International Journal of Biological Macromolecules 111 (2018) 208–218

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International Journal of Biological Macromolecules

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Mechanistic insights into the urea-induced denaturation of kinasedomain of human integrin linked kinase

Sunayana Begum Syed a, Faez Iqbal Khan b, Sabab Hasan Khan a, Saurabha Srivastava a, GulamMustafa Hasan c,Kevin A. Lobb b, Asimul Islam a, Faizan Ahmad a, Md. Imtaiyaz Hassan a,⁎a Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, Indiab Computational Mechanistic Chemistry and Drug Discovery, Department of Chemistry, Rhodes University, South Africac Department of Biochemistry, College of Medicine, Prince Sattam Bin Abdulaziz University, Al-Kharj, Saudi Arabia

⁎ Corresponding author at: Centre for Interdisciplinary RMillia Islamia, Jamia Nagar, New Delhi 110025, India.

E-mail address: mihassan@jmi.ac.in (M.I. Hassan).

https://doi.org/10.1016/j.ijbiomac.2017.12.1640141-8130/© 2018 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 21 November 2017Received in revised form 29 December 2017Accepted 30 December 2017Available online 05 January 2018

Integrin-linked kinase (ILK), a ubiquitously expressed intracellular Ser/Thr protein kinase, plays a major role inthe oncogenesis and tumour progression. The conformational stability and unfolding of kinase domain of ILK(ILK193–446) was examined in the presence of increasing concentrations of urea. The stability parameters of theurea-induced denaturation were measured by monitoring changes in [θ]222 (mean residue ellipticity at222 nm), difference absorption coefficient at 292 nm (Δε292) and intrinsic fluorescence emission intensity atpH 7.5 and 25 ± 0.1 °C. The urea-induced denaturation was found to be reversible. The protein unfolding transi-tion occurred in the urea concentration range 3.0–7.0M. A coincidence of normalized denaturation curves of op-tical properties ([θ]222, Δε292 and λmax, the wavelength of maximum emission intensity) suggested that urea-induced denaturation of kinase domain of ILK is a two-state process. We further performedmolecular dynamicssimulation for 100 ns to see the effect of urea on structural stability of kinase domain of ILK at atomic level. Struc-tural changeswith increasing concentrations of ureawere analysed, andwe observed a significant increase in therootmean square deviation, rootmean square fluctuations, solvent accessible surface area and radius of gyration.A correlation was observed between in vitro and in silico studies.

© 2018 Elsevier B.V. All rights reserved.

Keywords:Integrin linked kinaseUrea-induced denaturationProtein folding and stabilityMolecular dynamics simulation

1. Introduction

Integrin-linked kinase (ILK) is a member of Ser/Thr protein kinase,which interactswith the cytoplasmic domains of integrinβ1 andβ3 sub-units [1]. It acts as a key component of cell extracellular matrix (ECM)adhesion structures and implicates the regulation of anchorage-depen-dent cell cycle progression, cell growth and survival, epithelial–mesen-chymal transition (EMT), invasion, migration, cell motility, contractionand vascular development [2–5]. Structurally, ILK consists of two highlyconserved distinct domains. One is the C-terminal kinase domainwhichis having homology with other known kinase catalytic domains [6,7].The kinase domain consists of binding sites for integrin and calponin ho-mology domain of parvins [4]. Another one is N-terminal domainwhichcomprised of five ankyrin repeats that helps tomediate protein–proteininteractions [3,9].

Integrin linked kinase is the most predominant adapter protein ofintegrins [10]. ILK binding is crucial for many signalling pathways suchas actin rearrangement, cell polarisation, migration, spreading,

esearch in Basic Sciences, Jamia

proliferation and survival [11–13]. Despite of its focal adhesions andlocalisation function, it has also been reported that ILK contains someresidues in adhesion siteswhich involves in cell–cell adhesion in centro-some as well as in nucleus [14]. Before converting the focal complexes(FXs) to focal adhesions (FAs) and fibrillar adhesions (FBs), it bindswith LIM domain of PINCH1 and calponin binding domain of parvin toform IPP complex which ensures the stability of each component andtargets to the adhesion sites. Similarly, ILK forms different IPP com-plexes with other adaptor proteins associated with various signallingpathways [15,16].

In humans, the ILK encoding gene has been present at interphase ofthe bands 11p15.5 and 11p15.4 on chromosome 11. It shows a high de-gree of homology with mice, drosophila and C. elegans [17]. ILK is pre-dominantly expressed in various organs and tissues such as heart,brain, kidney, skeletalmuscle, platelet, skin, chondrocyte, keratinocytes,T cells and pancreas [18–22]. However, expression of the ILK has alsobeen reported to be increased in colon, melanoma, prostate, ovariancancer, non-small cell lung cancer, and head and neck squamous cellcarcinomas where it promotes the oncogenic transformation of cancercells through the regulation of several downstream targets. Further-more, ILK promotes cancer cell proliferation, survival, metastasis andangiogenesis [13]. Inhibition of ILK activity may reduces cancer

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progression, and thus it is being considered as a potential target for can-cer therapy [2,23].

There are a few reports available on the biophysical properties of ILK[24]. To understand the mechanism of folding, we have successfullycloned, expressed and purified the kinase domain of ILK193–446. Thermo-dynamic stability of the purified protein was measured in the presenceof increasing urea concentration using different spectroscopic tech-niques. To get deeper insight into themechanism of urea induced dena-turation, we have performed 100 ns molecular dynamics (MD)simulation of the kinase domain (ILK193–446) under explicit water andin increasing concentrations of urea. The combined spectroscopic andMD simulation approaches provided a clear insight into the stability aswell as the biophysical properties of ILK193–446. The term ILK in theman-uscript is used for the kinase domain representing residues from 193 to446.

2. Materials and methods

2.1. Materials

Plasmid pET-21c (Novagen,Wisconsin, USA)was used as expressionvector. NaCl, imidazole, Tris-HCl, isopropylβ-D-1-thiogalactopyranoside(IPTG), phenyl methyl sulphonyl fluoride (PMSF) and ampicillin werepurchased from Sigma (Saint Louis, MO, USA). Urea was acquiredfrom MP Biomedicals, Pvt. Ltd. (India). All chemicals and reagentsused for buffer preparation were of analytical grade.

2.2. Cloning, expression and purification

Human ILK clone was purchased from DF/HCC DNA Resource Core,Harvard Medical School (http://plasmid.med.harvard.edu/PLASMID).Kinase domain of ILK (residues 193–446) was amplified by using poly-merase chain reaction. A forward primer with NdeI site: 5′-GCTAGCATGAACAAGTATGGAGAGATGCCTGTGG-3′ and reverse primerwith XhoI site: 5′-CTCGAGAAGGATAGGCACAATCATGTC-3′ were usedfor the amplification of kinase domain. This domain was ligated intothe pET21c (+) expression vector contained 6X His-tag at the N-termi-nal (Fig. S1A and B). Plasmid was subsequently transformed into BL21(DE3) strain of E. coli, and positive clones were selected and grown inLB broth. Protein expression was induced with 0.5 mM IPTG at 37 °Cand incubated for 3 h in an orbital incubator shaker. After incubation,the culture was centrifuged at 6000 rpm for 15min at 4 °C, and the pel-letwas collected anddissolved in Tris buffer (50mM, pH8.0) containing1% of TritonX-100 and 20 mM EDTA. The suspend pellet was sonicatedfor 15min (15 s on, 15 s off) and centrifuged at 12,000 rpm. The pelletedcells were further resuspended in a buffer containing 50 mM Tris and20 mM EDTA. The sonication and centrifugation process were repeatedfor 2–3 times with milliQ water until we get a clear pellet. Finally, thepurified inclusion bodies (IBs) of ILK193–446 was dissolved in 3–5 ml ofmilliQ and stored at 4 °C.

Protein was purified from IBs by solubilizing in 50 mM Tris buffercontaining N-laurylsarcosine. The solubilised IBs were centrifuged at12,000 rpm, and the supernatant was used for the purification ofILK193–446 domain using Ni-NTA affinity column chromatography. Thebound protein was eluted by increasing the concentration of imidazole(20–250 mM). The eluted protein was dialyzed for 24 h (5 changes)against pH 7.5 buffer (50mMTris and 100mMNaCl) to get the refoldedprotein. This refolded protein was analysed by SDS-PAGE for purity andconfirmed by theWestern blot (Fig. S1C and D). Molar absorption coef-ficient at 280 nmwas used to determine the protein concentration [25].

2.3. Sample preparation

To know the effect of the chemical denaturant urea on ILK193–446,10.0 M urea stock solution was freshly prepared in 50 mM Tris buffer(pH 7.5). Refractive index measurement was used to calculate the

concentration of the stock solution. Protein samples of different ureaconcentrations in the range 0.2 to 9.5Mwere prepared. Protein concen-tration in these solutions were set at 0.3 mg/ml. Reversibility of dena-turation induced by urea was checked by following a proceduredescribed earlier [26]. Briefly, for each denaturation experiment, aknown amount of the buffer, and required amount of 10Murea solution(in the same buffer) were mixed followed by the addition of a knownamount of protein stock solution. This protein solution was incubatedovernight, a time long enough to complete the denaturation. For eachrenaturation experiment, protein was denatured first. To denature theprotein, a known amount of protein was mixed with known amountsof concentrated urea solution (in the same buffer), and this solutionwas incubated for 2 h, a time long enough to denature the protein.This denatured protein solution was diluted with required amount ofthe buffer, and solution was incubated for 2 h, a time long enough tocomplete renaturation of the protein. An observation of identical far-UV spectra of the protein from both denaturation and renaturation ex-periments is taken as a criterion for reversibility of denaturation of ILKdomain.

2.4. Measurements of secondary structure

Circular dichroism (CD) measurement of ILK193–446 was carried outin a Jasco spectropolarimeter (model J-1500) equipped with Peltiertemperature controller (PTC-517) interfaced with a computer. Far-UVCD spectra of the protein (0.3 mg/ml) in the presence of urea were col-lected from 250–215 nm using a quartz cuvette of 0.1 cm. For spectralanalysis, the raw CD data (millidegrees) were converted into mean res-idue ellipticity, [θ]λ (deg cm2 dmol−1) using the following relation,

θ½ �λ ¼ Moθλ=10lc ð1Þ

where θλ is the observed ellipticity in millidegrees,Mo is the mean res-idueweight of protein, c is the concentration of protein inmg/ml and l isthe path length of the cell (in centimetres). Each spectrum wascorrected by subtracting the contribution of blank solution. The urea-in-duced denaturation curvewas obtained byplotting [θ]222 (mean residueellipticity at 222 nm) as function of [urea], themolar urea concentration.

2.5. Absorption spectrum measurements

Absorption spectra of ILK193–446 were measured in Jasco UV–visiblespectrophotometer (Jasco V-660) facilitated with a temperature con-troller (ETCS-761). For spectral measurements, protein (0.3 mg/ml)samples containing different urea concentrationswere prepared in trip-licate independently. Spectra were recorded in 340–240 nm wave-length range at 25 ± 0.1 °C, using quartz cuvette of 1 cm. Theabsorption at 292 nm was converted into molar absorption coefficient,ε292, a probe to measure change in the environment of Trp residues.To generate denaturation curve, the difference molar absorption, Δε292(=value of ε292 in the presence of urea − value of ε292 in the absenceof urea) was plotted against [urea].

2.6. Intrinsic fluorescence measurements

Jasco spectrofluorimeter (Model FP-6200) equipped with an exter-nal thermostatedwater circulatorwas used tomeasure thefluorescencespectra of ILK in the presence of different urea concentrations at 25 ±0.1 °C. Both excitation and emission slits were set at 5 nm bandwidth.The entire experiment was carried out at a concentration of 0.3 mg/mlof the protein. Since the kinase domain ILK containing 7 tryptophans,so the proteinwas excited at 292 nm and the emission spectrawere col-lected between 300 and 400 nm.

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2.7. Analysis of transition curve

Denaturation curvesmonitored by [θ]222 (probe tomeasure changesin the secondary structure) and Δε292 and λmax of the emission spec-trum (probes to measure changes in the environment of Trp residues)were analysed for ΔGD

0, the value of ΔGD (change in standard Gibbsfree energy associated with the denaturation process, native (N) state↔ denatured (D) state) in the absence of urea; m, the slope (∂ΔGD/∂[urea]); and Cm (=ΔGD

0/m), the midpoint point of denaturation. Anon-linear least-squaresmethodwas used to fit the entire denaturationcurve obtained at constant temperature and pH according to the rela-tion [27],

y ¼ yN þ yD� Exp − ΔGD

0−m u½ �� �

=RTh i

= 1þ Exp − ΔGD0−m u½ �

� �=RT

h i� �ð2Þ

where y is the optical property used to follow the denaturation, yN andyD are the properties of native and denatured states of the protein undersame experimental conditions in which y has been measured, R is thegas constant, and T is temperature in Kelvin. In Eq. (2) it was assumedthat a plot of ΔGD versus [urea] is linear, i.e., ΔGD = ΔGD

0 − m[u] [28].It should be noted that the dependencies of yN and yD on [urea] arealso assumed to be linear (i.e., yN = aN + bN [urea], and yD = aD + bD[urea], where aN, bN, aD and bD are [urea]-independent parameters,and N and D subscripts represent for the N and D states of protein mol-ecules, respectively).

At a given [urea], the fraction of denatured protein, fD was estimatedusing the relation [29],

f D ¼ y−yNð Þ= yD−yNð Þ ¼ y− aN þ bN u½ �ð Þ= aD−aNð Þ þ bD−bNð Þ u½ � ð3Þ

2.8. MD simulations

MD simulations are effectively used to understand structure-func-tion relationships in a protein [30–33]. In order to study the effect ofurea denaturation on ILK domain and its stability, several MD simula-tions were carried out at the molecular mechanics level implementedin the GROMACS 5.1.2 [34] using the GROMOS96 43a1 force-field at dif-ferent concentrations of urea and 298 K. The PDB coordinates ofhuman ILK (PDB: 3KMW) were obtained from Protein Data Bank(https://www.rcsb.org/).

The topology and force-field parameter files for urea molecule weregenerated using PRODRG server [35]. The charges in the topology filewere manually corrected. The exact numbers of urea and water mole-cules were calculated for 2.0, 4.0, 6.0, 8.0 and 10.0 M urea

Fig. 1. Urea-induced changes in the secondary structure of ILK193–446 at pH 7.5 and 25 °C. (A). Faplotted with [θ]222 against [urea].

concentrations to fill a box of size 6 × 6 × 6 nm3 using standard pro-tocol [36,37]. The ILK molecule was soaked in a cubic box of watermolecules using the gmx editconf module for creating boundaryconditions. As a control, first we carried 100 ns MD simulation ofILK193–446 at 298 K in water. The simulation box for control containedone molecule of ILK193–446 placed at the centre of cubic box andwater molecules padding around the protein. The gmx insert-mole-cules module of GROMACS was used to add the calculated numberof urea molecules, and gmx solvate for the solvation. The parametersfor urea were included in the system topology. Six systems were pre-pared with different concentrations of urea such as 0.0, 2.0, 4.0, 6.0,8.0 and 10.0 M.

The Simple Point Charge (spc216) water model was used to solvatethe system. The charges on the ILK were neutralized by addition of Na+

and Cl− ions using gmx genionmodule to maintain neutrality. The sys-temwas thenminimized using 1500 steps of steepest descent. The tem-perature of all the systems was subsequently raised from 0 to 298 Kduring their equilibration period (100 ps) at a constant volume underperiodic boundary conditions.

Equilibration was performed in two phases: NVT ensemble (con-stant number of particles, volume, and temperature at 100 ps) andNPT ensemble (constant number of particles, pressure, and temperatureat 100 ps). In both equilibration steps Cα backbone atoms of the originalcrystal structure were restrained with all other atoms allowed to movefreely. After the equilibration phase, the particle-mesh Ewald method[38] was applied and the production phases consisting of 100 ns wereperformed at a temperature of 298 K. The resulting trajectories wereanalysed using gmx energy, gmx rms, gmx confirms, gmx rmsf, gmx gyrate,make_ndx, gmx hbond, gmx do_dssp, gmx covar, gmx anaeig, gmx sham,and gmx sasa utilities of GROMACS. All graphical presentations of the3Dmodels were prepared using PyMol and VMD (Visual Molecular Dy-namics) [39].

3. Results

3.1. Protein expression and purification

Kinase domain of ILK was successfully cloned in the pET21c vectorand expression has been done in the BL21 (DE3) cells followed by puri-fication usingNi-NTAaffinity column chromatography. A single bandonSDS-PAGE was observed which was further confirmed by Western blot(Fig. S1). In each culture we obtain ample amount of protein which issufficient to get the spectroscopic measurements. Concentration of ILKwas experimentally determined using ε value of 43,220 M−1 cm−1 (at280 nm), as discussed in Methods section.

r-UV CD spectra recordedwith the increasing urea concentration. (B). Denaturation curve

Table 1Thermodynamic parameters obtained from urea-induced denaturation of ILK at pH 7.5and 25 °C.

Probes ΔGD°, kcal mol−1 m, kcal mol−1 M−1 Cm, M

[θ]222 4.11 ± 0.19 0.81 ± 0.05 5.07 ± 0.12Δϵ292 3.96 ± 0.14 0.79 ± 0.06 5.03 ± 0.10λmax 3.71 ± 0.16 0.73 ± 0.05 5.08 ± 0.11

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3.2. Far UV-CD spectra

Fig. 1A shows the effect of different concentrations of urea on thesecondary structure of ILK193–446 domain in 50 mM Tris buffer(pH 7.5) at 25 ± 0.1 °C. It is seen in this figure that CD measurementsin the presence the denaturant are carried out only up to 215 nm. Thereason for this was that urea has very high absorbance beyond(215 nm) this wavelength. The value of ellipticity at 222 nm (signatureof α-helical content) is used to estimate the secondary structure ele-ments in the protein. Secondary structure content of protein at differenturea concentration was estimated by analyzing each CD spectrum onK2D2 server (http://dichroweb.cryst.bbk.ac.uk/html/links.shtml). Thisanalysis suggested that ILK193–446 domain contains 39% of α-helix and16% β-strands which are in excellent agreement with the crystal struc-ture analysis [7].

Fig. 1B shows the plot of [θ]222 (probe tomeasure change in second-ary structure) of the ILK domain as a function of [urea] at pH 7.5 and25 ± 0.1 °C. This illustrates a cooperative unfolding of the proteinwith an increasing concentration of urea. The denaturation curveshown in this was analysed for ΔGD

0 , mu and Cm using a non-linearleast-squares method according to Eq. (2). Table 1 shows the resultsof these thermodynamic parameters.

3.3. Absorption spectral measurements

Effect of urea on the tertiary structure of ILK193–446 domainwasmea-sured by recording the near-UV absorption spectra of the protein atpH 7.5 and 25 ± 0.1 °C. These spectra are shown in Fig. 2A. Fig. 2Bshows the urea-induced denaturation curve (plot of Δε292 versus[urea]). This denaturation curve was analysed to estimate values ofΔGD

0 , mu and Cm using non-linear least squares method according toEq. (2) and results of this analysis are given in Table 1.

3.4. Fluorescence spectrum measurements

After incubation of the protein in different urea concentrations, ILKdomain was excited at 292 nm and fluorescence spectrum was

Fig. 2. Urea-induced changes in the tertiary structure of ILK193–446 at pH 7.5 and 25 °C. (A). NILK193–446 obtained by plotting Δε292 as a function of [urea].

measured in the wavelength region 300–400 nm (Fig. 3A). It can beseen in Fig. 3A that remarkable change in λmax (red shift) was observedwith increasing concentration of denaturant in the range 3–7 M. Fig. 3Bshows plot ofλmax (wavelength atwhichmaximumemission occurs) asa function of [urea]. This plot (denaturation curve) was analysed forthermodynamic stability parameters (ΔGD

0,mu and Cm) using non-linearleast-squares analysis according to the Eq. (2). Values of these parame-ters are given in Table 1.

3.5. MD simulation

3.5.1. Average potential energy of systemTo establish the equilibration of the systems prior toMDanalysis, the

average potential energy of ILK protein was calculated at 0.0, 2.0, 4.0,6.0, 8.0 and 10.0 M urea concentrations. The average potential energyof ILK protein at 298 K was found to be −292,189.79, −272,599.01,−256,169.70, −242,901.77, −232,349.79 and −222,618.74 kJ/mol at0.0, 2.0, 4.0, 6.0, 8.0 and 10.0 M urea concentration, respectively(Fig. S2).

3.5.2. Structural deviations and compactnessIn order to investigate the effect of urea on the structural stability of

ILK, values of root mean square deviation (RMSD) were analysed withrespect to time. RMSD is one of the most significant fundamental prop-erties to establish whether the protein is stable and close to the experi-mental structure [40]. The average RMSD values of ILK at 0.0, 2.0, 4.0,6.0, 8.0 and 10.0 M urea concentrations were found to be 0.43, 0.54,0.75, 0.93, 1.06 and 1.05 nm, respectively (Fig. 5A). At 2.0 M urea con-centration, the ILK is slightly deviated from its native conformationswith stable RMSD trajectories throughout the 100 ns MD simulations.

To calculate the average fluctuation of all residues during the simu-lation, the rootmean square fluctuation (RMSF) of the ILK at 0.0–10.0Murea concentration were plotted as a function of residue number(Fig. 5B). RMSF plot suggested that ILK showed least residual fluctua-tions at 0.0 M urea concentration at 298 K. At 2.0 M urea concentration,there was slight increase in residual fluctuations. As the concentrationof urea increases from 4.0 to 10.0 M, the residual fluctuations increasewith maximum in the regions 200–250 aa, 340–380 aa.

Radius of gyration (Rg) is a parameter linked to the tertiary structuralvolume of a protein and has been applied to obtain insight into the sta-bility of the protein in a biological system. A protein is supposed to havethe higher radius of gyration due to less tight packing. The average Rgvalues for ILK at 0.0, 2.0, 4.0, 6.0, 8.0 and 10.0 M urea concentrationswere found to be 1.69, 1.88, 2.10, 2.12, 2.40 and 2.16 nm, respectively(Fig. 5C).

ear-UV absorption spectra with increasing urea concentration. (B) Denaturation curve of

Fig. 3. Intrinsicfluorescence emission spectra of ILK193–446 at pH 7.5 and 25 °C. (A). Fluorescence emission spectrawith the increasing urea concentration. (B). Denaturation curve obtainedby plotting change in λmax as a function of [urea].

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3.5.3. Solvent accessible surface areaThe solvent accessible surface area (SASA) is defined as the surface

area of a protein which interacts with its solvent molecules [41,42].The average SASA values for ILK at 0.0, 2.0, 4.0, 6.0, 8.0 and 10.0 Murea concentrations were found to be 131.21, 141.96, 145.86, 150.52,153.42 and 156.32 nm2, respectively (Fig. 5D). The SASA plot suggestedthat the addition of urea in the solvent leads to increase in SASA values.

3.5.4. Hydrogen bonds analysisDuring the simulations, hydrogen bonds were formed from the pro-

tein to both urea and water molecules as intra-protein hydrogen bondswere lost [43]. Ureawas found to compete efficientlywithwater as botha hydrogen bond donor and acceptor. In order to check the strength ofhydrogen bonds between ILK-water, and ILK-urea, the hydrogenbonds paired within 0.35 nm were calculated during the 100 ns MDsimulations. The number hydrogen bonds between ILK and water mol-ecules were found to be 420, 338, 297, 283, 274 and 262, at 0.0, 2.0, 4.0,6.0, 8.0 and 10.0 M of urea concentrations, respectively (Fig. 6A). Whilethe number of hydrogen bonds between ILK and urea molecules werefound to be 101, 149, 180, 204 and 221, at 2.0, 4.0, 6.0, 8.0 and 10.0 Murea concentrations, respectively (Fig. 6A).

3.5.5. Secondary structure changes upon ligand bindingThe purpose of estimating secondary structure changes upon ligand

binding is to see the structural content of a protein as a function of time.The secondary structure assignments in protein such asα-helix, β-sheetand turn were broken into individual residues for each time step. Theaverage number of residues participated in secondary structure forma-tion of ILK at different concentration of urea molecules were compared(Table 2). The percentage of residues participated in average structureformation during the 100 ns MD simulations were found to be lesswhen the concentration of urea was increased. It is found that at 6.0–10.0 M, the protein denatured at higher rates. As the concentration of

Table 2Percentage of residues participated in average structure formation in ILK193–446 at 0–10 M urea

Urea concentration Percentage of ILK193–446 secondary structure (SS %)

Structurea Coil β-Sheet

0.0 M 59% 25% 16%2.0 M 53% 30% 13%4.0 M 52% 34% 11%6.0 M 47% 35% 11%8.0 M 46% 39% 12%10 M 41% 42% 9%

a Structure = α-helix + β-sheet + β-bridge + Turn.

urea increases from 2.0 to 10.0 M, the percentage of coil increasesfrom 30 to 42%, respectively. While the β-sheet dropped from 16 to9%, and α-helix from 31 to 22%. The β-sheets and α-helices werefound to be lowest at 10.0 M urea concentrations. The secondary struc-ture plot suggested that there is large unfolding of protein ILK and in-crease in percentage of coil was found at 6.0–10.0 M ureaconcentrations (Fig. 7). The detailed unfolding of ILK at 30–40 ns and70–80 ns MD simulation were depicted in the Fig. S3. The simulationsshow that the unfolding in presence of urea destabilizes the β-sheetfirst [37].

3.6. Principal component analysis

The principal component analysis (PCA) or essential dynamics (ED)reflects the overall expansion of a protein during MD simulation [44].The PCA for ILKwere calculated at different concentrations of urea mol-ecules. In this method, the dynamics of ILK was calculated usinggmxcovar module with respect to the backbone. PCA identifies thelarge scale average motion of a protein thus reveals the structures un-derlying the atomic fluctuations [45]. The sum of the eigenvalues is ameasure of the total motility in the system. It can be used to comparethe flexibility of a protein under different conditions [46]. The trace ofthe covariance matrix and eigenvalues were found to be 201.62,823.62, 1728.47, 2225.47, 4266.91 and 4340.63 nm2 at 0.0, 2.0, 4.0,6.0, 8.0 and 10.0 M urea concentrations, respectively. The eigenvalueswere found to be increases as the urea concentration increased clearlyindicates that random fluctuations in ILK increases due to presence ofurea molecules. Higher eigenvalues indicate higher expansion of ILK,i.e., low compactness and denaturation. The multidimensional atomiccovariance matrix for each atom pair covariance was depicted (Fig. 8).

The Gibbs free energy landscapewas also calculated using gmxcovar,gmxanaeig, and gmx sham. The gmxanaeig module reads a set of eigen-vectors and eigenvalues as input files and returns to project an MD tra-jectory along a selected eigenvector values. The 2D projections of

concentrations during 100 ns MD simulations.

β-Bridge Bend Turn α-Helix 310-Helix

1% 15% 11% 31% 1%1% 16% 9% 30% 1%2% 13% 9% 30% 1%1% 15% 11% 23% 1%1% 14% 6% 26% 1%1% 15% 9% 22% 1%

Fig. 4. Plot of fraction denatured (fD) of ILK193–446 as a function of [urea].

213S.B. Syed et al. / International Journal of Biological Macromolecules 111 (2018) 208–218

trajectories on eigenvectors showed different projections of ILK at dif-ferent concentrations of urea molecules (Fig. 9). The overlapped regioncompletely changed at 6.0–10.0 M urea concentrations. The results alsosuggested a difference in the position of atoms due to unfolding of ILK atdifferent urea concentrations. The colour coded Gibbs free energy land-scape was also depicted in supplementary Fig. S3.

Fig. 5.Dynamics of ILK193–446 at different concentrations of urea molecules. (A) RMSD plot as aILK193–446. (C) Time evolution of radius of gyration (Rg) values obtained during 100 ns of MDyellow and brown colour represent values obtained at 0.0, 2.0, 4.0, 6.0, 8.0 and 10.0 M urea clegend, the reader is referred to the web version of this article.)

4. Discussion

Proteins are one of the most essential bio-molecules having im-mense importance in all living systems. They perform vital functionsby catalyzing chemical reactions, transport particles, controls cellgrowth and differentiation (such as transport of ions, catalysis, musclecontraction, transmission of information between specific cells and or-gans, activities in the immune system, passage of molecules across cellmembranes, etc.) Under normal physiological condition proteins ac-quire a well-defined compact three-dimensional native conformationto perform their biological function [47,48]. Thus three-dimensionalstructure of a protein is essentially important to investigate its function[49–54]. Their functionality can be achieved by its native structurewhich is marginally stable in contrast to the unfolded state [55–60].The native conformation or the folded state of proteins can be governedby a variety of physical conditions such as pH, temperature, ionicstrength, presence or absence of other regulatory proteins [32,55,58,61–64]. So, the aberrant change in these conditions may change theconformational stability of the proteins by modifying its secondary, ter-tiary or quaternary structures [65–69].

Urea and guanidinium chloride (GdmCl) are the two most com-monly used chemical denaturants to study the stability as well as theconformational changes of the native proteins [61,62,70]. However,the molecular mechanism of its unfolding is still controversial. Basedon the experimental and theoretical observations, urea may follow thedirect or indirect mechanism for the unfolding [71]. Tremendous re-search has been carried out for elucidating the molecular mechanismsof protein stability due to the effect of chemical denaturants. Urea and

function of time obtained for ILK193–446 at 0, 2.0, 4.0, 6.0, 8.0 and 10.0 M. (B) RMSF plot forsimulation. (D) SASA as a function of time obtained for ILK193–446. Black, red, green, blue,oncentrations, respectively. (For interpretation of the references to colour in this figure

Fig. 7. Secondary structure plot. Graphical representation indicating structural elements present in ILK193–446 during 100 nsMD simulations at (A) 0.0M, (B) 2.0M,(C) 4.0M, (D) 6.0M,(E)8.0 M, and (F) 10.0 M urea concentrations.

Fig. 6. The average number of hydrogen bonds as a function of time. The average number of hydrogen bonds formation calculated during 100 ns MD simulations between (A) ILK193–446

and water, and (B) ILK193–446 and urea molecules. Black, red, green, blue, yellow and brown colour represent values obtained at 0.0, 2.0, 4.0, 6.0, 8.0 and 10.0 M urea concentrations,respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

214 S.B. Syed et al. / International Journal of Biological Macromolecules 111 (2018) 208–218

215S.B. Syed et al. / International Journal of Biological Macromolecules 111 (2018) 208–218

GdmCl are the most commonly used indispensible chemical denatur-ants [72] interacts directly with the backbone of proteins, side chainsand due to interaction with water molecules it also effect indirectly[73–76]. Urea binds with polar and charged residues and significantlyinfluences polar interactions within the protein backbone. It bindswith carbonyl oxygen anddestabilizes the protein's secondary structure[77–80].

ILK is an evolutionarily conserved focal-adhesion protein which isexpressed intracellularly. This protein is considered as a potential drugtarget for cancer and other diseases. The conformational stability of pro-teins may change with the external environment (e.g., extreme pHvalues or temperature or the presence of organic solvents) that existsduring the activation process [81]. In order to design potential inhibitorsof ILK, a systematic understanding of its structural properties as well asits behaviour with different cellular microenvironments is essentiallyimportant. In the present study, we have cloned, expressed and purifiedthe kinase domain (193–446 residues) of ILK. Protein was purified withadmirable yield and identified using Western blot (Fig. S1). Despite,plenty of literature on the different biological aspects of ILK, scant

Fig. 8.Principal Component Analysis. PCA or essential dynamics calculated at (A) 0.0M, (B) 2.0Mlarge scale average motion of ILK193–446 thus revealed the structures underlying the atomic flu

information is available on its folding, stability and biophysical proper-ties. To see the effect of urea on these physicochemical properties, wehave carried out the far-UV CD, near-UV absorption and Trp fluores-cence spectroscopic measurements with the increasing concentrationsof urea. All spectroscopic results were further complemented by theMD simulations.

ILK193–446 domainwhichwas expressed in E. coli existed in IBswhichwas dissolved in lauryalsarcosine, a protein denaturant. The denaturedprotein was purified on Ni-affinity column. The purified denatured ILKdomain was refolded by dialysing it against 50 mM Tris buffer(pH 7.5). The far-UV CD spectrum of this purified domain showed thatit has 39% α-helix and 16% β-sheet. This observation is in excellentagreement with X-ray diffraction study on ILK domain bound to itscytoskeletal regulator, the C-terminal calponin homology domain ofα-parvin. A comparison of the near-UV and fluorescence spectra onthe native (refolded) protein with those of the urea-induced denaturedprotein suggested that the refolded protein exists in the folded state. Aswe will discuss later, this conclusion is supported by MD simulationstudied.

,(C) 4.0M, (D) 6.0M, (E) 8.0M and (F) 10.0Murea concentrations. The graph represents actuations at different urea concentrations.

216 S.B. Syed et al. / International Journal of Biological Macromolecules 111 (2018) 208–218

Few assumptions have been considered to derive thermodynamicparameters by use of Eq. (2). First, we considered the urea-induced de-naturation of ILK as a reversible process. So, we performed renaturationexperiments at different urea concentrations. We observed that at agiven [urea] spectra of all the three properties (CD, Absorbance andfluorescence) of protein samples from denaturation and renaturationexperiments overlap on each other. This observation was taken asproof for the reversibility of the urea-induced denaturation of ILKdomain.

Another assumption in the derivation of Eq. (2)was that theurea-in-duced denaturation of ILK domain is a two-state process. One of thecriteria for the two-state denaturation is the coincidence of normalizedtransition curves of different properties. The normalized curves of [θ]222,Δε292 and λmax of ILK193–446 are shown in Fig. 4. It is seen in this figurethat all denaturation curves (plots of fD versus [urea]) coincide witheach other. This observation is taken as a proof the two-state denatur-ation of ILK domain by urea. Furthermore, it is seen in Table 1 that allthermodynamic parameters (ΔGD

0, mu and Cm) obtained from the anal-ysis of denaturation curves of different optical properties are, within ex-perimental errors, identical, suggesting that urea-induced denaturationof ILK domain follows a two-state mechanism.

From our in vitro studies we conclude that (i) the ILK domain in thenative buffer exists the folded state, (ii) urea-induced denaturation ofILK domain is reversible and two-state process, and (iii) native ILK do-main is more stable than the denatured protein by about 4 kcal mol−1.

Structural changes with increasing concentration of urea weremon-itored at atomic level using MD simulations. We found that if the con-centration of urea in the solvent increases, the average potentialenergy also increases with time. At 4.0–10.0 M urea concentration, weobserved large structural deviations. This may be due to unfolding ofILK in this concentration range of urea. The ILK193–446 attained lesstight packing at higher urea concentration. Also, the internal residues

Fig. 9. 2D projection of trajectories. The 2D projections of trajectories on eigenvectors showed d(F) 10.0 M urea concentrations.

of ILK193–446 were exposed to solvent due to denaturation (conforma-tional change) of the protein on the addition of urea. Further, the hydro-gen bond plot suggested that the addition of urea decreases the numberof hydrogen bonds between ILK193–446 and water molecules. At thesame time as the concentration of urea increases, the ILK forms morenumbers of hydrogen bonds with urea molecules.

What urea does to proteins has been known for a very long time, e.g.,high concentrations of urea denature proteins leading to loss in theirfunctional activity. A question that has been asked since the early1950s is: How does urea denature proteins? A number of mechanismsof urea-induced denaturation have been proposed, which can be di-vided into two broad categories. One, urea interacts and denatures pro-teins by itself (direct mechanism), and second, urea acts indirectly byaltering the solvent environment, thereby mitigating the hydrophobiceffect and facilitating the exposure of residues in the hydrophobic core(indirect mechanism). Earlier studies suggested direct interaction be-tween urea and peptide backbone and between urea and side chains(e.g., see [73,82]). SomeMD simulation studies have also supported di-rect interaction between urea and all protein groups (e.g., see [83]). Aspointed out by Schellman [84], concept of specific binding is not logicalin the case of protein-urea system, for during denaturation/renaturationexperiments one uses very dilute protein solution and very high ureaconcentration. According to indirect mechanism, urea decreases thestructure of water, i.e., urea acts as a chaotrope. Hydrophobic groupsare said to increase the structure of water but this effect can becounteracted by the structure breaking effect of urea and thereforethere is a gain in entropy associated with the transfer of hydrocarbonsfrom water to a urea solution, leading to protein denaturation [85,86].This study supports the indirect mechanism of denaturation by urea.

At present, general mechanism of protein denaturation by urea isnot settled, for most of arguments regarding a mechanism there arecounter arguments. Thus, a picture for how urea denatures proteins is

ifferent projections of ILK193–446 at (A) 0.0M, (B) 2.0M, (C) 4.0M, (D) 6.0M, (E) 8.0M and

217S.B. Syed et al. / International Journal of Biological Macromolecules 111 (2018) 208–218

that it denatures proteins via both direct and indirectmechanisms. Ureaincorporates itself well in the hydrogen-bond network of water; and ithas a higher van der Waal site density than water and can accept anddonate hydrogen bonds. Urea accumulates in the vicinity of the proteindue to preferential interaction. The unfolded protein has morefavourable interactions (hydrogen bonding, hydrophobic interaction,electrostatic interaction, van derWaals interaction) than the folded pro-tein [71,86–89].

5. Conclusions

We have successfully cloned, expressed and purified the kinase do-main of ILK to see its thermodynamic stability during urea-induced de-naturation. Exploring the dynamics of folding pathway is useful tounderstand the mechanism of protein folding in natural or disease re-lated process. It would be helpful to understand and enhance ourknowledge towards the folding behaviour of ILK193–446 in different cel-lular environments. In the far-UV region, the native ILK193–446 showedtwo well resolved negative peaks at 208 and 222 nm. The signal at222 was more predominant indicating more structural integrity of theprotein. At nearly 7 M urea ILK193–446 loses its secondary structurewhichwas evident from the complete disappearance of the characteris-tic peak in far-UV spectra. When ILK protein was excited at 292 nm itshowed the fluorescence emissionmaximum(λmax) at 344nm in nativestate. However, it shifts to 356 nm with a decrease in fluorescence in-tensity (λmax) under complete denaturation. The structural changeswere further analysed by 100 nsMD simulation, and awell-defined cor-relation was found. This is the first report on the structural studies onthe kinase domain of ILK to get mechanistic insights into the structureand stability using a combined spectroscopic and MD simulationstudies.

Acknowledgements

SSB is thankful to Department of Biotechnology (India) for the awardof fellowship (DBT-RA). SHK is thankful to CSIR (India) for Senior Re-search Fellowship. MIH and FA thank to the Department of Scienceand Technology (India) (EMR/2015/002372) for financial support. FAthanks the IndianNational Science Academy for the award of Senior Sci-entist Position. FIK and KAL thank to the Centre for High PerformanceComputing (CHPC), South Africa. SS is thankful to University GrantsCommission (India), for the award of Dr. D. S. Kothari Fellowship. Har-vard University-plasmid facility is acknowledged providing the ILKgene. We thank Department of Science and Technology, India (SR/FST/LSI-541/2012) for FIST support.

Conflict of interest

Authors declare there is no conflict of interest.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijbiomac.2017.12.164.

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