liu, Cheng edge (see Table 3 ?). The presence of the mutations, Y115ECY117E, was verified using (Murshudov factor (2)13.9914.0Ramachandran plotMost favoured (%)97.296.7Allowed (%)2.83.3 Open in a separate window Manual rebuilding and modelling of the missing atoms into the electron density was performed with (Emsley & Cowtan, 2004 ?). The final model was inspected manually and checked with and (Laskowski (Krissinel & Henrick, 2007 ?; indicates that both the hQC-2X trimer and the dimer of trimers observed in the crystal packing might be stable in solution. However, hQC structures obtained from other sources show a different arrangement of subunits, indicating that the hexameric or trimeric quaternary structures are not physiological. Open in a separate window Figure 2 Crystal packing of the wild-type hQC structure. (conformation of the peptide bond between Asp159, involved in the coordination of the catalytic zinc, and Ser160 is maintained in our double Azatadine dimaleate mutant and is stabilized by a hydrogen-bonding network involving Asp248. The residue Trp207, which closes the catalytic site of the enzyme, forming a wall, assumes a different conformation to that observed in the wild-type structure. Namely, the indole ring is flipped by 180, as observed.