Expression, purification, stability optimization and characterization of human Aurora B kinase domain from E. coli

Archives of Biochemistry and Biophysics 503 (2010) 191–201

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Expression, purification, stability optimization and characterization of human Aurora B kinase domain from E. coli Payal R. Sheth a,⇑,1, Lata Ramanathan a,⇑⇑,1, Ashwin Ranchod a, Andrea D. Basso b, Dianah Barrett a, Jia Zhao c, Kimberly Gray b, Yan-Hui Liu c, Rumin Zhang a, Hung V. Le a a b c

Protein Science Department, Merck Research Laboratories, 320 Bent St., Cambridge, MA 02141, USA Tumor Biology Department, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA Chemistry Department, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 0Z033, USA

a r t i c l e

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Article history: Received 16 April 2010 and in revised form 5 August 2010 Available online 10 August 2010 Keywords: Aurora kinase, Cancer Protein purification Protein aggregation Circular dichroism Thermal-shift assay

a b s t r a c t Aurora B kinase plays a critical role in regulating mitotic progression, and its dysregulation has been linked to tumorigenesis. The structure of the kinase domain of human Aurora B and the complementary information of binding thermodynamics of known Aurora inhibitors is lacking. Towards that effort, we sought to identify a human Aurora B construct that would be amenable for large-scale protein production for biophysical and structural studies. Although the designed AurB69–333 construct expressed at high levels in Escherichia coli, the purified protein was largely unstable and prone to aggregation. We employed thermal-shift assay for high-throughput screening of 192 conditions to identify optimal pH and salt conditions that increased the stability and minimized aggregation of AurB69–333. Direct ligand binding analyses using temperature-dependent circular dichroism (TdCD) and TR-FRET-based Lanthascreen™ binding assay showed that the purified protein was folded and functional. The affinity rank-order obtained using TdCD and Lanthascreen™ binding assay correlated with enzymatic IC50 values measured using fulllength Aurora B protein for all the inhibitors tested except for AZD1152. The direct binding results support the hypothesis that the purified human AurB69–333 fragment is a good surrogate for its full-length counterpart for biophysical and structural analyses. Ó 2010 Elsevier Inc. All rights reserved.

Introduction Aurora kinases play a critical role in regulating mitotic processes including mitotic entry, centrosome maturation, and bipolar spindle formation [1,2]. Dysregulation of Aurora kinase functions results in aneuploidy and tumorigenesis, making this class of kinases as attractive oncology therapeutic targets [3–6]. The preclinical data on VX680 compound, a pan Aurora inhibitor, showed tumor regression in different animal models of cancer thus validating Aurora kinase as bonafide oncology targets. Several Aurora inhibitors patents have emerged in the recent years and ongoing recent publications from multiple companies highlight the high level of interest in Aurora as an anticancer biological targets [4,5]. There are three mammalian members in the Aurora protein family, Aurora A, B and C. The two major Aurora proteins, Aurora A and Aurora B, share high sequence conservation in the kinase domain (71% sequence identity). The residues involved in binding of ⇑ Corresponding author. Fax: +1 617 499 3814. ⇑⇑ Corresponding author. Fax: +1 617 499 3814. E-mail addresses: [email protected] (P.R. Sheth), [email protected] (L. Ramanathan). 1 Both authors have contributed equally to this publication. 0003-9861/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2010.08.004

the adenine ring in Aurora A and B ATP2 binding pocket are identical. In spite of the high sequence conservation in the catalytic regions, the two proteins have distinct subcellular localization and biological functions. Aurora A is implicated in centrosome maturation and separation, while Aurora B plays a critical role in cytokinesis, in addition to its role in mitosis. Activation of Aurora A is triggered allosterically by binding of an activator TPX2 [7,8]. Recent crystal structure determination of the Aurora A: TPX2 complex provided a structural basis for understanding the activation of Aurora A by TPX2 [9]. The N-terminal segment of TPX2 was shown to bind to the small lobe of Aurora A. In the presence of the activator, the Aurora A protein demonstrated an extended active conformation of the activation loop that harbors Thr288, a site that needs to be autophosphorylated for rendering the Aurora A protein fully active [10]. Similar to Aurora A, the activation of Aurora B occurs by binding of an 2 Abbreviations used: SDS, sodium dodecyl sulfate; ATP, adenosine 50 -triphosphate; CD, circular dichroism; DMSO, dimethyl sulfoxide; Hepes, N-(2-Hydroxyethyl)piperazine-N0 -(2-ethanesulfonic acid); DTT, dithiothreitol; ESI–MS Electro Spray Ionization Mass Spectrometry; LC–MS, liquid chromatography mass spectroscopy; TCEP, Tris(2carboxyethyl)phosphine hydrochloride; CD, circular dichroism; TdCD, temperaturedependent circular dichroism; TdF, temperature-dependent fluorescence; SEC, sizeexclusion chromatography; DLS, dynamic light scattering; AmOAc, ammonium acetate; NaCl, sodium chloride.

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activator, INCENP [11]. The highly conserved IN-box region of INCENP binds and activates Aurora B. Recent biochemical and structural studies have highlighted the differences in the activation mechanism of Aurora A and B [12–14]. INCENP was shown to activate Aurora B by a two-step mechanism wherein INCENP only partially activated Aurora B kinase, and the full activation was contingent on phosphorylation of a conserved Thr–Ser–Ser (TSS) motif at the C-terminus of the protein. The Xenopus Aurora B: IN-box segment structure that was recently solved corroborated the biochemical data that suggested differences in the activation mechanisms of the Aurora A and Aurora B proteins [15]. INCENP-bound Aurora B, in a binding mode that was distinct from TPX2 binding to Aurora A. INCENP was shown not to make any direct contacts with the activation loop of Aurora B making it likely that INCENP promotes the extended conformation of the Aurora B activation loop via an allosteric mechanism [15]. While the Xenopus structure of Aurora B has shed some light on the activation mechanism of the protein, the corresponding crystal structure of human Aurora B protein is still lacking. Furthermore, comparison of the human apo Aurora B structure versus human INCENP-bound Aurora B structure is needed to fully understand the structural basis of activation of Aurora B upon INCENP binding.

There are several well-characterized Aurora B kinase inhibitors that are under evaluation for their therapeutic potential (for example, VX-680, AZD1152, MLN8054, CYC116 and PF3814735) [4,5,16– 20]. The IC50 or apparent inhibition constant (Kiapp) values for some of the inhibitors have been reported utilizing the full-length Aurora B enzyme, however, the structural basis of the inhibitor binding to Aurora B is largely unknown due to the lack of structural data for the human enzyme. To our knowledge, no Aurora B direct binding studies have been reported for the inhibitors. A full understanding of Aurora B inhibition requires knowledge of structure as well as the thermodynamics of the ligands binding to the kinase domain of the protein. For these studies, however, it is imperative to have milligram quantities of purified protein. In order to address this gap in the field, we cloned a construct of human Aurora B kinase domain for Escherichia coli expression. The domain boundaries of the designed Aurora B construct were selected using the X-ray structure of the Xenopus ortholog as a starting point (Fig. 1) [15]. Initial protein preparations showed that the human Aurora B fragment had very poor solution behavior properties thus requiring buffer optimization. The thermal stability of Aurora B kinase domain (Aur69–333) was characterized over a wide variety of solution conditions to define its stability profile. The results of these studies

Fig. 1. Comparison of Xenopus and Human Aurora B and AurB69–333 construct design. (a) Sequence alignment of human and Xenopus Aurora B proteins. The sequence alignment was performed using CLUSTAL W [36]. (b) The schematic of Aurora B domains and the construct used for this study are highlighted. The area marked as T69-R333 corresponds to region retained in the AurB69–333 construct. The activation loop Thr232 in the human Aurora B and the corresponding Thr248 in the Xenopus Aurora B is highlighted.

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led to the identification of salting agents that confer optimal stability and solubility. Ammonium acetate was selected as the salt additive of choice taking into consideration its common use as a volatile buffer component for dissolution and chromatography of proteins. Its application facilitated the isolation, purification, concentration and storage of AurB69–333, and allowed for extensive characterization of inhibitors by biochemical and biophysical techniques. AurB69–333 bound known Aurora inhibitors with similar affinity as the full-length enzyme. AZD1152, a selective Aurora B inhibitor was the only compound that showed marked difference in the binding affinity between AurB69–333 and full-length Aurora B. Notably though, the compound bound the AurB69–333 with TdCD Kd of 82 nM while its affinity for full-length Aurora A was 10-fold lower, implying that certain degree of specificity is retained in the truncated kinase domain fragment. Our data point to the discovery of a human Aurora B fragment that can be used as a surrogate for its full-length counterpart for structural studies. The identification of such a fragment is especially significant in light of missing structural and biophysical data for the human Aurora B protein. Materials and methods Materials VX680, AZD1152, MLN8054, CYC116 and PF3814735 were synthesized at Merck Research Laboratory. Their identity was confirmed by NMR and LC–MS. These inhibitors were selected for study because they represent well-characterized Aurora inhibitors in the literature. ATP used in this study was obtained from Sigma (St. Louis, MO). The purity of the nucleotides was found to be >90% by LCMS. Expression and purification of Aurora B kinase domain fragment 69– 333 (AurB63–333) from E. coli The kinase domain fragment of human Aurora B (amino acids 69–333) was cloned into pDEST14 for bacterial expression as Nterminal hexahistidine fusion protein with a TEV protease site for cleaving the tag. The nucleotide sequence was confirmed by DNA sequencing (Genewiz Inc., S. Plainfield, NJ). The proteins were expressed in E. coli BL21 (DE3) (Novagen, San Diego, CA) cells for 18 h at 16 °C with 1 mM IPTG. Initial purification carried out in presence 0.3 M NaCl resulted in low to negligible amounts of AurB69–333 yields, therefore, all subsequent purification preparations were done at high salt concentrations as described below. For the purification, the bacterial pellet was lysed in 25 mM HEPES pH 7.5, 1 M NaCl, 10% glycerol, 1 mM TCEP, 10 mM MgCl2, 1 ml/L protease inhibitor cocktail III (EMD Biosciences, San Diego, CA). After lysis using a microfluidizer, the lysate was clarified by ultracentrifugation (100,000g for 1 h at 4 °C) and loaded onto a Ni–NTA agarose column prequilibrated with lysis buffer. The protein was eluted with 0–250 mM imidazole gradient. The fractions containing AurB69–333 protein were pooled and dialyzed against lysis buffer (without protease inhibitors). TEV protease was added to the dialyzed material at 1:50 M ratio (for cleaving the hexahistidine tag) and the cleavage reaction was allowed to proceed overnight at 4 °C. The cleaved AurB69–333 protein was separated from the uncleaved protein and the TEV-protease by Ni–NTA chromatography. The cleaved AurB69–333 did not bind the column, while the hexahistidine tagged TEV, and uncleaved AurB69–333 was retained on the Ni–NTA column. The AurB69–333 was further purified with S75 gel-filtration column (GE Healthcare, Piscataway, NJ). Fractions that showed >95% pure AurB69–333 based on SDS–PAGE analyses were pooled. The concentrations of AurB69–333 were determined

in 6 M GdnHCl using UV spectrophotometry and an extinction coefficient at 280 nm of 33140 M1 cm1 based on amino acid sequence. LC–MS analyses The purified Aurora B protein was buffer exchanged to 10 mM NH4HCO3 with 300 mM NaCl using 3 K MW cutoff filter (Millipore, Carrigtwohill, Ireland). The sample was then reduced by incubating with 10 mM DTT at 60 °C for 30 min. Sequencing grade trypsin (Promega, Madison, WI) was then added at 1:25 w/w to the protein sample for digestion. After incubation at 37 °C for 14 h, the samples were diluted for LC–MS analysis. Peptide mixtures were analyzed by nano LC ESI MS/MS in data dependent acquisition mode. Chromatography was performed using a nano 2D HPLC system (Ekisigent, Dublin, CA). The peptide samples were loaded by autosampler onto a C18 trap column (0.3  50 mm, Dionex, Sunnyvale, CA) with 5% B (0.1% formic acid in 97% ACN) at 10 lL/min for 5 min. The peptides were then separated by a nanobore picofrit column (C18, 75  150 lM, 100 Å, New Objective, Woburn, MA) using a 120 min gradient from 5% to 95% B at a flow rate of 350 nL/min, where solvent A was 0.1% formic acid with 3% ACN in HPLC grade water. Eluted sample was analyzed by LTQ-Orbitrap mass spectrometer (Thermo, Waltham, MA) equipped with nanoelectrospray ion source (Picoview PV500, New Objective, Woburn, MA). The spray voltage was set to 1.9 kV with sheath gas turned off. The data dependent acquisition mode was performed by acquiring one full scan mass spectrum in FT mode (Rs = 60,000), followed by MS/MS of the top five most intensive peptide peaks (2+ and 3+ ions) in ion trap with dynamic exclusion enabled. The m/z range is 300–1800. Eighty-three percentage of sequence coverage was obtained from proteolysis. Temperature-dependent fluorescence (TdF) measurements A 10-fold dilution was made of the NeXtal anions and cations suites (Qiagen, Hilden, Germany) (see Table 1) in 0.22 lm filtered

Table 1 Buffer and salt components of the thermal-shift assay screen. Buffer Cation screen Sodium acetate pH 4.6 MES pH 6.5 Sodium cacodylate pH 6.5 HEPES pH 7.5 Imidazole pH 7.5 BICINE pH 8.5 TRIS.HCl pH 8.5

Anion screen Sodium acetate pH 4.6 MES pH 6.5 HEPES pH 7.5 TRIS.HCl pH 8.5

Salt Ammonium acetate (3.5 M, 1.75 M) Ammonium chloride (3.5 M, 1.75 M) Calcium chloride (2.2 M, 1.1 M) Lithium acetate (2.5 M, 1.25 M) Lithium chloride (4 M, 2 M) Magnesium acetate (2 M, 1 M) Magnesium chloride (3.5 M, 1.75 M) Potassium acetate (4 M, 2 M) Potassium chloride (2.2 M, 1.1 M) Sodium chloride (3.2 M, 1.6 M) Zinc acetate (1.2 M, 0.6 M) Zinc Sulfate (1.6 M, 0.6 M) K/Na tartrate Sodium acetate (2.5 M, 1.25 M) Sodium bromide (3.5 M, 1.75 M) Sodium fluoride (0.6 M, 0.3 M) Sodium formate (3.5 M, 1.75 M) Sodium malonate (2.4 M, 1.2 M) Sodium nitrate (3.5 M, 1.75 M) Sodium phosphate, Potassium phosphate (0.9 M each, 0.45 M each) Sodium succinate (1 M, 0.5 M) Sodium sulfate (0.75 M, 0.37 M) Sodium thiocyanate (2.4 M, 1.2 M) Tri-sodium citrate (1.2 M, 0.6 M)

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HPLC grade water using a 1 ml deep well plate resulting in a 100 mM buffer and a 10-fold dilution of the salt. A working solution of 500 Sypro orange (Invitrogen Corp., San Diego, CA) in 100% DMSO was prepared from the stock 5000 solution. The screening buffer was further prepared by diluting 500 working solution of Sypro orange by 100 fold to obtain a screening buffer with 5 Sypro orange and 1% DMSO. The screening buffer was placed on ice. 100 lM of AurB69–333 protein in 25 mM HEPES, 500 mM NaCl, pH = 7.5 and 1 mM DTT was thawed from storage at 80 °C on an ice bath. The protein was spun at high speed for 5 min and the supernatant quantified with the Bradford assay. A 200-fold dilution of the stock protein was made into an aliquot of the above prepared screening buffer resulting in a sample consisting of 0.5 lM of protein, 100 mM of buffer, 10-fold dilution of the salt, 5 Sypro orange, 0.2 mM DTT and 1% DMSO. Twenty microliter of the sample was pipetted into a white 96-well PCR plate (Abgene, Epsom, UK) and sealed with flat ultra clear caps (Bio-Rad Laboratories Inc., Hercules, CA). The plate was kept on ice. Fluorescence based thermal-shift assays have been conducted with both customized and off the shelf RT–PCR instruments and the methods have been described previously [21–25]. The instrument used for these studies was Chromo4 RT–PCR instrument (Bio-Rad Laboratories Inc., Hercules, CA) equipped with a Peltier element block, four LEDs for illumination and four filtered photodiodes for detection. The instrument was programmed and data was acquired using the Opticon monitor 2 software. The prepared plate was removed from ice and placed into the programmed instrument and started immediately. The temperature was ramped from 20 to 80 °C in 0.2 °C increments. The temperature was allowed to stabilize with a 200 ms delay before reading. The fluorescence signals were acquired with excitation and emission wavelengths centered at 490 and 560 nm, respectively. A customized program using a non-linear least square method based on the generalized reduced gradient (GRG2) algorithm was used to fit the protein unfolding model published in Matulis et al. [24]. The fluorescence intensities of Sypro orange dye is generally linearly dependent on temperature. The following parameter were floated during the fitting process: Y intercepts for the intensity of Sypro orange in both the native and denatured protein (Yn and Yd), their slopes (Mn and Md), the midpoint of melting (Tm) and enthalpy at Tm (DHm). The heat capacity at Tm (DCp) was kept constant. Temperature-dependent circular dichroism (TdCD) For stability comparison, AurB69–333 protein in 25 mM HEPES, pH 7.4, 10% glycerol, 1 mM MgCl2, 1 mM TCEP with either 1 M AmOAc or 1 M NaCl was 10 lM with final AmOAc and NaCl concentration at 250 mM. Unless otherwise mentioned, AurB69–333 purified in presence of 1 M AmOAc was used for all the ligand binding analyses. For TdCD experiments, ellipticity was monitored at 227 nm as a function of temperature with a 1 mm pathlength cell (Jasco Inc., Great Dunmow, UK). The scan rate was 0.5 °C per min with a 4 s response time and 30 s equilibration between measurements. Stock protein was diluted to 8–10 lM with 25 mM HEPES, pH 7.4, 300 mM AmOAc, 10% glycerol, 1 mM MgCl2, 1 mM TCEP. Compound binding was tested at 50 lM. The final concentration of DMSO in TdCD assay was 1%. Data was analyzed using the Jasco software to calculate protein melting temperatures (Tm) and the enthalpy of unfolding DHu. The protein melting temperatures were reported as the average from two to three separate experiments. The relationship between ligand binding and protein stability as detected by changes in the midpoint of unfolding (Tm) has been well-documented [26] and [27], and Kd values can be estimated from the DTm determined by temperature-dependent circular dichroism (TdCD) [28]. Eq. (1) [28] was used to calculate Kd values

for inhibitor binding to Aurora B69–333. The ligand binding constant (KL (T)) can be calculated at any temperature (T) by the following equation:       DHL ðTÞ 1 1 DC pL T T ln K L ðTÞ ¼ K L ðT m Þexp þ1 þ  R R T Tm Tm Tm ð1Þ where Tm is the midpoint of unfolding in the presence of ligand, DHL is the enthalpy of binding, DCpL is the ligand binding heat capacity and KL (Tm) is the ligand binding constant at Tm. If estimates for both DHL and DCpL are available, then the ligand binding constant, KL (T), can be calculated at any temperature T (assuming that the heat capacity term is temperature independent). KL (Tm) can be calculated by the following equation: K L ðT m Þ ¼

fexpfðDHuðT 0 Þ=RÞð1=T m  1=T 0 Þ þ ðDC pu =RÞ½lnðT m =T 0 Þ þ ðT 0 =T m Þ  1g  1g ½LT m  ð2Þ

where T0 is the midpoint of unfolding for the unliganded protein, Tm is the midpoint of unfolding in the presence of ligand, DHu is the enthalpy of protein unfolding, DCpu is the heat capacity associated with protein unfolding and [LTm] is the free concentration of ligand at Tm. Unless otherwise specified, DHL values were assumed to be 7 kcal/mol and DCpL was set to zero for TdCD-determined KL (T) [29]. In non-ideal systems, the loss of secondary structure in TdCD as a function of temperature is due to both structural unfolding and irreversible protein aggregation. Large proteins such as AurB69–333 exhibit aggregation at high temperatures at high concentrations required for TdCD. As a result, the observed unfolding profile is a reflection of structural unfolding as well as aggregation. However, previous studies have suggested aggregation to be a slower process compared to the relatively faster native-to-unfolded reaction [30]. Therefore, in application to AurB69–333 unfolding, we assume the aggregation step is much slower than the native-to-unfolded reaction. Dynamic light scattering (DLS) The hydrodynamic radius of the AurB69–333 protein was measured by dynamic light-scattering (DLS) measurements. AurB69–333 protein purified in 25 mM HEPES, pH 7.4, 10% glycerol, 1 mM MgCl2, 1 mM TCEP with either 1 M AmOAc or 1 M NaCl was used for the DLS experiments. The DLS analyses were performed at 4 °C on 50 lL protein samples at 1 mg/ml concentration using a DynaPro DP801 instrument (Protein Solutions Inc., Charlottesville, MA). Molecular mass values were calculated based on 10 readings using the protein dynamics analysis software. Diffusion coefficients, particle radii and weights were corrected for buffer viscosity and refractive index. The buffer viscosity and refractive index estimates were made based on the values selected from the software. Molecular weight determination by size-exclusion chromatography (SEC) SEC analyses were performed at 4 °C on an analytical size exclusion column (Superdex 75, 1  30 cm) equilibrated in 25 mM HEPES pH 7.4, 300 mM AmOAc or 300 mM NaCl, 10% (v/v) glycerol, 1 mM TCEP, and 1 mM MgCl2. To determine the molecular size of AurB69–333, a gel-filtration calibration kit (Bio-Rad Laboratories, Herculus, CA) was used for molecular weight standards. The marker protein mixture was each injected onto the column and a standard curve between the molecular weight and the elution time was calculated. Based on the elution volume of AurB69–333, the solution molecular weight of the complex was calculated from the standard curves.

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IC50 measurements using Aurora B IMAP kinase assay The IMAP technology (IMAP, Molecular Devices) was used for the determination of substrate phosphorylation by Aurora B. Briefly, fluorescently labeled TAMRA-PKAtide peptides (5TAMRAGRTGRRNSI-NH2) were phosphorylated in a 384-well plate setup kinase reaction. Addition of the IMAP binding system induced specific binding of the phosphorylated substrates that were detected by fluorescence polarization or time-resolved fluorescence resonance energy transfer (TR-FRET). The full-length Aurora A and B enzymes were purchased from Invitrogen (Invitrogen Corp, San Diego, CA). The assay was setup as 20 lL reaction in 10 mM Tris pH 8, 10 mM MgCl2, 0.01% Tween 20, 1 mM DTT, 100 nM TAMRA-PKAtide and 25 nM Aurora B or 8 nM Aurora A. The reaction was initiated by the addition of 50 lM ATP. For IC50 measurements, the compounds were added to the assay mix at fixed concentration with final DMSO concentration of 1%. The reaction was allowed to continue for 2 h after which beads were added. The beads were incubated for additional 2 h before plate was read. All kinase reactions were performed in the linear range for reaction time and enzyme concentration and at an ATP concentration close to the Km of the Aurora B protein. Each kinase assay was validated with staurosporine as a positive control. For IC50 determinations, dose–response curves were plotted from inhibition data generated each in duplicate, from 8 point serial dilutions of inhibitory compounds. Concentration of compound was plotted against enzyme activity. To generate IC50 values, the dose–response curves were then fit to a standard sigmoidal curve and IC50 values were derived by non-linear regression analysis. Due to the unreliability of IC50 values below half the enzyme concentration, enzymatic IC50 values of potent compounds were reported as <13 nM and <4 nM for Aurora B and A enzymes, respectively. IC50 measurements using Lanthascreen™ binding assay IC50 values for test compounds were determined using the commercial Lanthascreen™ Eu Aurora kinase binding assay from Invitrogen. Assay set-up was done as described by the manufacturer (Invitrogen Corp., San Diego, CA). Briefly, the time-resolved fluorescence resonance energy transfer assay (TR-FRET) was performed in white, low volume 384 well plates (Corning). Each well contained 5 nM kinase, 2 nM Eu-anti-His antibody and 10 nM kinase tracer 236 in kinase buffer A (50 mM Hepes pH 7.5, 10 mM MgCl2, 1 mM EDTA, 0.01% Brij-35), varying amounts of test compounds and 1% residual DMSO. The binding assay was incubated for 1 h at room temperature. The signal was measured at 665/ 620 nm emission ratio over a 200 ls window following a 100 ls post-excitation delay on a PherastarPlus plate reader. All assays were performed using three replicates. The 12 point sigmoidal dose–response curves were each fitted using GraphPad Prism software from the inhibition data generated. Results Construct design and expression of AurB69–333 in E. coli Aurora B is an important oncology target. The structure of Xenopus Aurora B kinase domain in complex with IN-box region of INCENP was recently solved [15]. While Sessa et al. were successful in producing Xenopus Aurora B kinase domain using E. coli [15], reports of the corresponding human version are still lacking in the literature. As a result, the structural basis of regulation and inhibition of human Aurora B has remained largely elusive. The domain boundaries (amino acid 69–333) of the Aurora B kinase domain construct used for our studies were defined based on the crystal

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structure of its Xenopus counterpart (Fig. 1). The designed construct provides an opportunity to characterize human Aurora B protein, which in contrast to Aurora A, was relatively less-studied with respect to its biophysical and structural properties. Although there is high sequence conservation between the catalytic cores of Aurora A and Aurora B proteins, several inhibitors have shown surprisingly high specificity towards either Aurora A or Aurora B. The human AurB69–333 construct showed high expression levels in E. coli. However, our initial purification experiments using buffers containing 300 mM NaCl concentrations yielded AurB69–333 that was aggregated and unstable as a result of poor solubility. A significant effort aimed at solubilizing the protein using common detergents and other additives such as glycerol proved futile (data not shown). Based on these results, we concluded that AurB69–333 was a good candidate for sparse matrix buffer and salt optimization. The goal of the screen was to identify buffers and/or salts that would stabilize AurB69–333 and make it less susceptible to aggregation and precipitation. The thermal-shift assay is a high-throughput assay that can measure perturbations in protein thermodynamic stability. The high-throughput nature of the assay and low protein requirements made it an ideal choice for AurB69–333 buffer screening initiative. The thermal-shift assays were initially developed for drug discovery to allow rapid affinity ranking of ligands from compound libraries. The assays have also been consistently used as a secondary screen for measuring ligand binding during both lead identification and optimization stages of drug discovery. More recently, the use of the assay in buffer optimization for crystallography studies were reported [21,22]. Temperature-dependent fluorescence (TdF)-based optimization of AurB69–333 buffer conditions Fig. 2a shows typical denaturation curves obtained for AurB69– in the buffer screen. A total of 192 conditions with varying pH, anions and cations were tested in the temperature-dependent fluorescence (TdF) setup for AurB69–333. Our TdF measurements utilized Sypro orange as the fluorescent probe. Sypro orange is an environmentally sensitive dye that has low quantum yields in aqueous environments, but is highly fluorescent in non-polar environments with low dielectric constants such as hydrophobic areas within proteins. If the protein is largely folded and has no surface exposed hydrophobic patches, there would be low fluorescence emission at room temperature from the dye. Protein unfolding, as a function of temperature, would expose buried hydrophobic patches resulting in significant increase in fluorescence emission by the dye. The midpoint of the AurB69–333 folding-unfolding transition provided the Tm values. The thermal denaturation profiles of AurB69–333 in the presence of ammonium acetate (AmOAc), sodium chloride (NaCl), potassium chloride (KCl), magnesium and zinc acetates, MgOAc and ZnOAc, respectively, are depicted in Fig. 2a. The effect of various salts can be analyzed by inspecting the two important segments of the melting profile: the initial baseline near room temperature that represents the apparent ‘‘native” state, and the slope of thermal unfolding near Tm. At pH 7.5 and in the presence of either NaCl and KCl, AurB69–333 has high fluorescence baselines and a Tm of 38 °C (Fig. 2a). Under similar pH and buffers, ammonium acetate conditions show significantly higher Tm values (46 °C), a much sharper denaturation transition, and low baseline fluorescence, which is more typical of well-behaved proteins in TdF. While MgOAc showed the highest Tm of 53 °C, the denaturation transition was atypical with a very high baseline. ZnOAc conditions gave a non-discernible melting transition. Fig. 2b illustrates the effect of different salts on the Tm of AurB69–333. At pH 7.5, addition of chloride salts such as NaCl, LiCl, and KCl, resulted in lower Tm than acetate salts. The screens indicated the following general hierarchy for anions: 333

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a 300 2.2 M AmOAc

45

1.6 M NaCl 1.1 M KCl 1.0 M MgOAc

200

1.2 M ZnOAc

Tm (°C)

RFU (x1E3)

250

50

b

40

150

35

100 40

60

30

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K 2M Cl 3. LiC 2M l N a 1. 0 75 .6M Cl M N Na aF F 0. orm 75 at M e 2. 25 Na M S0 4 A 2. mO 5M A c Li O 1. 2M 2M Ac K K O N aT Ac ar ta ra te

20

c

2. 2M

Temperature (°C)

55

d

55

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Tm (°C)

50 45

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4.5M AmOAc 2.25M AmOAc

35

40 0

2

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[AmOAc]

7

9

8

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Fig. 2. Identification of optimal buffer for AurB69–333 protein. (a) Thermal melting curves for AurB69–333 protein. The individual curves AurB69–333 in the presence of AmOAc, NaCl, KCl, MgOAc, and ZnOAc are shown. (b) Thermal stabilization as measured by Tm in the presence of selected ions. (c and d) The effect of AmOAc concentration (c) and pH (d) on AurB69–333 Tm. The AmOAc Tms were measured in Hepes pH 7.5 buffer. The pH dependence was tested at 2.25 M and 4.5 M AmOAc as shown in the figure.

Folding assessment of AurB69–333 using temperature-dependent circular dichroism (TdCD) In order to ascertain whether the increased stabilization of AurB69–333 protein in AmOAc versus NaCl-containing buffers was not due to TdF assay-related artifacts, the Tm of AurB69–333 protein in the presence of AmOAc and NaCl were compared in a circulardichroism-based thermal denaturation assay (Fig. 3). In the TdF assay setup, the fluorescence is dependent on binding of the dye to the hydrophobic sites of the protein. Thus the dye binding equilibrium could have an impact on Tm measurements. The TdCD assay setup is free of such potential dye artifacts since the thermal denaturation monitoring probe in TdCD is intrinsic to the protein (circular dichroism signal from proteins secondary structure). Fig. 3 illustrates the thermal unfolding profile of AurB69–333 in buffers containing AmOAc and NaCl. Fig. 3 shows that the purified

0 0.25M NaCl 0.25M AmOAc

CD (mdeg)

  Tartrate > OAc > SO2 in inducing higher 4 > Formate > F > Cl Tm, lowering initial baseline fluorescence, and thus increasing the stability of AurB69–333. These studies allowed for the identification of ammonium acetate as an alternative to sodium chloride for Aur69–333 purification. In contrast to KNaTartrate or other acetate salts, ammonium acetate is a volatile buffer component that can be removed by lyophilization, and has been used extensively in protein formulation and chromatography. Consistent with what was observed for other salts, increasing AmOAc concentrations also increased protein stability (Fig. 2c). The thermal stability of AurB69–333 in the presence of ammonium acetate was pH-sensitive at lower AmOAc concentrations (Fig. 2d). The protein was most stable at 2 pH units below its calculated pI of 9, i.e. pH range of 6.5–7.5. In general, the results of the screen indicated the following: (1) the Tm of Aur69–333 increased with increase in salt concentrations; (2) the protein was mostly stable in the pH range of 7–8 as no changes in Tm could be detected; (3) decreasing pH and salt concentrations together had the most adverse effects on protein stability.

-10 Tm (ºC)

-20

NaCl

<30

AmOAc

35

-30 20

30

40

50

60

Temperature (°C) Fig. 3. TdCD analyses of purified AurB69–333. (a). CD thermal denaturation curve for AurB69–333 in ammonium acetate (h) and NaCl (s). Ellipticity (mdeg) was measured at 227 nm as a function of temperature (22–75 °C). Protein concentration was 10– 12 lM. The experiments were performed in 25 mM HEPES pH 7.4, 300 mM NaCl, 20% glycerol, 1 mM TCEP. The temperature was increased at 0.5 °C per min in a 1 mm path-length quartz cuvette.

AurB69–333 protein lost secondary structure in response to increasing temperature in a sigmoidal fashion as expected for a native-like protein that unfolds in a cooperative manner. The midpoint of the unfolding transition, Tm, was <30 °C and 35 °C in 250 mM NaCl and AmOAc, respectively. The absence of a steady initial baseline for NaCl precludes the calculation of an exact Tm. The data using an alternative assay (Fig 3) thus confirmed that the addition of ammonium acetate substantially increases the Tm for AurB69–333. Solution behavior of AurB69–333 Based on our TdF buffer screen results, AurB69–333 protein was purified in the presence of AmOAc and NaCl for comparison. The overall yields of the purified protein were 2-fold higher when ammonium acetate was used instead of sodium chloride in the gel-filtration and storage buffers. The total yield for AurB69–333

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1

2

3

4

5

6

7

shown). The hydrodynamic radius of AurB69–333 was measured by dynamic light scattering (DLS) measurements (Fig. 5b–c). DLS measurements indicated that AurB69–333 in the presence of ammonium acetate showed a hydrodynamic radius of 3.5 nm, which is 2-fold smaller than the 6.4 nm value observed with sodium chloride in the buffer conditions. The calculated solution molecular mass of AurB69–333 based on the DLS measurements, and assuming a globular shape, was 43 kDa and 260 kDa for AmOAc and NaCl conditions, respectively. The expected molecular mass of the hexahistidine cleaved version of AurB69–333 protein is 30, 955 Da. The AurB69–333 protein (in AmOAc buffer) was also subjected to sizeexclusion chromatography to assess its oligomerization state. The protein eluted at a volume corresponding to an apparent molecular mass consistent with a monomeric form. There was a trace amount of Aurora B protein in the fractions corresponding to the void volume of the Superdex gel-filtration corresponding to 2–5% of the total protein (Fig. 5a). Thus, the protein preparation in ammonium acetate yielded largely homogenous AurB69–333 protein with no significant aggregation.

8

200 116 97 66 45 31 21 14 6

Purification Step

Total Protein (mgs)

Fold Purification

Crude Lysate

10,000

1

Ni-NTA

60

100

Tev-cleavage and Size Exclusion

40

150

Mass spectrometry analyses of purified AurB69–333 Mass spectrometry results showed the purified AurB69–333 had a molecular mass of 31, 036 Da (Fig. 6a), which corresponds to 81 ± 10 Da greater than the expected molecular mass for the hexahistidine-tag cleaved version of the AurB69–333 protein (30, 955 Da). The mass difference was indicative of potential phosphorylation at a single site. In order to identify the phosphorylation site within the protein, phosphopeptide mapping analysis was performed. Phosphorylation was detected only on the residue Thr232 in peptide Arg230–Arg248 (Fig. 6b). No apo form of the peptide was observed implying the residue is fully phosphorylated.

Fig. 4. Coomassie-stained SDS–PAGE analysis of the various steps employed in the purification of AurB69–333 from E. coli. Lane 1: low molecular weight protein markers (Bio-Rad Broad Range). Lane 2: Crude lysate (30 lgs). Lane 3: Soluble lysate (30 lgs). Lane 4: Ni–NTA eluant. Lane 5–8: AurB69–333 from gel-filtration column with increasing loads (2.5 lgs, 5 lgs, 10 lgs and 15 lgs).

was 4 mg/L of E. coli culture at >95% purity by SDS–PAGE analyses (Fig. 4, lane 6–8). Purified AurB69–333 had the expected amino acid sequences based on N-terminal sequencing results (data not

a 100

0.5

mAU

Kav

0.4 0.3 0.2 0.1

50

0.0 3.5

3.9

4.4

4.9

Log MW

AurB Standards

0 0

10

20

30

Volume (mL)

AmOAc

c 100 % Mass

% Mass

b 100 50

0

NaCl

50

0

0.01

0.1

1

10

1000 10000 100000

100

0.01

0.1

1

Radius (nm)

10

100

1000 10000100000

Radius (nm)

Item

R (nm)

% Pd

MW-R (kDa)

% Mass

Item

R (nm)

% Pd

MW-R (kDa)

% Mass

Peak

3.5

14.8

43

100%

Peak

6.4

14.4

260

100%

Fig. 5. Determination of apparent molecular mass of AurB69–333 by size-exclusion chromatography and dynamic light scattering. (a) SEC elution profile as measured by absorption at 280 nm (red trace) of the purified AurB69–333 using the Superdex 200 (5  150 mm) column calibrated with known gel-filtration standards (blue trace). Molecular weight of the standards on the SEC trace, starting from left, 670 kDa (1), 158 kDa (2), 44 kDa (3), 17.5 kDa (4) and 1.35 kDa (5). (b and c) Size distribution histogram of AurB69–333 in ammonium acetate (b) and NaCl (c) obtained by dynamic light scattering. (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)

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Enzymatic analyses of purified AurB69–333 The AurB69–333 protein construct retains the intact kinase domain and the purified protein was phosphorylated on Thr232 on the activation loop. Using the IMAP assay setup to detect phosphorylation of fluorescently labeled TAMRA-PKAtide peptides (TAMRA-GRTGRRNSI-NH2), the enzymatic activity of AurB69–333 was compared with the full-length Aurora B. While the full-length Aurora B had significant catalytic activity at 26 nM, AurB69–333 was devoid of any measurable specific activity towards the TAMRAPKAtide peptide substrate at the concentrations tested (Fig. 7). These results are consistent with what has been reported for the AurB69–333 activation mechanism [11,15]. The differences in the specific activity of the full-length Aurora B and AurB69–333 could also be due to differential Km for the peptide substrate. This is consistent with what has been reported for the AurB69–333 activation mechanism [11,15]. Binding of inhibitors to AurB69–333 using TdCD and Lanthascreen™ binding assay Since the enzyme activity of AurB69–333 towards the exogenous substrate could not be measured, we sought alternative methods to verify appropriate folding and functionality of our construct. A test for proper folding is the ability of a protein to bind known cofactors or ligands. TdCD can be used to detect binding of such compounds. The additional stabilizing interactions created between the ligand and the protein allow a protein to become more resistant to thermal unfolding relative to the unliganded apo protein [24,26]. Therefore, changes in the protein Tm upon ligand bind-

250 DMSO Staurosporine

200 IMAP reading

150 100 50

D K

D 8

nM

K 20

4

nM

K 10

nM 52

26

nM

K

FL

D

D

0 nM

The residue Thr232 lies on the activation loop of Aurora B kinase domain and has been previously shown to be autophosphorylated. Thr232 of human Aurora B is equivalent of the activation loop Thr248 of Xenopus Aurora B. Activation loop phosphorylation is a common mechanism of controlling kinase activation. The equivalent Thr248 in Xenopus Aurora B kinase domain was also seen to be phosphorylated when purified from E. coli in complex in INCENP [15]. The Xenopus Aurora B kinase-dead mutant was shown to be unphosphorylated on Thr248, implying that the phosphorylation of the activation loop Thr248 was due to autocatalysis [15]. Thus, the AurB69–333 protein appeared to have undergone adventitious autophosphorylation during the expression or purification process in the absence of INCENP.

26

198

Fig. 7. Enzymatic activity of purified AurB69–333. The enzymatic activity of fulllength Aurora B (FL) and AurB69–333 (KD) was compared using the IMAP assay setup as described in the Methods section. For the full-length Aurora B enzyme, the IMAP reading was measured at 26 nM enzyme concentration in the absence (grey) and presence of 100 lM staurosporine (black). The IMAP reading for AurB69–333 was measured at 26 nM, 52 nM, 104 nM, and 208 nM enzyme concentrations for comparison.

ing (DTm) correlate with the affinity between the protein and the ligand. We hypothesized that the purified AurB69–333 should, if folded correctly, have the typical bilobular kinase domain fold with intact ATP site architecture capable of retaining some affinity for these inhibitors. We sought to investigate whether the truncated kinase domain fragment of Aurora B (AurB69–333) was capable of binding known Aurora inhibitors. Using TdCD we investigated the binding of Aurora inhibitors to AurB69–333. The five inhibitors PF3814735, VX680, MLN8054, CYC116 and AZD1152, were added at 5-fold concentration excess over AurB69–333 (10–12 lM) resulting in DTm of 12, 10, 9, 9 and 7 °C, which correspond to calculated Kd of 3, 17, 37, 37 and 82 nM, respectively (Fig 8a and Table 2). The affinity rank order was therefore PF3814735 > VX680 > MLN8054  CYC116 > AZD1152. The results indicate that the purified truncated kinase domain fragment was capable of binding the known inhibitors with nM affinity (Table 2). Since the TdCD Kds were calculated assuming a constant DHL of 7 kcal/mol, an alternative Lanthascreen™ direct binding assay was employed to generate binding affinities of the Aurora inhibitors for the AurB69–333 construct. The Lanthascreen™ binding IC50 for VX680 (20 nM), AZD1152 (98 nM), MLN8054 (22 nM), CYC116 (58 nM) and PF3814735 (9 nM) were comparable to the calculated TdCD Kd for these inhibitors for AurB69–333 (Table 2, Fig. 8b). The rank order observed for

Fig. 6. Mass spectrometry analyses of AurB69–333. (a) LC–MS of intact Aurora B protein. The deconvoluted mass is 31036 Da. The expected mass is 30955 Da. The mass difference is 81 Da (b) MS/MS spectrum (Orbi/IT) of peptide Arg230-Arg248, RKTMCGTLDYLPPEMIEGR, T232 is phosphorylated. Observed m/z = 764.0206 (3+), MH+ = 2290.0462. Theoretical mass with one phosphorylation: 2290.0430 (MH+). Delta mass 1.4 ppm.

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a

0

c

CD (mdeg)

VX680 -20

MLN8054 PF3814735 30

40

50

N

60 H3C

Temperature (°C)

N

N

N

N

Cl

N

F

O

S

H3C

0.08

AZD1152 F

N

N

O

VX680 N

AZD1152 MLN8054

N

N

N

H2N

CYC116

0.02

F

O

O

0.04

N

N

CYC116

0.06

N

O

N

b 0.10 Emission Ratio

NN

N

-40 20

OH N

N

N

O

F N

N

CYC116

-30

F

F

CH3

AZD1152

MLN8054

PF3814735

VX680

apo

-10

S

N

N

PF3814735

HO

N

N

CH3

N

O

N

H3C

0.00 -2

4 0 2 log[Compound] (nM)

6

Fig. 8. Inhibitor binding to purified AurB69–333 using TdCD and Lanthascreen™ binding assay. (a) CD thermal denaturation curves for AurB69–333 in the presence of inhibitors are shown. The plot curves are depicted as follows: no inhibitor (s); +VX680 (h); +AZD1152 (4); +MLN8054 (.); +CYC116 (e); +PF3814735 (x). Ellipticity (mdeg) was measured at 227 nm as a function of temperature (15–75 °C). Protein concentrations was 10–20 lM, inhibitor concentration was 50 lM. The temperature was increased at 0.5 °C per min in a 1 mm path-length quartz cuvette. (b) The affinities of the several ATP-competitive kinase inhibitors of Aurora B for AurB69–333 were obtained using a competitive displacement TR-FRET assay. Binding assays were performed with various concentrations of each inhibitor as the competitor. Data from a representative set of assays are shown for VX680 (h); AZD1152 (4); MLN8054 (.); CYC116 (e); PF3814735 (x). (c) The chemical structures of the inhibitors used in this study are shown.

Table 2

Direct binding analyses

Enzymatic analyses

TdCD (Aur BT69–R333)

apo VX680 AZD1152 MLN8054 CYC116 PF3814735

Lanthascreen™ IC50 (nM)

IMAP IC50 (nM)

Tm (°C)

DTm (°C)

TdCD Kd (nM)

Aur B T69-R333

Aur B full-length

Aur A full-length

Aur B full-length

Aur A full-length

35 45 43 44 44 47

10 7 9 9 12

17 82 37 37 3

20 98 22 58 9

13 12 21 27 3

7 >1000 1 117 13

<13 <13 30 52 <13

4 >3000 <4 64 <4

Standard error values were from two to three experiments: DTm, 5–10%; Lanthascreen™ IC50, 5–10%; IMAP IC50, 3–10%.

TdCD Kds PF3814735 > VX680 > MLN8054 > CYC116 > AZD1152 was also largely preserved for the Lanthascreen™ binding IC50 data. These results conclusively indicate that the purified AurB69–333 is capable of binding known Aurora inhibitors with nM affinity. Comparison of inhibitor binding affinities of AurB69–333 and the fulllength Aurora B Although the observed inhibitor-mediated Tm shifts for AurB69–333 were large and significant and the calculated TdCD Kds were comparable with the Lanthascreen™ binding IC50s for AurB69–333, we sought to determine whether inhibitor binding affinities measured for AurB69–333 were representative of the full-length Aurora B protein. The commercially available active Aurora B protein that was purified from insect cells provided an opportunity for benchmarking. Therefore, we sought to compare the inhibitor TdCD Kd and Lanthascreen™ IC50s of AurB69–333 to the IMAP IC50s and Lanthascreen™ binding IC50s in the presence of inhibitors with the full-length version of the protein as a way to determine the equivalency of the two constructs in inhibitor recognition. The inhibitor

IC50 data from the IMAP assay and the Lanthascreen™ binding assay for the full-length human Aurora B are shown in Table 2. Consistent with the TdCD and Lanthascreen™ binding assay results for AurB69–333, the compounds bound and inhibited full-length Aurora B with IC50s in the nanomolar range. In the enzymatic assay, VX680, AZD1152 and PF3814735 showed the lowest IMAP IC50 values (<13 nM) with the full-length Aurora B enzyme. The Lanthascreen™ binding IC50s of the full-length Aurora B were also consistent with the enzymatic IMAP IC50 values for the inhibitors (Table 2). Furthermore, the affinities of the inhibitors for the AurB69–333 were mostly comparable with the full-length Aurora B in the Lanthascreen™ binding assay. The only compound that showed differential binding affinity in the Lanthascreen™ binding assay for the full-length Aurora B and AurB69–333 was AZD1152. AZD1152, which bound AurB69–333 with TdCD Kd of 82 nM and Lanthascreen™ IC50 = 98 nM demonstrated enzymatic IMAP IC50 of <13 nM and Lanthascreen™ IC50 = 12 nM for the full-length Aurora B protein. These results indicate that certain key interactions for AZD1152 that are present in context of the full-length Aurora B protein are lost in the AurB69–333 construct, although the com-

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pound does bind the truncated kinase domain with double-digit nM affinity. The exact source of these interactions is unknown and is a subject for future structural studies with the enzyme inhibitor complexes. It is noteworthy that AZD1152 is a selective Aurora B inhibitor [31]. Quite possibly, the binding modes are sufficiently distinct for AZD1152 in the active full-length and the truncated inactive AurB69–333 proteins. Specificity of the Aurora inhibitors In order to compare the inhibitors specificity, the IMAP IC50 and Lanthascreen™ binding IC50 values for the five inhibitors were measured with the full-length Aurora A enzyme (Table 2). AZD1152, which is an Aurora B-specific inhibitor, bound Aurora A with Lanthascreen IC50 of >1000 nM, a >10-fold higher value compared to AurB69–333 Lanthascreen™ IC50 of 98 nM, and an >80-fold higher value compared to full-length Aurora B enzyme Lanthascreen™ binding IC50 of 12 nM (Table 2). The IMAP IC50 of AZD1152 for full-length Aurora A was >3000 nM consistent with low affinity binding of the compound to the Aurora A enzyme (Table 2). These results indicate that the purified AurB69–333 kinase domain fragment retains certain specificity for AZD1152 albeit some key interactions are lost compared to the full-length Aurora B enzyme. MLN8054, which has been reported as an Aurora A-specific inhibitor showed 20-fold lower Lanthascreen™ IC50 for full-length Aurora A (1 nM) compared to the full-length Aurora B (21 nM) and the AurB69–333 (22 nM) construct (Table 2). VX680, and PF3814735 showed comparable binding affinities between the full-length Aurora A, Aurora B and the truncated AurB69–333 construct. Similarly, the Lanthascreen™ IC50 values for CYC116 binding to Aurora A full-length, Aurora B full-length, and AurB69–333 were within 2-fold of each other implying comparable affinity for the compound between the different proteins. These results further confirm that the truncated AurB69–333 produced from E. coli cells is fully functional with respect to recognition of well-known inhibitors. Discussion Aurora kinases play a critical role in mitosis and completion of cell division. Although Aurora A and B have high sequence conservation in their kinases domains and the residues lining the ATP binding pocket, their functions in mitosis are quite distinct [32]. Aurora B is important for chromosome condensation via phosphorylation of histone H3, bipolar spindle formation, and cytokinesis. Many Aurora inhibitors cause the characteristic loss of phosphohistone H3, mitotic arrest and cytokinesis failure. Accordingly, the effect of pan Aurora inhibitors is believed to be a result of inhibition of Aurora B [33]. Thus, Aurora B is an important oncology therapeutic target, and yet information on the molecular basis of inhibition of human Aurora B kinase activity is largely lacking. The present study describes, for the first time, the preparativescale expression and purification of human Aurora B protein using E. coli expression system. The recombinant protein provides a versatile tool for understanding the architecture of the kinase domain and for deciphering the mechanism of inhibition of Aurora B protein. The human Aurora B construct (AurB69–333) that was designed based on the Xenopus ortholog [15] was overexpressed in E. coli, albeit as aggregated and unstable protein. The differences in solution behavior of Xenopus and human Aurora B constructs is especially intriguing considering high sequence identity between the two constructs. The purification and crystallization of truncated kinase domain fragment of Aurora A have also been extensively described in the literature and the protein has good solution behavior properties. The high-throughput buffer screening strategy using thermal-shift assay yielded acetate salts as AurB69–333 stabilizers, and

thus enabled production of a well-behaved protein preparation that was suitable for biophysical analyses. The screens indicated + + + the following hierarchy, in general, for cations NHþ 4 > Na > Li > K 2    and Tartarate > OAc > SO4 > Formate > F > Cl for anions. The results are mostly consistent with Hofmeister series where the effect of anions predominate [34]. The utility of thermal-shift assay in buffer optimization of problematic proteins is thus highlighted. The simplicity and high-throughput nature of the assay makes it especially attractive for such formulation studies. Aurora B protein has basal kinase activity that is amplified several-fold in the presence of its binding partner INCENP [11]. We found that, although AurB69–333 purified from bacterial cells was phosphorylated on Thr232 of the activation loop, the protein was not catalytically competent in phosphorylating the exogenous peptide substrate that was tested. The peptide substrate could however, be phosphorylated by the full-length Aurora B enzyme. The difference in the enzymatic activity of the full-length Aurora B and the truncated AurB69–333 remains be understood. Nonetheless, two hypotheses could be formulated to explain the activity differences between the two constructs. The activity differences could either stem from differential affinity for the peptide substrate between full-length Aurora B or AurB69–333 or due to differences in the catalytic site conformation or kcat of the enzymes. If the lack of activity in AurB69–333 is indeed due to differential peptide substrate binding and not ATP binding and catalysis, the construct could still function as a valid surrogate for the full-length for interrogating the inhibitor binding site. Therefore, we sought alternative techniques to determine appropriate folding and functionality of the purified protein with respect to inhibitor binding. Direct binding assays that do not require protein to be enzymatically active, such as thermal denaturation and Lanthascreen™ binding assay, can provide valuable information of the affinity of inhibitors [28,35]. The ability to measure the binding of inhibitors to truncated enzyme constructs that are not amenable for enzymatic characterization is especially important in being able to identify smaller fragments of the protein that would be suitable for structural studies such as X-ray crystallography. Although several Aurora inhibitors have been described in the literature, the direct binding parameters associated with these inhibitors are largely unknown. Using TdCD, we determined that the isolated kinase domain of Aurora B, AurB69–33, was capable of binding a panel of known Aurora inhibitors with nanomolar affinity. The relative potencies of these inhibitors were also investigated using this assay setup. TdCD analyses confirmed that the AurB69–33 was capable of binding the known inhibitors as seen by 7–12 °C increase in the Tm of the protein in the presence of the compounds. The dissociation constants can be calculated accurately using the observed Tm values if the binding enthalpy of the different chemotypes is available. Due to inadequate solubility of the compounds, ITC experiments aimed at measuring binding enthalpy were not feasible. Therefore, assuming a constant DHL of 7 kcal/mol, the TdCD-derived Kd (20 °C) values, for the inhibitors, were calculated for comparison with the IC50 values that were derived using the full-length Aurora B protein. The binding enthalpy value of 5 to 7 kcal/mol gives TdF/TdCD Kd (20 °C) values that are within 2–3-fold of the ITC Kd values [23,28]. The AurB69–333 was also used in the Lanthascreen™ binding assay setup to measure the binding affinity of the five inhibitors to the truncated kinase domain. Indeed, the Lanthascreen™ binding IC50’s for the inhibitors using the AurB69–333 protein correlated with the calculated TdCD Kd values obtained using the same construct. The results indicate the binding enthalpy value approximation for TdCD Kd calculation was reasonable. Furthermore, the Lanthascreen™ inhibitor binding IC50s for AurB69–333 were compared with the binding IC50s and IMAP IC50s obtained using the full-length Aurora B protein. Interestingly, all but one compound, AZD1152, showed strikingly comparable inhibitor binding affinities between the

P.R. Sheth et al. / Archives of Biochemistry and Biophysics 503 (2010) 191–201

full-length Aurora B and AurB69–333. These results imply that the AZD1152 binding mode between the truncated AurB69–33 and the full-length Aurora B protein is distinct. The published Ki = 0.36 nM [18] for AZD1152 is consistent with our IMAP IC50 data of <13 nM (Table 2) for the compound obtained using the full-length Aurora B enzyme. However, the compound showed smallest Tm shifts in our TdCD setting and highest Lanthascreen™ IC50 using AurB69–333. The differences seen in the TdCD Kd values obtained using AurB69–333 and IMAP IC50 values obtained using the full-length Aurora B protein for AZD1152 compound could be due to the loss of key interactions between the inhibitor and the protein that are present only in context of the full-length activated protein. The source of these interactions could be speculated to be within the kinase domain or outside the kinase domain. It is worthwhile to note that AZD1152 compound has been shown to possess exceptional selectivity towards Aurora B compared to Aurora A [18]. Furthermore, the Lanthascreen™ IC50 for AZD1152 binding to full-length Aurora A was measured to be >1000 nM, >10-fold higher than the Lanthascreen™ IC50 value of 98 nM that was obtained for AurB69–333, implying certain specificity is retained in the truncated kinase domain construct for the AZD1152 compound. Crystal structure of AurB69–333AZD1152 and full-length Aurora BAZD1152 would be able to shed light on the structural basis of binding and selectivity of this compound. Since there is high sequence conservation within the ATP binding pockets of Aurora A and B, it is tempting to speculate that the compound is stabilized by residue(s) interactions outside the kinase domain, although further studies need to be done to confirm this hypothesis. The presence of PF3814735 resulted in the largest Tm shifts for AurB69–333 amongst all inhibitors tested. The trifluoromethylpyrimidine compound is a potent reversible Aurora A and Aurora B inhibitor currently in Phase I clinical trials. The published IC50 (0.8 nM) value for Aurora B inhibition by PF3814735 is consistent with our calculated TdCD Kd value of 3 nM for AurB69–333 [20]. Similarly, the published inhibition data for VX680 (Ki = 18 nM) [16] and CYC116 (IC50 = 19 nM) [19] are comparable to the calculated TdCD Kd (VX680 Kd = 17 nM; CYC116 Kd = 37 nM, Table 2) and measured Lanthascreen™ IC50 (VX680 IC50 = 20 nM; CYC116 IC50 = 58 nM) values obtained for AurB69–333 in this report. MLN8054 (enzymatic IC50 = 172 nM) [17] showed TdCD Kd of 37 nM with AurB69–333, which is 4-fold different from the published IC50 values. Although it should be noted the compound showed an Aurora B IMAP IC50 of 30 nM in our hands (Table 2). Acknowledgements We thank Dr. Charles McNemar and Dr. Cheng-Chi Chuang for performing N-terminal sequencing and mass spectrometry, respectively. References [1] D.M. Glover, M.H. Leibowitz, D.A. McLean, H. Parry, Cell 81 (1995) 95–105. [2] E. Hannak, M. Kirkham, A.A. Hyman, K. Oegema, J. Cell. Biol. 155 (2001) 1109– 1115. [3] S. Rojanala, H. Han, R.M. Munoz, W. Browne, R. Nagle, D.D. Von Hoff, D.J. Bearss, Mol. Cancer Ther. 3 (2004) 451–457.

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