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Substrate Specificity In Human Monomeric Carbonyl Reductases - Pilka et al .
Material and Methods
Cloning and Mutagenesis: A human CBR3 clone was obtained from the MGC clone collection, and a synthetic, codon-adapted CBR1 clone was obtained from GenScript Corporation. CBR1 and CBR3 expression construct were cloned by PCR into pNIC28-Bsa4 or p11-Bsa4, which are T7/pET21a derived expression vectors containing Tobacco Etch Virus (TEV) protease cleavable N-terminal hexahistidine tags. All CBR1 and CBR3 mutants were generated from the vector template encoding the wild type (WT) gene by using a site-directed mutagenesis kit (Quick change, Stratagene). Sequences of all wild-type and mutant constructs were verified by DNA sequencing.
Expression and purification of CBR proteins : Expression plasmids were transformed into competent Rosetta E. coli cells. Protein expression was induced at 18°C at an OD 600 = 1 by adding isopropyl-1-thio-β-D-galactopyranoside to a final concentration of 0.5 mM to cultures grown in Terrific Broth, supplemented with 50 µg/ml kanamycin or 100 µg/ml ampicillin at 37°C. Induced cultures were then incubated overnight with shaking at 18°C. Cell pellets were resuspended in 50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM imidazole, 5% glycerol and protease inhibitors (EDTA-free Complete, Sigma). Cells were lysed using a high pressure homogenizer (EmulsiFlex-C5, Avestin), followed by centrifugation at 37,000 x g for 45 min. The supernatant was loaded on an AKTA-Express system (GE/Amersham) and purified using nickel-affinity chromatography on a HisTrap FF 1 ml column (GE/Amersham) and gel filtration on a Superdex 200 column (GE/Amersham) equilibrated in 10 mM HEPES pH 7.5, 500 mM NaCl, 5 % glycerol, 0.5 mM TCEP. For crystallization purposes, CBR3 wild-type fractions from gel filtration were subjected to TEV cleavage overnight at 4°C and the cleaved protein was purified on IMAC-Sepharose (GE/Amersham). The final step of this purification was ion-exchange chromatography on a QHP column (GE/Amersham) using a 0.05- 2 M NaCl gradient in 50 mM HEPES pH 7.5, 0.5 mM TCEP, followed by a subsequent buffer-exchange into gel filtration buffer (as above). All purification steps were analyzed by SDS-PAGE and the molecular weight of purified protein was verified by electrospray mass ionization-time-of flight mass spectrometry (Agilent LC/MSD time-of-flight). Proteins were concentrated to 5- 10 mg/ml in an Amicon Ultra-15 concentrator with a 10 kDa mass cut-off and the final concentration was measured by UV-spectroscopy (Labtech, Nanodrop 1000 spectrophotometer). Proteins were flash-frozen in liquid nitrogen until further use.
Substrate screening of CBR1 and CBR3 proteins: Frozen aliquots of CBR1 and CBR3 enzymes were thawed quickly in water of RT immediately placed on ice. Assays were performed at 30°C in buffer S (50 mM sodium phosphate, pH 6.8, 150 mM NaCl, 1 mM MgCl 2 , 2% (v/v) DMSO). The final assay solution contained 200 nM of protein, 200 µM NADPH and 200 µM of compound. Prior to the start of the experiment, each protein was incubated in buffer containing 1 mM NADPH at 2 µM concentration for 10 minutes at room temperature. A solution comprising NADPH in buffer alone was used for the setup of control experiments for each tested compound. Dilutions of compounds at 10 mM concentrations in DMSO were prepared in 96-well plates and used to set up the assay plate (384-well white PCR plate, Bio-rad), by adding 200 nl of each solution into 7.8 µl of buffer S (STARlet nano, Hamilton). The assay plate was centrifuged (1 min, 1,000 rpm, RT) to collect all solutions in the bottom of the wells. Reactions were performed on 24 wells at a time in a filter-based fluorescence reader (Omega Polarstar, BMG Labtech). After one minute of monitoring the fluorescence intensity (excitation, 355 nm; emission, 460 nm) the reactions were started with injections from the instrument-controlled syringe, of 2 µl/well of protein/NADPH solution (see above). The fluorescence intensity in all 24 wells was then monitored for additional 10 minutes. The next set of reactions were afterwards automatically started and measured via the instrument’s script mode until all wells of the plate were read. In total the time for the measurement of a complete set of triplicates for 96 conditions was approximately 90 minutes. Data were analyzed for the initial rates of activity, by regression in the linear region of the curves as appropriate. Protein-independent, ‘background’ activities were subtracted and corrected for compound effects, e.g. quenching, by normalization to the fluorescence offset that resulted from the injection of NADPH. Specific activities (in µmol/min/mg) were calculated using the molecular weight of the protein, respectively.
Kinetic analysis of CBR proteins: The kinetic measurement for oracin was performed employing a HPLC method (Agilent 1100 Series, Agilent Technologies, Waldbronn, Germany). Samples were incubated for 60 min and reactions were stopped by the addition of 80 µl of 30% ammonia and cooling on ice. The mixtures were extracted twice with 500 µl of ethylacetate and the combined organic phases were evaporated under vacuum. The residue was dissolved in the mobile phase and analyzed by HPLC (mobile phase, 10 mM hexanesulfonic acid and 50 mM triethylamine adjusted to pH 3.3 with H 3 PO 4 ; flow, 1.5 ml/min; 5µM BDS; Hypersil C18 column (4 x 250 mm, 5 µm, Thermo Electron Corporation, Cheshire, UK)). The fluorescence emitted at 418 nm was monitored upon 340 nm excitation. The increase of the product concentration was linear over the measurement time. Catalytic properties for isatin were determined by measuring the decrease in absorbance at 340 nm (Cary 100 scan photometer, Varian, California, USA). A reaction mixture consisted of substrate, 500 μM NADPH, 100 mM Tris–HCl pH 7.4, and enzyme. The enzyme solution was diluted in the corresponding elution buffer, a 7:3 mixture of 10:500 (mM) imidazole buffer, to ensure that substrate consumption was linear over time. Each concentration was measured at least three times. The reaction temperature was held constant at 25°C. After a preincubation time of 2 min 10 μl of enzyme solution were added to 790 μl of reaction mixture. A reference cuvette contained the reaction solution without enzyme. Isatin stock solution was prepared in DMSO. The final concentration of DMSO in the reaction mixture was 10% (v/v). A maximum of 4000 μM isatin was used in the kinetic measurement as the change in absorbance of this concentration still follows Lambert–Beer’s law and no precipitation of isatin occurred. The kinetic constants were calculated by nonlinear regression (Gnuplot 4.2) with a molar extinction coefficient for NADPH of 6.22×103 M −1 cm −1 . For the determination of the kinetic constants for the activity of the enzymes on 1,2-naphthoquinone and 1,4-naphthoquinone a modified version of the protocol used for substrate screening (see above) was applied: In a 96-well pre-plate, 12 concentrations each substrate, covering ranges from 0 to 4 mM or 0 to 7.5 mM of 1,2- and 1,4-naphthoquinone, respectively, were set up in two rows and then aliquoted into the remaining rows to fill the entire plate. This was then used as pre-plate for the assay as described above. The resulting assay concentrations of the two substrates spanned, thus, ranges from 0 to 1 mM or from 0 to 1.875 mM, respectively. Linearity in the protein-independent reduction of compound was observed up to the applied maximum concentrations, thus showing that the compounds were soluble up to that concentration. The resulting data were fitted to the Michaelis-Menten equation using non linear regression (Levenberg-Marquardt) calculated with Gnuplot 4.0 (http://www.gnuplot.info)or by Prism 5.0 (GraphPad Software, Inc.). In cases where a fit was not possible, due to a failure to plateau, a value of k cat / K m was estimated from a linear regression over the initial part of the curve. Very small activites (compared to background) were regarded noise below a threshold for the goodness of fit (R 2 ) of 0.5 (for all other data values of R 2 were 0.9 or higher)
Crystallization of human CBR3: Frozen protein was quickly thawed, and 5 mM NADP was added to the protein aliquot prior to crystallization. A sitting drop consisting of 50 nl of protein solution and 100 nl of well solution was equilibrated against a well solution containing 1.8 M tri-ammonium citrate pH 7.0 at 20°C. Large, irregular crystals that appeared after 24 hrs, were cryo-protected in a mixture of well solution with 25 % glycerol in the presence of NADP before flash-cooling in liquid nitrogen.
Data Collection, Phasing and Refinement: The native dataset was collected on a Rigaku FRE-Superbright generator with R-AXIS HTC area detector. Initial phases were calculated by molecular replacement using the crystal structure of human CBR1 (PDB 1wma) as a model for PHASER [16]. Before the refinement commenced, 5 % of the data was flagged during processing for the calculation of Rfree. The final model was created by alternating rounds of the refinement using REFMAC5 [17] and model building with adding ligand and solvent molecules using COOT [18]. The final statistics for the CBR3 binary complex structure are given in Supplementary information (SI) Table 2.
CBR3 loop modelling: The active site loop of CBR3 was identified and submitted to a search against an ICM v.3.4-9d [28] built-in loop library containing suitable loops with matching loop ends and as close to the loop sequence as possible. The algorithm then inserts the matched loops into the model and modifies the side-chains according to the model sequence. The next step then adjusts the best loops found and keeps a stack of loop alternatives. We have manually browsed through the loop alternatives until identifying a suitable conformation that satisfied the condition of being part of the cofactor binding cavity (as seen in CBR1) and not bearing major atom clashes. The suitable loops were then submitted to local minimisations, with the side chains allowed to move along the chi angles in order to solve the remaining clashes. Upon solution of clashes, the modelled loop was accepted and the resulting model was saved.
Substrate docking: Docking procedures were performed according to the methodology described and implemented in the program ICM v.3.4-9d [28]. Three different protein structures were used in the docking procedure as receptors: human CBR1 (PDB 1wma), human CBR3 (PDB 2hrb) and human CBR3 with the active site loop modelled as a variant of the conformation adopted in human CBR1. Each of the receptors was docked with seven ligands: 1,2-naphthoquinone, isatin, oracin, menadione, metyrapone, oxononenal and NNK. In each docking procedure, grid maps representing different properties of the receptor were computed. During the docking, either one of the torsional angles of the ligand was randomly changed or a pseudo-Brownian move was performed. Each random change was followed by 100 steps of local conjugate-gradient minimization against the grid maps. The new conformation was accepted or rejected according to metropolis criteria using a temperature of 600 K. The length (number of Monte Carlo steps) of the docking run as well as the length of local minimization was determined automatically by an adaptive algorithm, depending on the size and number of flexible torsions in the ligand. Visual inspection was performed for the lowest energy conformations satisfying the absence of clashes after docking.
Note: For further details about this strutcure, please refer to SGC Material and Methods entry for CBR3 .
References
16. McCoy AJ, Grosse-Kunstleve RW, Storoni LC, Read RJ (2005) Likelihood-enhanced fast translation functions. Acta Crystallogr D Biol Crystallogr 61: 458-464.
17. Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53: 240-255.
18. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60: 2126-2132.
28. Abagyan R, Totrov M, Kuznetsov DA (1994) A new method for protein modelling and design: applications to docking and structure prediction from the distorted native conformation. J Comput Chem 15:488-506.
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