Evaluation of Maltose Binding Protein-Tagged hATR Kinase

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Evaluation of MBP tagged-hATR kinase domain catalytic activity with p53 Ser-15 phosphorylation Rashmi Bhakuni, ALTHAF SHAIK, and Sivapriya Kirubakaran Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00845 • Publication Date (Web): 31 Oct 2018 Downloaded from http://pubs.acs.org on November 6, 2018

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Evaluation of MBP tagged-hATR kinase domain catalytic activity with p53 Ser-15 phosphorylation Rashmi Bhakuni1, Althaf Shaik2 and Sivapriya Kirubakaran1,2 1Discipline 2Discipline

of Biological Engineering, Indian Institute of Technology Gandhinagar, Gujarat, India. of Chemistry, Indian Institute of Technology Gandhinagar, Gujarat, India.

*Corresponding Author: Dr. Sivapriya Kirubakaran, Discipline of Biological Engineering and Chemistry, Indian Institute of Technology Gandhinagar, Simkheda, Palaj, Gandhinagar-382355, Gujarat, India Email: [email protected]

Keywords: DNA damage response, ATR, genome integrity, cell cycle checkpoint, inclusion bodies

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ABSTRACT DNA damage response (DDR) pathways form an integral part of the body’s repair machinery and ATR (ataxia telangiectasia and Rad-3 related) protein kinase is one of the key mediators in the DDR pathway that helps in maintaining genomic integrity. Increasing evidence suggests that inhibition of ATR can help sensitize tumor cells towards combinatorial treatment. However, specific ATR kinase inhibitors have largely remained elusive till date. Despite much interest in the protein for more than a decade, there has been little characterization available for the kinase domain alone, an essential target site for a variety of ATR inhibitors. Here, we report our findings for the bacterial expression, purification and biological characterization of this potentially important recombinant kinase domain, which have further prospects of being considered for structure elucidation studies. Introduction of a solubility partner i.e., maltose binding protein (MBP), at N-terminus of the ATR kinase domain generated a soluble form of the protein i.e., MBP tagged-hATR kinase domain (MBP-ATR-6X His) which was found to be catalytically active, as assessed by substrate p53 Ser-15 phosphorylation (EPPLSQEAFADLWKK). Our results also highlight the prospect of overexpressed recombinant ATR kinase domain utilization in characterization of kinase domain specific inhibitors.


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INTRODUCTION The genome of our body goes through a number of cell cycle events to ensure proper growth, replication, and cell division. As the cell proceeds through different phases of the cell cycle, various checkpoints get activated in response to any kind of genotoxic insult (like replication stress, UV light, ionizing radiation, reactive oxygen species) to the cell1. These checkpoints are essentially specialized regulatory signal transduction pathways responsible for maintaining the integrity of our genome by allowing time for repair 2.

DNA damage response pathways form a part of such complex network signaling at checkpoints while

dealing with detection and repair of damaged DNA by ensuing transient cell cycle arrest 1. Defects in DDR (DNA damage response) components of cancer cells have shed light on the regulators of such pathways to be considered for selective sensitization of cancer cells 3, 4. ATR (ataxia telangiectasia and Rad-3 related kinase) is one such important apical kinase intricately involved in DNA damage response to counter stalled replication forks or single strand breaks5. Reports have demonstrated that knockout of ATR can result in early embryonic lethality in homozygotes suggesting the essentiality of the gene 6. ATR belongs to the large phosphatidylinositol 3 kinase-related kinase (PIKKs) family of serine/ threonine protein kinases, displaying a high level of sequence homology in their kinase, FAT (FRAP-ATM-TRRAP) and FATC (FAT Carboxyl-terminal) domains5,7. FAT and FATC domains act as flanking sites to the Cterminal kinase domain and aid in the regulation of kinase activity 5. ATR gene essentially codes for 2644 amino acids protein having an N-terminus ATRIP (ATR interacting protein) binding site present for its localization and activation 2. Following activation, the 301KDa ATR kinase phosphorylates its immediate downstream target Chk1 (Checkpoint kinase 1) at Ser-317 and Ser-345 residues to initiate cell cycle arrest and DNA repair process5, 3. The 274 (2293-2567 amino acids) amino acid residues long ATR kinase domain acts as the catalytic region of ATR, responsible for phosphorylating major downstream substrates which help in initiating the DNA repair process. Reports indicate that expression of the kinase-dead version


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of ATR leads to abrogation of DNA damage-induced G2/M cell cycle arrest and sensitization of such cells to chemo- or radiotherapy 8, 9, 10. Accordingly, inhibition of ATR using specific kinase inhibitors has been gaining wide importance with ATP-competitive inhibitors constituting a major part of such inhibition strategy 7,

11, 12.

Such inhibitors target active conformation of the kinase and render kinase inactive by

prohibiting its phospho-transfer activity. However, a high degree of sequence homology between the kinase domains of ATR and other PIKK family members pose a significant risk of selectivity while considering the design of such inhibitors 12, 7. Additionally, the large size of the protein kinase makes it difficult to be expressed in bacteria with good yield, to be used further for kinase assays and crystallography study 11. Since the kinase domain (2293- 2567 amino acids) is an essential aspect for assessing ATR activity, we focused on its expression in bacterial system. The characterization of ATR kinase domain can provide insights on its active site, required for considering structure-based drug design of domain-specific inhibitor/s. In this elaborate study, we report the cloning, expression, purification and characterization conditions of the ATR kinase domain from Escherichia coli to overcome ATR kinase size and expression yield concerns. While expression and purification of the full ATR kinase have been widely reported in mammalian cells 13, there still has been no study reported to express the protein in E. coli for its use in characterization of potential specific ATR substrate/inhibitors. A solubility partner, such as MBP, was introduced to overcome solubility and stability issues because of the propensity of kinase domain to go into inclusion bodies when expressed alone 14, 15. The protein of interest was expressed as a recombinant MBP tagged-hATR kinase domain i.e., MBP-ATR-6X His protein (with an N-terminal MBP and a Cterminal 6X Histidine tag), which was further characterized for its catalytic activity using p53 substrate. Studies have validated the role of ATR kinase in DNA damage induced phosphorylation of p53 as the overexpression of a catalytically inactive ATR kinase in human fibroblasts inhibited p53 phosphorylation


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at Ser-15 in response to DNA damage 16. Hence, Ser-15 represents a functionally important residue in the amino-terminal region of p53 protein as a downstream substrate of ATR kinase. The kinase activity of the purified recombinant ATR kinase domain was assessed using commercial ADP-Glo kinase assay kit 17, 18. Our results indicate that the overexpressed recombinant ATR kinase domain was catalytically active and can be potentially used for characterization of kinase domain specific inhibitors.


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Materials and Methods Materials pMalc5X His vector (Cat No. N8114) was obtained from New England Biolabs (Ipswich, MA, USA). The ATR kinase domain, 837 bp long (amino acids 2293-2567) was commercially purchased as ATR kinase domain-pUC57 (ATRkd-pUC57) construct from Merck Miilipore (Darmstadt, Germany). Restriction endonucleases BamHI and EcoRI, Phusion DNA polymerase, T4 DNA ligase, Factor Xa, and amylose resin were purchased from New England Biolabs (Ipswich, MA, USA). Plasmid DNA Purification kit and Gel Extraction kit were purchased from Qiagen (Hilden, Germany). Escherichia coli DH5α (Cat. No. 18265-017) and Rosettagami 2 (DE3) plysS (Cat. No. 71352) competent cells were procured from Invitrogen Corporation (Carlsbad, CA, USA) and Novagen (Darmstadt, Germany), respectively. Ampicillin, Chloramphenicol, Phenylmethyl sulfonyl fluoride (PMSF), Isopropyl β-D-1-thiogalactopyranoside (IPTG), D-glucose, Tris base, Sodium chloride (NaCl), Triton X-100,

Tween

20,

Imidazole,

Acrylamide,

N,N′-Methylenebisacrylamide,

Tetramethylethylenediamine (TEMED), Magnesium chloride, Ammonium Per Sulphate (APS), Sodium Dodecyl Sulphate (SDS), β-Mercaptoethanol (BME), Bromophenol Blue, Coomassie Brilliant Blue R-250, Non-Fat Milk were purchased from Sigma-Aldrich (Darmstadt, Germany). Nuvia Immobilized Metal Affinity chromatography (IMAC) resin, Macro Prep High Q support (ion exchange resin), Immun-Blot PVDF western blotting membrane and Clarity Western ECL substrate were purchased from Bio-Rad (Hercules, California, USA). PD-10 columns and HiLoad 16/600 Superdex 75pg gel filtration chromatography column were purchased from GE Healthcare (Chicago, Illinois, USA). Amicon Ultra concentrator with a 3KDa cut-off filter was obtained from Merck Millipore (Darmstadt, Germany). Anti-MBP antibody (mouse monoclonal; E8032S) was purchased


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from New England Biolabs (Ipswich, MA) and anti-His antibody (rabbit monoclonal; 12698P) was purchased from Cell Signaling Technology (Danvers, MA). Anti-rabbit IgG HRP-linked secondary antibody (Cat. 7074) and anti-mouse IgG HRP-linked secondary antibody (Cat. 7076) were obtained from Cell Signaling Technology (Danvers, MA, USA). ADP Glo-kinase assay kit was purchased from Promega Corporation (Madison, WI, USA). Recombinant p53 substrate (EPPLSQEAFADLWKK) 19 for the kinase assay was custom synthesised from Biotech Desk Pvt. Ltd. (Hyderabad, India). Staurosporine (QG-1880) was purchased from Combi-Blocks (San Diego, USA).

Cloning and Construction of vector containing MBP- and His-tagged form of ATR kinase domain ATR kinase domain corresponds to 274 residues (amino acids 2293-2567) of the C-terminus of ATR kinase protein (2644 amino acids), present between FAT and FATC domains. The DNA sequence coding for ATR kinase domain (ATR, NCBI GenBank: CAA70298.1) was commercially synthesized and codon optimized for expression in E.coli using Merck Millipore services. The kinase domain DNA fragment was cloned in pUC57 vector with BamHI and EcoRI restriction sites at the ends. To construct the C-terminal His tagged construct, ATR kinase domain DNA was PCR amplified using universal M13 forward and reverse primers from ATRkd-pUC57 construct. The amplified fragment was digested with BamHI and EcoRI restriction enzymes and then cloned into the pMalc5X His vector in-frame with the N-terminus maltose binding protein (MBP), Factor Xa site (for protein cleavage) and C-terminus hexahistidine residues (Supplementary figure, S1). The presence of ATR kinase domain insert in the expression construct was confirmed by restriction digestion and DNA sequencing (Supplementary figure, S2).


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Expression of ATR kinase domain in E. coli The prepared construct was chemically transformed with calcium chloride into Escherichia coli Rosettagami 2 (DE3) plysS competent cells, which were plated onto Luria-Bertani (LB) agar plate containing ampicillin (100μg/ml) and chloramphenicol (35μg/ml) and then incubated overnight at 370C. A single colony was picked to inoculate a 10ml primary culture of LB media containing 100μg/ml of ampicillin and the culture was then allowed to grow overnight at 370C at 250rpm. For the ATR kinase domain-pMalc5X His construct expression, starter culture (10ml) was inoculated into 1 liter of LB secondary culture media containing 100μg/ml of ampicillin and 0.2% glucose and cells were allowed to grow until OD600 reached 0.6.-0.8. Recombinant protein expression was then induced using 0.3mM and 0.5mM of isopropyl-β-D-1-thiogalactopyranoside (IPTG) at 370C. The cells were harvested 3 hours after induction by centrifugation and stored in deep freezer (-80ºC) until further use.

Purification of recombinant MBP-ATR-6X His fusion protein The cells harvested by centrifugation were weighed, thawed on ice for 20min and re-suspended using 5ml lysis buffer per gram-wet weight of the cells (40mM Tris-Cl, 500mM NaCl, 10% glycerol, 1% Triton X-100, 5mM imidazole and 1mM PMSF, pH 8.4). The re-suspended cells were lysed by ultrasonication using Vibracell VCX-130 cell disruptor (Sonics and Materials Inc., Newtown, CT, USA) at 40% amp (10sec ON/ 15 sec OFF) while being maintained on ice for 10 minutes. The soluble and insoluble cell lysate were fractionated by centrifugation at 8000rpm for 30 minutes at 40C. The soluble fraction of the cell lysate (prepared after cell lysis) was collected and loaded onto the 4ml NiNTA resin containing column (Bio-Rad Laboratories, CA, USA) pre-equilibrated in 40mM Tris-Cl, 500mM NaCl, 5mM imidazole and 10mM β-ME, pH 8.4 at a flow rate of 1ml/min. The resin was incubated with supernatant for one hour at 40C and then washed with 30-35 column volumes of buffer


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containing 40mM Tris-Cl, 500mM NaCl, 20mM imidazole and 10mM β-ME, pH 8.4 to remove any non-specific binding of proteins. The recombinant fusion protein was eluted using elution buffer containing 40mM Tris-Cl, 500mM NaCl, 300mM imidazole and 10mM β-ME, pH 8.4 at a flow rate of 1ml per minute (Supplementary figure, S3A). The purified fractions were pooled to a volume of around 5ml using Amicon Ultra concentrator unit (Millipore) with a 3KDa cut off filter and then subjected to ion exchange chromatography after performing buffer exchange (20mM Tris-Cl and 50mM NaCl, pH 8.4) with PD10 column (GE Healthcare, Chicago, Illinois, USA). Recombinant MBP-ATR-6X His fusion protein has a theoretical isoelectric point (pI) of 6.42 (as calculated using ExPasy ProtParam online tool). At a pH 8.4 (~2 units greater than its pI), the protein was present majorly in an anionic form and thus anion exchange chromatography was considered as a next purification step. The anion exchange resin was pre-equilibrated in 20mM Tris-Cl and 50mM NaCl buffer before loading the concentrated sample onto the resin. The protein-resin slurry mixture was incubated at room temperature for an hour after which the resin was washed with 10-15 column volumes of buffer containing 20mM Tris-Cl, 50mM NaCl, pH 8.4. Finally, protein was eluted using gradient buffer containing 100mM, 150mM, 250mM, 350mM and 500mM NaCl along with 20mM Tris-Cl (pH 8.4), respectively (Supplementary figure, S3B). Fractions corresponding to the purified protein were concentrated using 3KDa Amicon Ultra concentrator. Next, the fusion protein was purified using size exclusion chromatography. The concentrated protein was loaded on to the preequilibrated HiLoad 16/60 Superdex 75pg gel filtration chromatography column (20 mM Tris-Cl, 50 mM NaCl, pH 8.4). The protein peak was monitored at UV 280 and 260 nm and accordingly the purified protein was eluted, maintaining a flow rate of 1 ml/min throughout the purification. The purified protein fractions were concentrated and analyzed by Coomassie blue staining of a 10% SDSPAGE gel (Supplementary figure, S3C). The protein concentration was quantified using Bradford’s


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assay as well as extinction coefficient method (using the calculated extinction coefficient i.e., 98780 M-1cm-1) 20 21. Protein fractions purified by size exclusion chromatography were concentrated and the final purified protein sample was rapidly frozen using liquid nitrogen for its use in further studies.

Mass spectrometry The expressed recombinant MBP tagged-hATR kinase domain protein sample was analyzed using mass spectrometry (LC-ESI MS/MS) to validate the sequence of the expressed protein with human ATR kinase domain (NCBI database). The protein expressed from ATR-pMalc5X construct was run on a 10% SDS-PAGE gel. The electrophoresed protein sample was stained using 1% Coomassie Brilliant Blue R-250 to visualize the protein. The concerned protein band at 77.29 KDa was excised and subsequently analyzed using LC-ESI MS/MS facility (Proteomics facility, IISc Bangalore). Results were analyzed using Mascot Peptide Mass Fingerprint Server (Matrix Science Inc., MA, USA).

Western blotting Expression of the recombinant ATR kinase domain was confirmed by western blotting using both rabbit monoclonal anti-His antibody and mouse monoclonal anti-MBP antibody. Recombinant purified MBP tagged-hATR kinase domain (~20 μg) was run under denaturing conditions using 10% SDS-PAGE gel along with broad range Precision Plus Protein Kaleidoscope Protein Standards (BioRad Laboratories, CA, USA). The gel was run with 1X Tris-Glycine SDS running buffer. The electrophoresed protein sample was transferred onto an Immun-Blot PVDF western blotting membrane (Bio-Rad Laboratories, CA, USA) using western blotting (90 minutes at 100V, 300mA).


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The membrane was incubated overnight in blocking buffer (5% w/v fat-free milk in 1X Tris-Buffered Saline with Tween-20, TBST) at 40C to reduce non-specific binding. The blotting membrane was then washed 3 times with 1X TBST (for 5 minutes each) and incubated with His-Tag D3I10 XP Rabbit Monoclonal antibody (Cell Signaling Technology, MA, USA; 1:1000 in blocking buffer) at 40C overnight/ anti-MBP monoclonal antibody (New England Biolabs; Ipswich, MA, USA; 1:10000 in blocking buffer) for 4-5 hours at 40C. The membrane after washing with 1X TBST was finally incubated with anti-rabbit IgG HRP-linked secondary antibody (Cell Signaling Technology, MA, USA; 1: 2500 in blocking buffer)/ anti-mouse IgG HRP-linked secondary antibody (Cell Signaling Technology, MA, USA; 1: 2500 in blocking buffer) for 1 hour at room temperature. The protein was visualized using Clarity ECL luminescent substrate according to the manufacturer’s instructions (BioRad Laboratories, CA, USA).

Factor Xa cleavage The recombinant purified MBP-ATR-6X His fusion protein was subjected to Factor Xa treatment to characterize the presence of ~35 kDa ATR kinase domain after protein cleavage. The reaction mixture containing 800μg of fusion protein was incubated with 0.5μg/50μl of Factor Xa enzyme (New England Biolabs, Ipswich, MA, USA) in 20mM Tris-Cl, 50mM NaCl, 1mM CaCl2, pH 6.5 reaction buffer for different incubation periods (maximum of 72 hours) at room temperature. The reaction was terminated by transferring the samples to -200C. Result of the experiment was analyzed using SDS-PAGE gel electrophoresis.


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Circular dichroism (CD) spectroscopy Recombinant MBP-ATR-6X His fusion protein (with a concentration of 100μg/ml) was purified as mentioned above and was finally present in buffer containing 20mM Tris-Cl and 50mM NaCl; pH 8.4 (after buffer exchange). The blank and protein sample were passed through a 0.22μm syringe filter (Merck Millipore, Darmstadt, Germany) before data collection. Far UV CD spectra (190-260nm) was recorded using 1mm path length quartz cell at 250C on a Jasco (J-815) circular dichroism spectropolarimeter (Jasco, Inc., MD, USA). Data from at least three scans was averaged together and blank corrected. The recorded CD spectrum was analyzed using SOPMA and DICHROWEB secondary structure prediction servers for the assessment of its secondary structure composition 22, 23. Alpha helix (positive band at 193 nm and negative bands at 222 nm and 208 nm), beta sheet (positive band at 195nm and negative band at 218 nm) and random coil content (positive band at 212 nm and negative band at 195 nm) give rise to a characteristic spectrum, used to analyze the secondary structure of a protein24.

ADP-Glo Kinase assay The biological activity of the recombinant MBP tagged-hATR kinase domain was estimated using ADP-Glo kinase assay (Promega Corporation, Madison, WI, USA) and the kinase assay was adapted according to the given guidelines 17, 18. In brief, the kinase assay was performed in an opaque white 96-well plate with a reaction volume of 25 µl containing 1 μΜ of recombinant MBP tagged-hATR kinase domain, 1μM of p53 peptide substrate (EPPLSQEAFADLWKK), 100 μM ATP (Promega, Madison, WI) and 1X Kinase Reaction buffer (10mM HEPES-pH 7.5, 50mM NaCl, 10mM MgCl2). The kinase reaction was initiated by adding ATP, incubated for 60 minutes at room temperature, and terminated by adding 25 µl of ADP-Glo reagent (and deplete the unconsumed ATP). After an


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incubation of 45 minutes at room temperature (RT), 50 μl of kinase detection reagent was added to each well and incubated for another 45 minutes in dark at RT, before measuring the newly synthesized ATP using a luciferase reaction. The readout of the experiment was recorded using EnVision multilabel plate reader (Perkin Elmer, Inc., MA, USA). Appropriate control reactions such as without recombinant protein kinase, substrate and ATP were included in the kinase assay. Standard curves with defined ADP/ATP molar ratios were performed and used to convert relative light units into reaction velocities to determine the percentage of ATP-ADP conversion in a kinase reaction.

Statistical analysis The data presented is the mean ± standard error (SE) of n ≥ 2 of two independent experiments. Statistical analyses were performed using GraphPad Prism 6 statistical software program (GraphPad Software Inc., CA, USA). The Michaelis-Menten constant (Km) was determined using MichaelisMenten curve fitting. Dose-response values (EC50) were calculated using a four-parameter logistic nonlinear regression model.


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RESULTS Cloning expression and purification of recombinant ATR kinase domain ATR-pMalc5X construct (ATRkd-pMalc5X His) was successfully cloned for the expression of ATR kinase domain with an N-terminal MBP tag and a C-terminal 6X His tag i.e., MBP-ATR-6X His fusion protein (Figure 1A). The kinase domain was fused to a MBP solubility tag to provide stability to the otherwise unstable kinase domain (as calculated by Expasy ProtParam online tool) 25. Prepared construct was confirmed by DNA sequencing and then over expressed in Escherichia coli RosettaGami2 (DE3) pLysS cells. The human ATR kinase has been overexpressed in minimal yields in mammalian cells for studying its structure and binding properties 13, 26. However, efforts towards obtaining a homogenously soluble and purified ATR kinase domain in a prokaryotic system such as E. coli have not been reported till date. The overexpression of ATR kinase domain will be helpful in studying the inhibition profile of newly designed ATP-competitive inhibitors. The expression level of recombinant MBP-ATR-6X His fusion protein expressed form ATR-pMalc5X His construct was tested using different IPTG concentrations (0.3mM and 0.5mM). Expression of the protein at 37ºC for 3 hours with 0.3mM IPTG induction showed maximum localization of the fusion protein in soluble fraction with comparable amounts of the protein being localized as inclusion bodies, as observed on SDS-PAGE gel (Figure 1B).


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Figure 1: Optimization of recombinant MBP tagged-hATR kinase domain expression in Escherichia coli: (A) Schematic representation of human ATR kinase domain cloned in pMalc5X His expression vector i.e., ATRkd-pMalc5X His construct. (B) The expression of recombinant MBP tagged-hATR kinase domain was induced by 0.3 mM and 0.5 mM IPTG concentration at 37ºC. The control sample was kept induced (i.e., without IPTG) at 37ºC. MW: Molecular weight; kDa: kilo Dalton; IPTG: isopropyl-β-D-1thiogalactopyranoside; S: supernatant; P: pellet.

The expressed recombinant ATR kinase domain was purified from the soluble fraction of the cell lysate using Ni-NTA affinity, anion exchange and then size exclusion chromatography at a yield of ~1mg protein per 4 liters of secondary culture (Figure 2). The purified recombinant protein was analyzed using mass spectrometry and identity of the recombinant MBP-ATR-6X His protein was confirmed by in-gel trypsin digestion (Proteomics facility, IISc Bangalore, Karnataka, India). Results were analyzed by submitting the raw data files to MASCOT Peptide Mass Fingerprint server (Matrix Science Inc., MA, USA) (Figure 3). Cysteine carbamidomethylation and methionine oxidation were included as a fixed and variable modifications, respectively. Tryptic searches were executed with one missed cleavage permissible. With our results, we observed that the expressed recombinant ATR kinase protein was in complete alignment with the native human ATR kinase domain sequence (Supplementary figure, S5A and S5B). The purified protein was also characterized through western blotting using anti-MBP and anti-His primary antibody to affirm the identity of the overexpressed protein (Supplementary figure, S4). The blot with anti-MBP showed two characteristic bands hinting at the spontaneous N-terminal truncation of the recombinant protein. However, such observation remained largely case dependent and was observed only few times. No such additional bands were observed on analyzing the same sample with anti-His antibody.


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Additionally, purification of the fusion protein performed using amylose resin (affinity for the MBP tag) showed no significant binding of the MBP-ATR-6X His fusion protein, indicative of a conformation which does not support MBP (of the fusion protein) binding to amylose (data not shown).


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Figure 2: Purified recombinant MBP tagged-hATR kinase domain from Escherichia coli (after Ni-NTA IMAC, anion exchange chromatography and size exclusion chromatography): (A) Chromatogram of recombinant MBP tagged-hATR kinase domain purified using size exclusion chromatography. (B) The purified protein was run on a 10% SDS-PAGE gel and analyzed by Coomassie blue staining. The recombinant protein gets expressed as 77.29 kDa protein (as indicated on the picture). MW: Molecular weight; kDa: kilo Dalton.

Figure 3: Mass spectrometry of the recombinant MBP tagged-hATR kinase domain fusion protein: The represented figure explains the mass spectrometry analysis of the recombinant MBP tagged-hATR kinase domain using MASCOT Peptide Mass Fingerprint Server (Matrix Science Inc., MA, USA). Cysteine


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Biochemistry

carbamidomethylation and methionine oxidation were included as a fixed and variable modifications, respectively. Tryptic searches were executed with one missed cleavage permissible. The resulting peptide fragment showed complete alignment with native human ATR kinase domain.

Factor Xa cleavage The 77.29 kDa fusion protein was also subjected to factor Xa based cleavage reaction in order to remove the MBP tag from the overexpressed recombinant MBP tagged-hATR kinase domain. Factor Xa, which cleaves after the arginine residue in its preferred cleavage site Ile-(Glu/Asp)-Gly-Arg, was used to cleave the fusion protein into 42.5 kDa MBP and ~35 kDa ATR kinase domain fragment. With this experiment, we observed that the cleavage of the fusion protein increased gradually with extended incubation periods and was found to be the maximum at 72 hours, as analyzed on SDSPAGE gel (Figure 4A). Further, presence of the ATR kinase domain from the cleavage reaction mixture was validated using anti-His antibody (Figure 4B). Attempts to purify the ATR kinase domain (with C–terminal 6X His tag) after factor Xa cleavage resulted in precipitation issues of the protein (data not shown). This result further supported the role of MBP solubility tag in assisting kinase domain stabilization as a fusion protein.


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Figure 4: Cleavage of the recombinant fusion protein using Factor Xa protease: (A) The fusion protein was incubated with Factor Xa (10 μg/ml) for different time periods and cleavage of the fusion protein into MBP (42.5 kDa) and ATR kinase domain (~35 kDa) was analyzed on a 10% SDS-PAGE gel. Control: MBP tagged-hATR kinase domain; hrs: hours. (B) The cleaved MBP and ATR kinase domain (with Cterminal 6X His tag) from Factor Xa reaction were also analyzed with western blotting using anti-MBP (mouse monoclonal) and anti-His (rabbit monoclonal) antibody.


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Biochemistry

Secondary structure determination of the recombinant MBP tagged-hATR kinase domain fusion protein by circular dichroism (CD) We attempted to understand the secondary structure composition of the purified recombinant overexpressed fusion protein using circular dichroism (CD). Circular dichroism is widely used to estimate the alpha helix and beta sheet content of the protein. Far-UV CD spectra (below 260nm) helps to apprehend the secondary structure content in a certain protein structure. Each protein has a unique characteristic spectra depending on their individual conformation and hence, CD can be used to estimate the structure of unexplored proteins such as recombinant ATR kinase domain. Far UV CD data collected for the fusion protein revealed a positive and negative dip at 195 nm and 220 nm, respectively (Figure 5A). Analysis of the observed CD spectrum using DICROWEB online software (Department of Crystallography at Birkbeck College, University of London) suggested that the fusion protein is largely helical (40%), However, it also showed the presence of beta sheet (9%). random coil (20%) and turns (5%) in its secondary structure. The secondary structure composition of the recombinant fusion protein was also assessed using secondary structure prediction server SOPMA, which also revealed the alpha helix and beta sheet content to be 44% and 19%, respectively (Figure 5B). It also calculated the random coil and turns content to be around 31% and 9%. The values obtained from experimental calculations and theoretical predictions were mostly in agreement with each other indicating that recombinant MBP-ATR-6X His fusion protein is largely alpha helical with stretches of beta sheet, random coil or unstructured loop present between helices (Figure 5C). The ability of the recombinant MBP-ATR-6X His fusion protein to fold into a stable secondary structure could be attributed to the addition of the N-terminal Maltose binding protein (MBP) solubility tag which helps to stabilize the otherwise unstable ATR kinase domain (when expressed without a solubility tag).


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Biochemistry


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Figure 5: Estimation of recombinant MBP tagged-hATR kinase domain secondary structure using CD spectroscopic data: The data represented here is an average of at least three scans and blank corrected. Mean residue ellipticity (θ) was analyzed using GraphPad Prism software (GraphPad Software Inc., CA, USA). (A) CD of the recombinant MBP tagged-hATR kinase domain reveals that the protein contained 40% alpha helices, 20% beta sheet, 31% random coil and 9% turns. (B) Theoretical secondary structure analysis of the recombinant MBP tagged-hATR kinase domain using SOPMA software. (C) The table represents the comparison of experimental and theoretical secondary structure calculations.

Assessment of recombinant MBP tagged-hATR kinase domain bioactivity with p53 substrate The activity of the purified recombinant MBP tagged-hATR kinase domain (overexpressed in E.coli) was assessed using a novel ADP-Glo kinase assay. The kinase assay was essentially carried out in two steps, with the first step comprising of kinase reaction termination and depletion of unused ATP. The second step utilizes the ADP generated from kinase reaction for ATP production which is further quantified with a luminescent signal produced as a result of luciferase/luciferin reaction. The quantification of luminescent signal can be directly correlated with the kinase activity (ADP produced from luciferase/luciferin reaction). The entire kinase reaction was carried out at room temperature. An ATP standard curve pertaining to the conversion of ATP to ADP was generated using ADP-Glo kinase assay. The standard curve was used to estimate the amount of ADP produced in the kinase reaction and represent the amount of ATP and ADP available in the reaction at the specified conversion percentage (Supplementary figure, S7).


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Biochemistry

A pilot experiment was performed to monitor the purified recombinant hATR kinase domain activity using

1

μM

MBP

tagged-hATR

kinase

domain,

1

μM

p53

peptide

substrate

(EPPLSQEAFADLWKK) and 100 μM ATP for 60 minutes at room tempature in a solid, white, 96 well plate (lane 1, Figure 6). Also, appropriate negative controls such as recombinant kinase domain with ATP (lane 2, Figure 6), MBP protien (1 μM) with ATP and p53 peptide (lane 3, Figure 6), p53 peptide without MBP tagged-hATR kinase domain (lane 4, Fig 6), and only ATP (lane 5, Figure 6) were included in the experiment. We found that the purified recombinant hATR kinase domain was enzymatically active in presence of p53 peptide as its substrate, with a luminiscence intensity of 11621400 RLUs, when compared with negative control with out the peptide substrate having a luminiscence signal of 3238600 RLUs (Figure 6). Based on this result, we carried out sets of optimization experiments to determine an optimum enzyme concentration for inhibitor screening, Vmax and Km values with respect to p53 and ATP. In the first step towards MBP tagged-hATR kinase domain assay development, we standardised the optimum recombinant hATR kinase domain concentrations required for further screening. Various concentrations of hATR kinase domain (5.377 μM to 0.00525 μM) were incubated with 1 μM of p53 peptide susbtrate and 100 μM ATP for 60 minutes at room temperature. Sufficiently robust signal was achieved with 1.283 μM of recombinant MBP tagged-hATR kd, which resulted in a signal to background ratio of 3 (S/B=3) with 40% ATP hydrolysis (Figure 7A and Supplementary Figure, S8).


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Figure 6: Determination of MBP tagged-hATR kinase domain catalytic activity under various conditions: The overexpressed recombinant MBP tagged-hATR kinase domain was tested for its bioactivity using p53 substrate (EPPLSQEAFADLWKK), containing Ser-15 phosphorylation site. The kinase assay was performed using ADP-Glo reagent (Promega Corporation, Madison, WI, USA) and the quantified luminescent signal was directly correlated with the kinase activity. MBP tagged-hATR kd: Maltose binding protein tagged-human ATR kinase domain; p53: EPPLSQEAFADLWKK; ATP: Adenosine triphosphate; MBP: Maltose binding protein; RLU: Relative light units.


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Biochemistry

Figure 7: Determination of MBP tagged-hATR kinase domain catalytic activity with varying enzyme concentrations: (A) MBP tagged-hATR kinase domain activity assay was performed with various concentrations of hATR kinase domain (5.377 μM to 0.00525 μM), 1 μM p53-peptide substrate (EPPLSQEAFADLWKK) and 100 μM ATP for 60 minutes at room temperature. Curve fitting was done in GraphPad Prism 6.0 software using sigmoidal–dose response, four-parameter logistic nonlinear regression model. (B) Determination of optimum p53 substrate concentration for recombinant hATR kinase domain assay: MBP tagged-hATR kinase domain activity assay was performed with various concentrations of p53-peptide substrate (EPPLSQEAFADLWKK) concentrations (9 to 0.562 μM), 1.283 μM of the fusion protein and 100 μM ATP incubated for 60 minutes at room temperature. The Km value


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of p53 peptide was found to be 0.138 μM. (C) Determination of Km value for ATP. To determine the Km for ATP in recombinant hATR kinase domain assay, the reactions were performed in total volume of 25 μL and incubated at room temperature for 60 minutes. The ATP titration contained 1.283 μM of MBP taghATR kinase domain, 1 μM p53-peptide, and a serial dilution of ATP (0 to 1000 μM). The data for (A) and (B) was ﬁtted to the Michaelis-Menten equation using GraphPad prism 6.0 software, to obtain the indicated Km values for ATP; RLU, relative light unit. Luminescence values (presented in RLUs, or relative light units) in (A), (B) and (C) represent the mean of 2 replicates.

Similarly, different p53 peptide (9 to 0.562 μM) concentrations were incubated with 1.283 μM of recombinant MBP tagged hATR kinase domain. The Km of the substrate was noticed to be 0.138 μM (Figure 7B). For the determination of Vmax and Km values for ATP, 1.283 μM of MBP tagged-hATR kinase domain (fusion protein) was used. The p53 peptide substrate concentration was fixed to 1 μM while different ATP concentrations were used. Michaelis-Menten curve fit resulted in a Km of 389.9 μM for ATP and turnover of ~ 4% ADP for 1.283 μM of kinase domain within 60 minutes (Figure 7C). These values were used to calculate the specific activity of MBP tagged-hATR kinase domain with respect to ATP. The specific activity and kcat values were observed to be 457.25 nmol/min/mg and 0.5196 min-1, respectively. From these observed results, we can conclude that the purified MBP tagged-hATR kinase domain was moderately active with low catalytic efficiency kcat (

Evaluation of Maltose Binding Protein-Tagged hATR Kinase

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