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Curcumin Specifically binds to the Human Calcium-calmodulin Dependent Protein Kinase IV: Fluorescence and Molecular Dynamics Simulation Studies a

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Nasimul Hoda , Huma Naz , Ehtesham Jameel , Ashutosh Shandilya , Sarmishtha Dey , b

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Md. Imtaiyaz Hassan , Faizan Ahmad & B. Jayaram a

Department of Chemistry, Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India b

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Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India c

Department of Chemistry, Babasaheb Bhimrao Ambedkar Bihar University, Muzaffarpur, Bihar 842002, India d

Department of Chemistry, Supercomputing Facility for Bioinformatics & Computational Biology, Indian Institute of Technology, Hauz Khas, New Delhi - 110016, India e

Department of Chemistry, Indian Institute of Technology, Hauz Khas, New Delhi 110016, India f

Department of Biophysics, All India Institute of Medical Sciences, New Delhi 110029, India Accepted author version posted online: 01 May 2015.

To cite this article: Nasimul Hoda, Huma Naz, Ehtesham Jameel, Ashutosh Shandilya, Sarmishtha Dey, Md. Imtaiyaz Hassan, Faizan Ahmad & B. Jayaram (2015): Curcumin Specifically binds to the Human Calcium-calmodulin Dependent Protein Kinase IV: Fluorescence and Molecular Dynamics Simulation Studies, Journal of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2015.1046934 To link to this article: http://dx.doi.org/10.1080/07391102.2015.1046934

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Publisher: Taylor & Francis Journal: Journal of Biomolecular Structure and Dynamics DOI: http://dx.doi.org/10.1080/07391102.2015.1046934

Running head: Binding of Curcumin to CAMK4

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Curcumin Specifically binds to the Human Calcium-calmodulin Dependent Protein Kinase IV: Fluorescence and Molecular Dynamics Simulation Studies Nasimul Hoda1, Huma Naz2, Ehtesham Jameel3, Ashutosh Shandilya4,5, Sarmishtha Dey6, Md. Imtaiyaz Hassan2,*, Faizan Ahmad2 and B. Jayaram4,5 1

Department of Chemistry, 2Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India

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Department of Chemistry, Babasaheb Bhimrao Ambedkar Bihar University, Muzaffarpur, Bihar 842002, India.

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Supercomputing Facility for Bioinformatics & Computational Biology, 5Department of Chemistry, Indian Institute of Technology, Hauz Khas, New Delhi - 110016, India 6

Department of Biophysics, All India Institute of Medical Sciences, New Delhi 110029, India.

*Correspondence Md. Imtaiyaz Hassan, Ph.D. (Assistant Professor) Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India E-mail: [email protected] 1

Abstract

Calcium-calmodulin dependent protein kinase IV (CAMK4) plays significant role in the regulation of calcium-dependent gene expression, and thus it is involved in varieties of cellular functions such as cell signaling and neuronal survival. On the other hand, curcumin, a naturally

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occurring yellow bioactive component of turmeric possesses wide spectrum of biological actions, and it is widely used to treat atherosclerosis, diabetes, cancer, and inflammation. It also acts as an antioxidant. Here, we studied the interaction of curcumin with human CAMK4 at pH 7.4 using molecular docking, molecular dynamics (MD) simulations, fluorescence binding and surface plasmon resonance (SPR) methods. We performed MD simulations for both neutral and anionic forms of CAMK4-curcumin complexes for a reasonably long time (150 ns) to see the overall stability of the protein-ligand complex. Molecular docking studies revealed that the curcumin binds in the large hydrophobic cavity of kinase domain of CAMK4 through several hydrophobic and hydrogen bonded interactions. Additionally, MD simulations studies contributed in understanding the stability of protein-ligand complex system in aqueous solution and conformational changes in the CAMK4 upon binding of curcumin. A significant increase in the fluorescence intensity at 495 nm was observed (λexc=425 nm), suggesting a strong interaction of curcumin to the CAMK4. A high binding-affinity (KD = 3.7 x 10-8 ± 0.03 M) of curcumin for the CAMK4 was measured by SPR further indicating curcumin as a potential ligand for the CAMK4. The present study will provide insights into designing a new inspired curcumin derivatives as therapeutic agents against many life threatening diseases.

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Keywords: Curcumin; calcium-calmodulin dependent protein kinase IV; molecular dynamics simulation; docking; drug target; high affinity ligand

Introduction

Calcium-calmodulin dependent protein kinase IV (CAMK4) is a multifunctional enzyme that belongs to the Ser/Thr kinase family (Soderling 1999; Hook & Means 2001) and present in the

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nucleus of a cell (Heist & Schulman 1998). This protein has wide range of tissue expression especially in the brain (Bland, Monroe & Ohmstede 1994), thymus (Jang et al. 2001), neuronal subpopulations (Ohmstede & Bwl. Chem. 264 1989), spleen and testis (Wu et al. 2000). CAMK4 playing critically important role in the cell proliferation, gene expression, apoptosis, muscle contraction and neurotransmitter release (Bachs, Agell & Carafoli 1992; Nicotera, Zhivotovsky & Orrenius 1994; Lu & Hunter 1995; Bito, Deisseroth & Tsien 1997). Since CAMK4 is expressed at high concentration in the nucleus and binds with CREB, it is therefore, considered as a major factor that regulates gene transcription process (Sandoval, Pigazzi & Sakamoto 2009) which in turn regulates several biological processes.

Crystal structure of CAMK4 has been recently determined, offering a new approach for designing potential ligands (Protein Data Bank Code: 2W4O). It is a 473-residue long protein consisting of a 255-residue long kinase domain, a 17-residue long autoinhibitory domain (305 – 321), a 18-residue long PP2A-binding domain (306-323) and a 18-residue long calmodulinbinding domain (322 – 341). CAMK4 has many critical residues such as Lys64 in the active site (proton acceptor), Lys75 in the ATP binding site, a nine-residues long nucleotide binding domain (52LGRGATSIV60), seven glycosylation sites (Thr57, Ser58, Ser137, Ser189, Ser344, Ser345 and Ser356) and five potential phosphorylation sites (Ser12, Ser13, Thr200, Ser336 and 3

Ser360). Any mutation in Thr200 may lead to the complete loss of activation by CaMKK1 or CaMKK2 (Enslen, Tokumitsu, Stork, Davis & Soderling 1996; Anderson, Noeldner, Reece, Wadzinski & Means 2004). Ser189 is also essentially important for the CAMK4 activity because mutation in this residue increases the phosphorylation of CREB1 (2-fold) and decreases the total O-linked glycosylation (2-fold). However, the ATP-binding affinity has increased significantly (Anderson et al. 2004).

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Overexpression or mutation in the CAMK4 causes disruption of transcription activation and signaling which leads to ovarian and lung cancer (Knapp & Sundstrom 2014). Beside disruption of CAMKIV activation, it also causes memory loss and systemic lupus erythematosus (Juang et al. 2005; Fukushima et al. 2008; Ichinose et al. 2011). It was found that increase in intracellular Ca+2 level leads to the activation of calmodulin, a calcium binding protein. Binding of this complex enhances exposer of the activation loop, Thr200 which is a unique feature of CAMK4 (Anderson et al. 2004; Chow, Anderson, Noeldner & Means 2005). This autophosphorylated form of CAMK4 directly phosphorylates CREB at Ser133 and binding with CREB to the promoter accelerates the transcription (Jensen, Ohmstede, Fisher & Sahyoun 1991). Hence, the phosphorylation and activation of transcription factors are the cardinal of proper cell signaling and cell cycle regulation. CAMK4 activates MAP kinases MAPK1/ERK2, MAPK8/JNK1 and MAPK14/p38 and stimulates transcription through phosphorylation of ELK1, a member of ETS oncogene family and cyclic AMP-dependent transcription factor ATF-2. On the other hand, CAMK4 is inactivated by protein phosphatase 2A (PPP2CA/PPP2CB) which dephosphorylates Thr200. This causes termination of autonomous activity and helping to maintain the enzyme in its autoinhibited state.

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CAMK4 offers a new target for the structure based drug designing because of its direct association with neurodegenerative diseases and varieties of cancer. Hence, we tried to find a better binding partner which restricts the activation loop of CAMK4. Here, we used curcumin as a potential ligand to do so. It is a constituent of turmeric extract and has gained significant interest as a plant-based compound with anti-cancer properties (Lee et al. 2014). The anticancer effect of curcumin has already been demonstrated in many cell and animal studies (Wang et al.

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2009; Zheng et al. 2014). Recent research has shown that curcumin can target cancer stem cells (Li & Zhang 2014). On the other hand, curcumin has been reported to possess antiamyloidogenic, anti-inflammatory, anti-oxidative, and metal chelating properties that may result in potential neuroprotective effects (Yang et al. 2013). Furthermore, the hydrophobicity of the curcumin molecule hints at the possibility of blood-brain barrier penetration and accumulation in the brain (Chin, Huebbe, Pallauf & Rimbach 2013).

Docking approaches has widely been used to know the mode of binding between protein and ligand and their application towards drug design and discovery (Hassan et al. 2007; Hassan, Kumar, Singh & Yadav 2007; Thakur & Hassan 2011; Thakur, Kumar, Ray, Anjum & Hassan 2013; Thakur et al. 2013; Singh et al. 2014). Docking studies suggest that curcumin binds effectively to the CAMK4 in its substrate binding pocket. We performed 120 ns molecular dynamics (MD) simulation and found that curcumin remains inside the binding pocket of CAMK4. Furthermore, fluorescence binding studies supplemented by surface plasmon resonance (SPR), further provides an insight for interaction of curcumin to the CAMK4.

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Materials and Methods

Materials

Media for bacterial culture Luria broth, Luria agar, ampicillin, kanamycin, monoclonal anti-His antibody and DNA preparation kits were purchased from Sigma (St. Louis, MO). Agarose was

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purchased from Biobasic (Ontario, Canada). Restriction enzymes (NdeI and XhoI) PCR-Taq DNA polymerase and phusion polymerase, cloning-quick DNA ligase were purchased from New England Biolabs. Ni-HF column and gel filtration column Superdex-75 were purchased from GE healthcare (GE Healthcare Life Sciences, Uppsala, Sweden).

Methods

Docking

Docking of curcumin with CAMK4 was carried out using Pardock (Gupta, Gandhimathi, Sharma & Jayaram 2007). Scoring of these docked structures was done by Bappl scoring function (Jain & Jayaram 2005). A complete protocol for docking was described elsewhere (Singh, Biswas & Jayaram 2011). These docked structures were taken as input for MD simulations.

Molecular dynamics simulation

Partial atomic charges for ligands were obtained after optimization at the Hartree-Fock level with 6-31G* basis set using Gaussian 03 algorithm and subsequent single-point calculation of the electrostatic potential to which the charges are fitted using RESP procedure (Bayly, Cieplak,

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Cornell & Kollman 1993; Cornell, Cieplak, Bayly & Kollman 1993). Force field parameters of the ligands were assigned based on the atom types of force field model developed by Cornell et al. (Cornell et al. 1995). All MD simulations were carried out using AMBER 14 (Berryman et al. 2014). Complexes obtained from Pardock were solvated in a box of TIP3P water molecules with a 10 Å distance between the protein surface and the box boundary (and 20 counter ions to ensure electro neutrality), and minimized for an additional 2500 steps (1000SD and 1500CG). All these

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system size comprised around 125,000 atoms. MD simulations were started by slowly heating the solvent to 300 K over a period of 500 ps keeping the complex fixed. After this, the entire system was gradually brought to 300 K over a period of 300 ps. The simulation was then carried out under NPT conditions for 150 ns. A2 femto second time step was used for integrating the equation of motion. Periodic boundary conditions were applied throughout MD simulations, along with PME summation for treating electrostatics. The temperature was kept constant by coupling heat bath through the Berendsen algorithm using separate solute and solvent scaling. Pressure was adjusted by isotropic position scaling using a Berendsen like algorithm (Berendsen, Postma, Vangunsteren & Dinola 1984). Covalent bonds to hydrogen atoms were constrained by the SHAKE algorithm (Ryckaert, Ciccotti & Berendsen 1977). Convergence of energy, density were monitored. The initial 10 ns was treated as equilibration and the subsequent 150 ns was treated as production phase for structural and energy analyses. MD simulations were performed on eight node Nvidia GPU cards at Supercomputing Facility (SCFBio) at Indian Institute of Technology Delhi, New Delhi, India.

Cloning, Expression and Purification

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Human CAMKIV gene was purchased from DF/HCC DNA Resource Core, Harvard Medical School http://plasmid.med.harvard.edu/PLASMID). A 1.4 Kb CAMKIV gene was cloned in PDNR-Dual plasmid. The CAMK4 gene was amplified using forward primer with NdeI site: 5` AATCATATGCTCAAAGTCACGGTGCCC3` and reverse primer with Xho1 site: 5` TACATCTCGAGTTAGTACTCTGGCAGGATC3`. Maximum amplification was observed when annealing was kept at 65 oC. The amplified product was run on 1% agarose gel. Band of

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amplified product in phusion was eluted using gel extraction kit. Gene was ligated into pET28a(+) vector.

The His-tag cloned cDNA was transformed into BL21(DE3) strain of E. coli. After transformation in BL21(DE3) cell, primary culture was grown by picking a fresh full grown colony from the culture plate. Secondary culture of BL21(DE3) was done at 37 °C. Culture was induced with 0.5 mM IPTG once the optical density of cells were reached to 0.6 at 600 nm followed by overnight growth at 16 °C. Full grown culture was centrifuged at 4000 rpm for 30 minutes at 4 °C. Supernatant was discarded and pellet was dissolved in 20mM phosphate pH 7.8 buffer containing 500 mM NaCl, RNAase (20 µl), DNAase (20 µl), lysozyme, protease inhibitor cocktail. After sonication pellet was further centrifuged at 8000 rpm for 40 minutes at 4 °C. Both supernatant and pellet fractions were run on SDS-PAGE to check protein expression. Ammonium sulphate (30%) was added in the soluble fraction, and centrifuged at 8000 rpm (30 minutes) then pellet was dissolved in 20mM phosphate pH 7.8 buffer containing 500 mM NaCl and dialyzed in the same buffer and subsequently loaded to the Ni-HF affinity chromatography column which was preequilibrated with 20 mM sodium phosphate buffer (pH 7.8). Bound protein was eluted with 50-100 mM imidazole in the 20 mM sodium phosphate buffer pH 6.0. Eluent was concentrated and loaded the gel filtration column chromatography to get the highly 8

purified protein. Fractions were collected and purity was checked by SDS-PAGE. The bands of SDS-PAGE was transferred to PVDF membrane for Western blotting experiment.

Measurements of fluorescence spectra

Fluorescence measurements were carried out in the Jasco spectrofluorimeter (Model FP-6200) using 5 mm quartz cuvette. Experiment was carried out at 25 ± 0.1 °C maintained by an external

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thermostated water circulator. Curcumin was dissolved in the DMSO and then diluted to 1 mg/ml in the 50 mM phosphate pH 7.4 buffer containing 150 mM NaCl (PBS). Both CAMK4 and curcumin were further dialyzed in the PBS before the experiment. An increase in curcumin fluorescence upon binding to the CAMK4 was used to determine the binding of curcumin to CAMK4. We set two experiments separately. In one case, CAMK4 (1 µM) was incubated with increasing concentrations of curcumin (1 to 10 µM) in PBS (pH 7.4) at 25 °C for 30 min in the dark. In other case, we incubated curcumin (1 µM) with increasing concentrations of CAMK4 (1, 2, 4, 6 and 12 µM) in the PBS. A similar blank was set for both experiment. The fluorescence spectra of the samples were recorded using a λex of 425 nm. The fluorescence intensities at 495 nm were used for determining the binding constant as described (Rai, Singh, Roy & Panda 2008). Final spectrum was collected after deducting their corresponding blank. Three independent experiments were performed and their average was taken for data analyses.

Surface Plasmon Resonance The experiment was performed in biosensor-based system for real time specific interaction analysis BIAcore-3000 apparatus (GE Healthcare Life Sciences, Uppsala, Sweden). CAMK4 was immobilized on the surface of nickel NTA sensor chip. For this, nickel chloride was passed over the dextran matrix of the chips to activate the surface. After this 222 ng/μl of purified 9

CAMK4 in HEPES buffer of pH 7.4 was passed at a flow rate of 5 μl/min across the activated surface. Six histidine-tag are attached to the terminal position of CAMK4 was an ideal tag for immobilization due to strong rebinding effect caused by the high surface density of immobilized Ni 2+–nitriloacetic acid (NTA) on the chip. Three different concentrations of curcumin (0.5, 1.5, 1.8ng/μl) in 1% DMSO were passed at a flow rate of 10 μl/min over the immobilized CAMK4 and the corresponding RUs were obtained. The RU for each curcumin sample was recorded and

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used to calculate the binding affinity using the SPR signal for immobilized CAMK4 using Bia evaluation 3.0 software.

Results and Discussion

Medicinal values of curcumin molecule found in the turmeric root, are known since ages (Dulbecco & Savarino 2013; Fan, Zhang, Liu, Yan & Liang 2013; Witkin & Li 2013; Pescosolido, Giannotti, Plateroti, Pascarella & Nebbioso 2014). We performed docking of curcumin both in anionic and neutral forms into modelled structure of CAMK4. Curcumin is supposed to be in anionic form at physiological pH. Curcumin's anionic and neutral structures differ significantly. The neutral form is planer and straight and its both keto groups of β-diketone are in the syn conformation to make a six membered ring. However, due to electrostatic repulsion in the deprotonated curcumin two hydroxyl and ketooxygens are in anti-position forming a overall curved structure. Hence, we have considered both the structures of curcumin for the docking and MD simulation.

Modeling and docking To predict the binding pattern of curcumin with CAMK4, we have analyzed its crystal structure available in the protein data bank (PDB id: 2W4O). We found that few important amino acid 10

residues (187 -203) are missing in the crystal structure. Hence, using homology modeling based on the protein's primary sequence (Uniprot id: Q16566) we build a good quality model of CAMK4, on Bhageerath server (Jayaram et al. 2006). The energy of final model was minimized using AMBER force field (ff99SB-ILDN) for removal of clashes and side chain optimization. The energy minimized structure was used for molecular docking and MD simulations. We subsequently predicted the structure and orientation of the ligand in the binding cavity by using

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molecular docking. A comprehensive examination of CAMK4 structure reveals a novel binding orientation of curcumin in the binding site of CAMK4, which has not been reported so far in the literature. Docking poses were obtained using Pardock (Gupta et al. 2007), which is an all atom energy-based monte carlo docking protocol. Docking using Pardock requires a reference complex (target protein bound to a reference ligand) and a candidate molecule along with specific motion of the center of mass of the cavity on which the ligand is to be docked. Hence, the crystal structure of human CAMK4 in complex with 4-Amino (sulfamoyl- phenylamino)triazole-carbothioic acid (2,6-difluoro-phenyl)-amide) was used for reference. We performed docking of curcumin both in the anionic and neutral forms into the structure of CAMK4. We found that curcumin binds to the critical residues such as Asp164, Leu52, Glu119 and Val121 of the protein, which are important for its kinase activity. The curcumin forming several H-bonded interactions to the crucial residues of CAMK4. Amino hydrogen of Val121 forms hydrogen bond with anionic oxygen of curcumin. Other terminal hydroxyl of curcumin bonds up with carboxyl group of Asp164. Apart from H-bonds, curcumin was also found forming extensive hydrophobic interactions. Curcumin forms several van der Waals interactions with Lue52, Glu119 and Glu168. MD simulation 11

To understand the effect of solvent, flexibility of ligand, side chain of protein and therein any conformational changes, we performed MD simulations on the protein-ligand complex in neutral and anionic forms. MD simulations introduce theoretical rigor into binding free energy estimates and eliminate some of the limitations inherent in the single point free energy calculations. Also dynamics simulations with explicit solvent and small ions yield an enhanced view of recognition from structural and dynamic perspectives. Docking does not show any significant binding-energy

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differences as the interacting residues were mostly same for both the neutral and anioic forms of curcumin. We performed MD simulations for both the complexes for reasonably long time (150 ns) of production run to see the overall stability of the ligand protein complex.

Thermodynamic stability of trajectories were analyzed by total energy calculation for both the complexes, which shows that anionic curcumin complex has lower energy than that of neutral curcumin complex (Table 1 and 2). To study the effective conformation sampling, the stability of complex in terms of root mean square deviation (rmsd) was analyzed. The rmsd values for heavy atoms of all the structures were calculated by aligning all frames to initial docked structure using the mass-weighted least-squares fitting method. Rmsd plot shows the structures are stable during the course of MD simulations (Figure 1). Furthermore, this plot shows a remarkable increasing trend in the first few nanoseconds of the equilibration. However, during the production phase, rmsd of the complexes has attained a stationary phase showing no significant change in overall topology. A significant difference in the structure of complex before and after MD simulations clearly indicates that a conformation change in the protein structure has occurred to fit the curcumin in the binding pocket of CAMK4 (Figure 2).

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To comprehend the progression in dynamic adaptability of the regions of protein structure due to the curcumin binding, B-factors for the Cα atoms were calculated. As shown in Figure 2, the calculated B-factor values for the neutral curcumin docked complex protein apart from starting and terminal residues which are loop regions, show a major peak in the region of residues Lys75, Asp164, residue number 175-201 and reside near the active site. Analyzing from these high values of neutral curcumin bind complex, these buildups appear to assume a critical part in the

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enzymatic capacity of CAMK4 through a high adequacy movement in the enzymatic function of CAMK4. With anionic curcumin complexed these residues fluctuations were subdued. The binding of negatively charged curcumin seems to cause an overall reduction in B-factor values of CAMK4 complex indicating an increase in conformational rigidity of the protein. Decrease in Bfactor for critical residues Lys75 and Asp164 in particular implies that such a high amplitude motion of these flexible residues dampens out upon binding of the inhibitor in the active site, affirming the inhibitory activity of curcumin against CAMK4.

Radius of gyration is a measure of the stability/extent of conformational change of the protein. Hence, we plotted radius of gyration for both the systems. Figure 3 shows that after initial change during equilibration there was no major adjustment in the protein structure because of the presence of ligand indicating the stability of complexes throughout the trajectories.

In anionic curcumin-CAMK4 complex, changes in the structure of the enzyme are accompanied by a new interacting residue environment. Curcumin was found to bind in the active site region of CAMK4 (Figure 4A). Anionic curcumin moves towards the helical region forming hydrogen bonded interactions with Asp164 and Val121 residue allowing the ring part of curcumin to interact favorably with Leu52, Glu127, Glu168. Whereas, neutral curcumin showed a lateral

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displacement to interact with Arg54 and Pro167 along with common residues Val121 and Glu127 (Figure 4B and 4C). As evident from the Figure 4B, in the anionic form of curcumin enolic oxygens are on opposite side of the backbone and has a curved structure which enabled one of the oxygen to form electrostatic bond with main chain amino hydrogen and snug fit into the cavity. But that was not the case with neutral curcumin being elongated and coming out of the cavity. Anionic curcumin binds relatively strongly to CAMK4 as compared to its neutral

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form because of shape complementarity and additional electrostatic interactions of negatively charged curcumin. Hydrogen bonds are considered to make significant contribution in the binding of ligands to the enzyme. We observe that in neutral curcumin enzyme complex, switching off a hydrogen bond resulted in a loss in the net binding free energy.

Binding free energy calculation

Binding free energies for both the complexes were calculated using molecular mechanics generalized born surface area [MM/GBSA] methods using MM/GBSA implementation in AMBER14 (Mena-Ulecia, Vergara-Jaque, Poblete, Tiznado & Caballero 2014; Sun et al. 2014). Binding free energy (ΔG) is given by the following equation: ΔGbinding= ΔGMM + ΔG(polar,solvation) +ΔG(nonpolar,solvation)–TΔS where ΔGMM is the molecular mechanics interaction energy between protein and ligand. ΔGMM is given as ΔGMM = ΔEint + ΔEelectrostatic + ΔEvdW where ΔEint, ΔEelectrostatic and ΔEvdW are differences in internal, electrostatic, and van der Waals energies respectively. MD simulation trajectories from 100 to 150 ns were considered for binding free energy calculations (Table 1 and 2). 14

Conformation of significant residues

CAMK4 active site consists of Asp164 which acts as proton acceptor (Tokumitsu et al. 1994). Terminal hydroxyl group hydrogen of anionic curcumin is accessible to the acetate group of

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Asp164 at around ~1.5 Å throughout the trajectory (Figure 5), which is not in case of neutral curcumin. Initial docked poses do not show any interaction with the Asp164. During the simulation, ligand molecule showed a lateral displacement in such a way that the acetate group can abstract hydrogen from the hydroxyl group connected to the benzene ring of anionic curcumin which gives an additional stability to the complex. For the analysis of van der Walls contribution with the hydrophobic residues in the active site pocket such as Leu52 and Val121 with respect to ligand molecule was also plotted (Figure 6A and B). Thr200 is thought to be phosphorylation site which is essential for several biological activity of CAMK4 (Krebs 1998). In fact, CAMK4 gets switched on by phosphorylation of Thr200, leading to a conformational change in the structure causing it to get activated. The lateral displacement of anionic curcumin adjusted in such a way that it blocked the phosphorylation site Thr200. Curved shape of anionic curcumin allowed snug fitting into the phosphorylation site in such a manner that it would not allow any phosphate near the active site. Major electrostatic contribution was not only limited to one of the aspartate residue. But Asp185 also played a key role in the binding of negatively charged and neutral curcumin (Figure 7). To examine in detail we calculated the time evolutions of the associated interatomic distances of the ligand-receptor interactions and to estimate the dynamic stabilities of hydrogen bonds facilitating the inhibitor in the active site. 15

Protein expression and purification

To perform binding studies of CAMK4 to the curcumin, we need pure protein. CAMK4 gene construct was cloned between the NdeI and XhoI sites of pET28a(+) (Novagen), placing a 6His-

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tag on N-terminus. Transfected E. coli BL21(DE3) cells were transformed with plasmid grown in terrific broth at 37 °C, induced with 0.5 mM IPTG overnight at 16 °C. Cells were lysed, centrifuged and cleared lysate was loaded on NiNTA resin and protein was eluted with increasing concentration of imidazole (Figure 8A). Protein fraction eluted on HF nickel affinity gel column was showing contamination with other proteins. Hence, we concentrated the eluent and loaded to the Sephacryl S-200 column (GE Healthcare Life Sciences, Uppsala, Sweden). for further purification. The elution profile of gel filtration chromatography is shown in Figure 8B. The purity of CAMK4 was further confirmed by SDS-PAGE (Figure 8C). To further confirm the protein is CAMk4, we performed Western blotting experiment and found that purified protein is actually CAMK4 (Figure 8D). Finally, we got pure CAMK4 which was extensively dialyzed in the PBS for further binding studies.

Florescence study Figure 9A and 9B represent fluorescence emission spectra of increasing concentration of curcumin (1 to 10 μM) to the fixed concentration of CAMK4 (1 μM) and increasing protein concentration (1, 2, 4, 6 and 12 μM) in a fixed concentration of curcumin (1 μM), respectively. A significant increase in fluorescence intensity of curcumin at 495 nm was observed with 16

increase in concentration of CAMK4, clearly indicating that it binds to CAMK4. The binding affinity (KD value) of curcumin to the CAMK4 was calculated using the values of the fluorescence intensity change against the concentration of curcumin by fitting (Rai et al. 2008). KD value was found to be 1.89 μM, a reasonably high value, and indication of strong bindingaffinity between CAMK4 and curcumin.

We also performed circular dichroism (CD) study of CAMK4 titrated with same amount of

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curcumin. A marginal change in the dichroic signal was observed (data not shown), clearly indicating a conformation change in the CAMK4 occurred while binding to the curcumin. All these observations clearly indicates that curcumin efficiently binds to the CAMK4 and has ability to change the conformation and biological activities of this protein considerably.

SPR-binding studies of curcumin-CAMK4

The sensitive and specific interaction of curcumin to the CAMK4 was determined in the form of binding capacity on to immobilized protein as the RU changed with different concentrations of curcumin denoting the change in bound mass on the sensor chip with time giving the dissociation constant (KD) value of the curcumin. We observed the RU value as 3074 for the CAMK4 immobilized under these conditions (Figure 10). A sharp increase in the response while increasing the curcumin concentration, shows an efficient binding to the CAMK4. We found the KD value of this binding is 3.7 x 10-8 ± 0.03 M indicating that curcumin binds effectively to the CAMK4.

We observed a significant difference in the KD values obtained using fluorescence emission (1.89 μM) and SPR (0.037 μM). Although such values are indicative of the formation of a

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transient complex, the difference could be due to the different conditions of the two experimental setup. In the SPR measurement, the ligand molecules are immobilized on the substrate and the analyte is in solution. However, in the fluorescence experiment, both partners are free in solution, and this could lead to a somewhat different binding response between the two partners. Conversely, SPR monitors refractive index change upon binding interaction, and when proteins interact, the signal is generated independently from the binding site localization. It is thus

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possible that the KD bound in the fluorescence quenching experiment is the weighted average of complexes having different affinity (perhaps encounter and stable final complex) and that the binding sites of these complexes does not always involve a fixed residue in the interaction. The fact that the kinetic results depend, at least in part, on the experimental technique used, should be taken into account when kinetic data from molecules in solution are extrapolated to immobilized biomolecules, as those reacting in biosensors.

Conclusions

CAMK4 plays central roles in cellular signaling pathways, and its abnormal phosphorylation activity is directly linked with various human diseases including cancer, diabetes and neurodegenerative disorders. Therefore, modulation of kinase activity using potent inhibitor is an attractive strategy for the treatment of associated diseases. With the growing understanding of structural information, we first time observed a novel mode of binding to curcumin to the ATPbinding site and other functional motifs of CAMK4. Our docking studies followed by MD simulation, indicates a significant complexation of curcumin to the CAMK4. These in silico studies were further validated by fluorescence and SPR based binding studies which clearly indicate a remarkable association of curcumin to the CAMK4 in the physiological condition.

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These studies will be helpful for the identification of potent CAMK4 inhibitors and more towards issues of target selectivity, cellular efficacy, therapeutic effectiveness and tolerability. Furthermore, our study has demonstrated a unique structural property of the CAMK4 which provides a molecular basis for understanding towards the distinct selectivity. Inhibition of CAMK4 activity may be useful for the blocking of phosphorylation activity or by disrupting protein-protein interactions. Finally, this work gives information about the formation of a stable

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complex (curcumin-CAMK4) which further opens a promising channel towards to application of curcumin and its derivative for the treatment of ailments associated with CAMK4. However. further optimization is necessary.

Acknowledgement: We sincerely thank Dana-Farber/Harvard Cancer Center (DF/HCC) Boston, MA 02215 for providing the clone of CAMK4. FA and MIH is thankful to the Department of Science and Technology (Government of India) and Indian Council for Medical Research (ICMR) for financial support. HN thanks ICMR (Government of India) for the award of Senior Research Fellowship.

Conflict of interest: Authors declare no conflict of interest regarding any financial and personal relationships with other people or organizations that could inappropriately influence (bias) this work.

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Figure 1: RMSD plot of anionic (red) and neutral (green) curcumin complexed with the CAMK4. Figure 2: RMSF plot for neutral- (green) and anionic curcumin (blue)-CAMK4 complex. Loop region and terminus residues show high degree of fluctuation in CAMK4. Figure 3: Radius of gyration plot of anionic (red) and neutral (black) curcumin complexed with

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the CAMK4. Figure 4: (A). Overall structure of CAMK4 complexed with curcumin. Residues forming hydrogen bonded interaction (green) and functionally important residues (yellow) are shown in ball and stick. Structure of curcumin is shown in light red (ball and stick). Residues forming close interactions with the (B). Anionic curcumin and (C). Neutral curcumin are shown in ball and stick. Figure 5: Interatomic distance between terminal oxygen of negatively charged curcumin and side chain oxygen atom of Asp164 shown in red. Distances plot between terminal oxygen of curcumin and side chain of oxygen atom of Asp164 is shown in blue. Figure 6: (A). Interatomic distances between carbon atom of Cα Leu52 and carbon atom of phenyl ring of negatively charged curcumin and neutral curcumin shown in red and blue respectively. (B). Distance plot of side chain carbon atom of Val121 and center of mass of phenyl ring of negatively charged and neutral curcumin shown in red and blue respectively. Figure 7: (A). Interatomic distances between carbon atom of Cα Thr200 and carbon atom of phenyl ring of negatively charged curcumin and neutral curcumin shown in red and blue respectively. (B). Distance plot of side chain carbon atom of Asp185 and center

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of mass of phenyl ring of negatively charged and neutral curcumin shown in red and blue respectively. Figure 8: Purification and identification of CAMK4. (A). SDS-PAGE pattern of different steps of purification. Lane 1 represents SDS-PAGE pattern of crude cell lysate, lane 2 is CAMK4 after NiNTA column, lane 3, 4, 5 is washing, lane 6 is unbound fraction of NiNTA column and M represents marker of different size. (B). Gel-filtration elution

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profile of CAMK4 obtained as eluent of Ni-NTA column was loaded on Sephacryl S-200 attached with Akta Purifier. (C). SDS-PAGE of purified CAMK4 showing a single band. (D). Western blot of CAMK4. Figure 9: CAMK4 (1 μM) was incubated with different concentrations of curcumin (1–10 μM) and the fluorescence spectra were monitored as described in the Materials and methods section. The λex and λem values were 425 and 495 nm, respectively. (A). Fluorescence emission spectra of increasing concentration of curcumin (1 to 10 μM) to the fixed concentration of CAMK4 (1 μM). (B). Fluorescence emission spectra of increasing protein concentration (1, 2, 4, 6 and 12 μM) in a fixed concentration of curcumin (1 μM). All spectra were corrected for baseline with the corresponding blanks.

Figure 10: The sensorgram shows concentrations of curcumin passed over the immobilized CAMK4 on the Ni-NTA chip for the calculation of binding constant.

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Table 1: Average binding energy for negatively charged curcumin with the complex Differences (Complex - Receptor - Ligand) Energy Component

Average

Std. Dev.

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----------------------------------------------------------------VDWAALS

-54.6289

2.9691

EEL

-12.8274

4.2383

EGB

34.5365

3.6747

ESURF

-6.4442

0.2389

DELTA G gas

-67.4563

3.9579

DELTA G solv

38.0922

3.7034

DELTA TOTAL

-29.3641

2.9443

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Table 2: Average binding energy for neutral curcumin with the complex Differences (Complex - Receptor - Ligand) Energy Component

Average

Std. Dev.

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----------------------------------------------------------------VDWAALS

-53.6127

2.6678

EEL

-11.6785

4.2383

EGB

48.8589

6.1554

ESURF

-5.8019

1.2979

DELTA G gas

-67.4563

3.9579

DELTA G solv

46.1974

6.5117

- 21.2589

5.4104

DELTA TOTAL

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Curcumin specifically binds to the human calcium-calmodulin-dependent protein kinase IV: fluorescence and molecular dynamics simulation studies.

Calcium-calmodulin-dependent protein kinase IV (CAMK4) plays significant role in the regulation of calcium-dependent gene expression, and thus, it is ...
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