- Email: [email protected]
Investigating the folding pathway and substrate induced conformational changes in B. malayi Guanylate kinase
Accepted Manuscript Title: Investigating the folding pathway and substrate induced conformational changes in B. malayi Guanylate kinase Author: Smita Gupta Sunita Yadav Venkatesan Suryanarayanan Sanjeev K. Singh Jitendra K. Saxena PII: DOI: Reference:
S0141-8130(16)31891-8 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.10.008 BIOMAC 6588
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
13-2-2016 1-10-2016 5-10-2016
Please cite this article as: Smita Gupta, Sunita Yadav, Venkatesan Suryanarayanan, Sanjeev K.Singh, Jitendra K.Saxena, Investigating the folding pathway and substrate induced conformational changes in B.malayi Guanylate kinase, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.10.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Investigating the folding pathway and substrate induced conformational changes in B. malayi Guanylate kinase Smita Gupta1, Sunita Yadav1, Venkatesan Suryanarayanan2, Sanjeev K. Singh2, Jitendra K. Saxena1* 1
Division of Biochemistry, CSIR- Central Drug Research Institute, Lucknow-226031, Uttar
Pradesh, India 2
Computer Aided Drug Design and Molecular Modeling Lab, Department of Bioinformatics, Alagappa University, Karaikudi, Tamilnadu, India
Abbreviations NMP kinase- Nucleoside monophosphate kinase BmGK - Brugia malayi Guanylate kinase SEC- Size Exclusion Chromatography GdnCl- Guanidine hydrochloride MS- Molecular Simulation
*
Corresponding author:
Jitendra K. Saxena Chief scientist Division of Biochemistry CSIR- Central Drug Research Institute BS10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow- 226031, Uttar Pradesh, India Telephone: +91 0522 2625932 Fax: +91 0522 2623405 E-mail: [email protected]
Highlights
Detailed biophysical characterization of BmGK carried out for the first time. Folding pathway of BmGK is altered upon binding of substrates (GMP & ATP). Binding of substrates stabilises BmGK. The binding interaction of Arg145 with GMP shows a switch over to ATP after 40ns simulation of BmGK-GMP-ATP ternary complex. Addition of ATP also results in increase in fluctuation in BmGK-GMP-ATP complex in the region from 130-150 residues.
ABSTRACT Guanylate kinase is one of the key enzymes in nucleotide biosynthesis. The study highlights the structural and functional properties of Brugia malayi Guanylate kinase (BmGK) in the presence of chemical denaturants. An inactive, partially unfolded, dimeric intermediate was observed at 1-2M urea while GdnCl unfolding showed monomer molten globule like intermediate at 0.8-1.0M. The results also illustrate the protective role of substrates in maintaining the integrity of the enzyme. The thermo stability of protein was found to be significantly enhanced in the presence of the substrates. Furthermore, binding of the substrates, GMP and ATP to BmGK changed its GdnCl induced unfolding pattern. Docking and molecular dynamic simulation performed for native BmGK, BmGK bound to GMP and GMP+ATP showed change in the fluctuation in the region between 130-150 residues. Arg134 lost its interaction with GMP and Arg145 interaction shifted to ATP after 40ns simulation upon binding of ATP to BmGK-GMP complex. We, thus, propose the importance of specific rearrangements contributed by binding of substrates which participate in the overall stability of the protein. The work here emphasizes on detailed biophysical characterization of BmGK along with the significant role of substrates in modulating the structural and functional properties of BmGK.
Keywords: B. malayi Guanylate Kinase; unfolding; Molten globule
1. Introduction Understanding the structure-function relationships of an enzyme under different conditions is fundamentally important for both theoretical and applicative aspects. Such studies may provide insight into the molecular basis of the stability of the enzyme. Denaturation is a useful means for studying the characteristics of protein folding and has given a better insight into the mechanism of the folding process [1-3]. Exposure of proteins to denaturing agents such as extremes of pH, temperature, high ionic strength, chemical denaturants and organic solvents has been utilized to study the unfolding/folding pathway [4-6]. Though partially folded forms of numerous proteins have been studied, it has yet not been possible to generalize the concept of „intermediate states‟ as universal equilibrium folding intermediates. However, characterization of protein folding intermediates is helpful in identifying and understanding various transition steps and forms [7]. A compact intermediate state, which possesses a high degree of secondary structure and a fluctuating tertiary structure, has been described in several recent studies under various denaturing conditions [8-11]. Ligand binding to proteins is a subject of interest to explain one of the most intriguing and perplexing question in structural and molecular biology i.e. the problem of protein folding into a unique, compact, highly ordered and functionally active form and especially the role of ligands in the structure and in the stabilization of proteins in their native states. The interaction of proteins with small ligands is associated with increase in protein thermostability due to the coupling of binding with unfolding process [12-15]. The binding of proteins to small molecules and protein– protein interactions are key processes in cellular biochemistry. Ligand binding results in change in the conformation of the target protein which in turn produces a given response. Since biological processes are carried out through binding events, therefore the induced structural changes upon ligand binding have been found to be a matter of extensive studies [12]. Guanylate kinase, GK (ATP:GMP phosphotransferases, EC 2.7.4.8) , a nucleoside monophosphate kinase (NMP kinase), catalyzes the reversible transfer of the terminal phosphoryl group from ATP to guanosine 5- monophosphate (GMP) substrate, subsequently releasing guanosine diphosphate (GDP) as product. This is a crucial intermediate step in RNA or DNA synthesis foregoing the formation of the key nucleotide guanosine triphosphate (GTP) or its deoxy form dGTP [16]. The structure of Guanylate kinase comprises of NMP binding region, CORE and the LID region. Crystal structures have been solved for enzymes from many organisms including Saccharomyces cerevisiae GK which is available in both nucleotide free form and in complex with GMP and also for mouse GK in an abortive complex with ADP and GMP (mGK-GMP-ADP) [17, 18]. This investigation describes the biophysical characterization of BmGK, as an endeavour of understanding the structure-function relationship, the basis and rationale of its distinct physicochemical properties and the folding unfolding mechanism. The study also reports the changes in the conformation and dynamic behaviour of BmGK on binding to substrates. Very little information about the folding aspects of Guanylate kinase is available; hence a detailed analysis of native, intermediate and denatured states of BmGK was carried out by measuring the intrinsic tryptophan fluorescence measurement, near & far-UV CD
analysis, 1- aniline-8- naphthalene sulphonic acid (ANS) binding and quenching of intrinsic tryptophan fluorescence. Large-scale conformational alterations are thought to mediate allosteric regulations, which are related with protein function in signal transduction, immune response, and enzymatic activity [19]. Thus, a fundamental problem is to understand the mechanism for the conformational transitions in BmGK upon binding of substrates which is not known. The relationship between protein stability upon substrate binding and the role of substrate in modulating the intrinsic flexibility of the enzyme using various spectroscopic techniques and bioinformatics tools such as molecular dynamic simulation have been carried out. Such a comprehensive and consistent description for BmGK is largely complementary to previous experimental studies carried out on this enzyme. 2. Materials and methods 2.1. Materials All the reagents were purchased from Sigma–Aldrich Chemical, USA unless specifically mentioned. Molecular weight markers for SDS–PAGE were purchased from MBI Fermentas, Maryland, USA. Ni–NTA agarose matrix was procured from Qiagen, Germany. 2.2. Protein expression, purification and activity assay Guanylate Kinase was expressed in E. coli BL21 (DE3) and purified to homogeneity by NiNTA affinity chromatography as described earlier [20]. The purity of the protein was checked on 12% SDS PAGE. Enzyme activity was measured according to the method of Agrawal et. al [21]. For studies involving denaturation with urea or GdnCl, the enzyme was incubated in assay buffer containing desired concentrations of the denaturants for 1 h. It was determined that the auxiliary enzymes (pyruvate kinase and lactate dehydrogenase) during assay remained active under the experimental conditions. The reversibility of the transitions was determined by incubating 20μM BmGK for 1 h in the presence of the different concentrations of denaturants. The samples were then diluted with 50mM Tris buffer pH 7.5 to attain the final concentration of 10mM urea and 2.5mM GdnCl respectively. The diluted enzyme mixture was then immediately used for the activity. The loss of enzyme activity as a function of temperature was followed, in the presence and absence of substrates in 50mM Tris-Cl buffer (pH 7.5). The enzyme samples were incubated for 15 min at different temperatures 25°C to 55°C and after cooling to 4°C, the residual activity was measured on spectrophotometer at 25°C. 2.3. Fluorescence spectroscopy Fluorescence spectra of BmGK were recorded in Perkin-Elmer LS 5B luminescence spectrometer with a 5mm path-length quartz cell. Samples containing different concentrations of urea or GdnCl were equilibrated for 5h in 50mM sodium phosphate buffer (pH 7.0) at 25°C. The spectra were normalized for the solvent contribution to fluorescence. Titrations for binding of nucleotides (GMP and ATP) with BmGK were carried out by adding small aliquots (0-2mM) to 5μM of BmGK in buffer containing 50mM phosphate buffer (pH 7.0)
after an equilibration period of 2 h. For tryptophan fluorescence measurements, an excitation wavelength of 290 nm was used and the emission was recorded between 300-400 nm. ANS binding was estimated by incubating BmGK previously denatured at different concentrations of urea or GdnCl with optimised concentration (100μM) of ANS at 25°C. The excitation wavelength was 380 nm and emission was recorded between 400 nm- 600 nm. 2.4. Fluorescence quenching experiment Acrylamide quenching experiments were performed by adding acrylamide to BmGK denatured at different concentrations of urea or GdnCl for 5h and the fluorescence quenching data were analyzed according to the Stern–Volmer equation [22]. 2.5. Circular dichroism measurements CD experiments were performed with a JASCO J810 spectropolarimeter (equipped with peltier temperature controller system) in a 1cm path length cell at 25oC. The far UV CD spectra were measured for 5μM BmGK treated with desired concentration of denaturants for 5h in 50mM sodium phosphate buffer (pH 7.0) at 25°C. Readings obtained were normalized by subtracting the baseline observed for the buffer with similar denaturant concentration under similar conditions. The tertiary structure of the enzyme (20μM) was monitored in the near UV region in wavelength range 250–350 nm using a cell of path length of 0.5 cm. Each spectrum was recorded as an average of three scans. For binding experiments, the CD spectra of 5μM BmGK were measured in buffer containing 50mM phosphate buffer (pH 7.0) with 0.25mM GMP and (0.25mM GMP+0.25mM ATP). The values obtained were normalized by subtracting the baseline recorded for the buffer having same concentration of substrate under similar conditions. Thermal denaturation was studied by monitoring changes in molar ellipticity at 222 nm as a function of temperature from 30oC to 85oC. 2.6. Limited proteolysis BmGK (0.5 mg/ml) was subjected to limited proteolysis with trypsin for 1 h at 37oC with protease to protein ratio at 1:500 (w/w). Samples were preincubated with 0.25mM substrates for 1hr before trypsin digestion. The reaction was stopped by adding PMSF (1mM) in the reaction mixture and samples were analyzed on 15 % SDS-PAGE. 2.7. Size Exclusion chromatography Gel filtration experiments were carried out on Superdex TM 75, 10/300GL column on AKTA FPLC (GE Health care) pre-calibrated with standard molecular weight markers. The column was pre-equilibrated with 50mM sodium phosphate buffer (pH 7.0) and protein was eluted with or without the desired denaturant concentration at 25oC at a flow rate of 0.3 ml/min with detection at 280 nm. 2.8. Crosslinking using glutaraldehyde 100μg of protein denatured with desired concentration of urea or GdnCl were incubated with 5μl of 1% glutaraldehyde in a 100μl reaction volume in 50mM sodium phosphate buffer
(pH7.0) for 30 min at 37◦C. The reactions were stopped by adding 5μl of 1M Tris–HCl pH 8.0 in the reaction mixture and the samples were analysed on 10% SDS-PAGE. 2.9. Binding site prediction and Molecular Docking BmGK protein docked with GMP was taken from the previous studies [20] and the protein was prepared through Protein Preparation Wizard (Schrodinger, LLC, New York, NY, 2014), implemented in Maestro 9.6. GMP and ATP were downloaded from Pubchem and they were prepared through LigPrep (Suite 2014: LigPrep, version 2.9, Schrodinger, LLC, New York, NY, 2014) module from Schrodinger. Binding site of GMP was noted from the previous studies and it was docked in the same site after getting the equilibrated structure of BmGK from Molecular dynamics simulation using „extra precision‟ glide docking (Glide XP) method (Suite 2014: Glide, version 6.2, Schrodinger, LLC, New York, NY, 2014). Further, the binding site for ATP was investigated through SiteMap (Suite 2014: SiteMap, version 3.0, Schrodinger, LLC, New York, NY, 2014), which generates binding site‟s characteristic information using novel search and analytical facilities. ATP molecule was also docked in the predicted binding site of BmGK protein using Glide XP docking method. In docking calculations, to soften the potential for non polar parts of the receptor, we scaled the van der Waals radii of receptor atoms by 1.00 with a partial atomic charge of 0.25. The default grid size was adopted from the Glide program, and no constraints were applied for all the docking studies. Glide generates conformations internally and passes these through a series of filters. The lowest-energy docked complex was found in the majority of similar docking conformations, which was selected for further study. 2.10. Molecular Dynamics Simulation BmGK Apoprotein and its complexes with GMP and GMP-ATP were carried for computing MD simulation by using Desmond (Suite 2014: Desmond, version 3.7, Schrodinger, LLC, New York, NY, 2014) with Optimized Potentials for Liquid Simulations (OPLS) 2005 force field. All the three systems were imported in Desmond‟s System Builder panel and solvated in an orthorhombic box of TIP3P water molecules and neutralized using appropriate number of counter ions and 0.15M of salt concentration. The system was subjected to the local energy minimization using a hybrid method of the steepest decent and the limited-memory Broyden– Fletcher– Goldfarb–Shanno (LBFGS) algorithms with a maximum of 5000 steps until a gradient threshold (25kcal/mol/Å) was reached. The simulation system was relaxed by constant NPT (number of atoms N, pressure P, temperature T) ensemble condition to generate simulation data for post-simulation analyses. The temperature value was defined as 300 K for the whole simulation process using Nose–Hoover thermostats and stable atmospheric pressure (1atm) carried out by Martina– Tobias–Klein barostat method. The multi-time step RESPA integrator algorithm was used to investigate the equation of motion in dynamics. The time step for bonded, “near” non bonded, and “far” non bonded interactions were 2, 2, and 6fs, respectively. SHAKE algorithm was employed to constrain the atoms which are involved in hydrogen bond interaction. The short range electrostatic and Lennard– Jones interactions were estimated by setting up the cutoff value as 9- Å radius. The longrange electrostatic interactions were evaluated by using particle mesh Ewald (PME) method with the simulation process using periodic boundary conditions (PBC). Energy and trajectory
analysis data were documented at 1.2- and 4.8-ps intervals respectively for statistical analysis. The Final production MD was carried for 100000ps for all three systems and the results were analyzed using Simulation event analysis and Simulation Interaction diagram available in Desmond module [23, 24]. 3. Results & Discussion 3.1. Unfolding of BmGK with urea and GdnCl 3.1.1. Activity is lost prior to structural denaturation Activity of the enzyme is considered the most sensitive probe to study the changes in an enzyme‟s conformation as it reflects subtle readjustments at the active site, allowing very small conformational variations in structure to be detected [25,26]. Fig. 1 shows the activity of BmGK with increasing concentrations of denaturants (urea/GdnCl). The activity was lost at much lower concentration of urea or GdnCl at which no detectable changes in either secondary structure or intrinsic fluorescence occurred. An early loss of enzymatic activity (<0.1M GdnCl) has been detected prior to other changes which may be due to binding of guanidinium ions to BmGK disturbing the electrostatic interactions involved in the stabilization of the active-site conformation, as observed in several cases [11]. It has been reported that restored enzymatic activity is the real marker for assessing the refolding not only the structural properties. Fig. S1 summarizes the refolding efficiency of GdnCl or urea treated BmGK. The observations demonstrated that complete refolding to native BmGK can be achieved after denaturation of BmGK with low denaturant concentrations, whereas denaturation with high denaturant concentration is irreversible. 3.1.2. Changes in tertiary and secondary structure of BmGK upon unfolding Fluorescence spectra provide a sensitive means of characterizing proteins and their conformation. The spectrum is determined by the polarity of the tryptophan and tyrosine residues‟ environment and by their specific interactions [27]. The tryptophan fluorescence emission maxima of BmGK showed red shift in wavelength with increasing concentration of urea or GdnCl (Fig. 2a & b). Two separate transitions were observed for BmGK unfolding in presence of urea or GdnCl. An intermediate state was found to be populated at 1-2M urea. Above 4M urea, the complete unfolding marked by a prominent red shift in wavelength was observed. The results indicated that the single tryptophan residue of BmGK is significantly exposed to the solvent. Similarly, a two state transition was observed during GdnCl unfolding of BmGK. An initial transition between 0 and 0.8M GdnCl and the plateau region at 0.8- 1M GdnCl followed by sharp transition ending into complete unfolding of BmGK at 3M GdnCl was observed. BmGK showed a typical α/β type secondary structure composition as observed by the far-UV CD spectrum of the protein. Changes in the secondary structure of BmGK induced by urea or GdnCl were examined by far UV- CD measurements (Fig. 2c & 2d). Addition of 2M urea and 0.5M GdnCl caused only a minor decrease in ellipticity at 222 nm. However, a significant drop in ellipticity was observed at 4M urea and 2.5M GdnCl. The near-UV CD spectrum of BmGK was characterized by several positive peaks and shoulders suggesting that
the aromatic residues are incorporated into a rigid tertiary structure giving rise to aromatic CD signals (Fig. 2e & 2f). The CD signal in the near-UV region at 1M GdnCl is significantly lower than native state of BmGK suggesting that the addition of 1M GdnCl induces a looser and more flexible environment nearby aromatic residues. Similarly at 1-2M urea there was change in the tertiary structure of BmGK as evident in near UV region of CD spectra. Addition of 5M urea and 3M GdnCl abolished the far and near UV CD signals, indicating extensive or complete unfolding of BmGK. During unfolding, the non-coincidence of the ellipticity curve with those monitoring changes in enzymatic activity and tertiary structure also suggest the existence of intermediate states during urea or GdnCl denaturation. Although at 1-2 M urea, a 40-50% loss of tertiary structure was observed in tryptophan fluorescence spectra but it still retained 75-85% secondary contents as can be seen in far UV CD spectra. All the denaturation experiments were also performed as a function of enzyme concentration (1μM, 2μM 4μM, 6μM) and there was no change in the denaturation profile of BmGK either with urea or GdnCl (data not shown). 3.1.3. ANS binding to BmGK ANS is a fluorescent dye that binds to hydrophobic regions within the proteins. Moltenglobule (MG) intermediate states expose a significant hydrophobic core to the solvent [10, 28]. Hence, ANS binds strongly to the MG state of proteins and shows increased fluorescence intensity. To gain an insight into the unfolding intermediate state, the binding of ANS to BmGK was studied as a function of increasing concentration of urea or GdnCl (Fig. 3a & b). It was observed that ANS binding did not show any increase in fluorescence intensity at lower concentration of urea. This further strengthened the absence of MG like properties for intermediate at 1-2M urea while an increase in ANS fluorescence intensity could be observed at 1M GdnCl as shown in Fig. 3b. Thus, the presence of an intermediate state possessing the characteristics of molten globule could be speculated during the unfolding of BmGK with GdnCl showing compact secondary as well as loose tertiary structure as observed by CD spectroscopy and enhanced ANS fluorescence intensity. 3.1.4. Fluorescence quenching of tryptophan residue of BmGK Fluorescence quenching of the tryptophan residues by quenchers has been shown to be useful in getting information about the solvent accessibility of these residues in proteins and the polarity of their microenvironment [29]. The quenching of fluorescence intensity of tryptophan residue under varying concentrations of urea or GdnCl was monitored in order to probe accessibility of acrylamide to the protein core at different conditions (Fig. 3c & d). It was observed that urea below 4M concentration did not bring any prominent change in fluorescence intensity while at 7M concentration significant quenching was monitored. BmGK treated with 0.6 and 1M GdnCl showed increase in fluorescence quenching as compared to native protein indicating an increased exposure of aromatic hydrophobic residues. The presence of molten globule state at 1.0 M GdnCl with characteristics compact structure and exposed hydrophobic residues as indicated by enhanced ANS fluorescence intensity further supports the quenching result. The Stern–Volmer quenching constants for the urea and GdnCl denatured states are shown in Table 1.
3.1.5. Changes in the quaternary structure of BmGK upon urea and GdnCl induced unfolding Size Exclusion chromatography (SEC) experiments were performed to analyze changes in the quaternary structure of the enzyme in the presence of increasing concentration of urea or GdnCl as shown in Fig. 4. For native BmGK, single peak at 12.27 ml was observed. This is similar to the peak observed for ovalbumin corresponding to retention volume of 12.3 ml with molecular weight of 43KDa. This indicates BmGK is dimeric in nature. For 0.2M GdnCl treated BmGK, single peak with retention volume of 12.8 ml was observed. Two peaks have been observed for 0.8M GdnCl corresponding to retention volume of 9.1ml and 13.0ml for aggregated protein species and compact monomer species of BmGK, respectively. However at 1M GdnCl single peak with retention volume 13.5 ml has been observed corresponding to monomer BmGK. Molten globule monomers have been reported as intermediates in the dissociation of dimeric proteins such as human superoxide dismutase [30], rabbit muscle creatine kinase [11], yeast glutathione reductase [31]. Fig. 4b shows urea induced changes in BmGK. For 1M urea denatured BmGK, two peaks at 12.27 ml and 10.9 ml were observed designated as peak A and B, respectively. The peak A corresponds to native dimeric form of enzyme while peak B represents the slight expansion of the enzyme structure forming highly solvated structure, thus enhanced hydrodynamic radii as determined by size exclusion chromatography. Such a situation occurs only when the dimeric enzyme undergoes progressive unfolding without dissociation into monomers [32]. The preferential interactions at the external regions of BmGK might be the principal reason for a local unfolding of the protein structure, far from the dimeric interface [31]. Further increases in urea concentration to 2M resulted in single peak at 10.5ml with loss or complete vanishing of dimeric form peak of BmGK at 12.27 ml. However, at 4 M urea and 3M GdnCl the protein eluted in void volume at retention volume of 7.8 ml, which was due to complete unfolding of the enzyme resulting in significant enhancement in the hydrodynamic radii of the enzyme. The results were supported by glutaraldehyde crosslinking data as evident in Fig. 4c&d. 3.2. Substrate induced changes within BmGK 3.2.1. Binding of substrate to BmGK alters the folding pathway of BmGK The protein having several domains and each contributing to the parameter being followed shows a multi transitional denaturation curve depending on the method of following the unfolding process, reflecting the variable stabilities of the different domains. If a ligand is found to stabilize the overall structure of the protein, the denaturation curve will be shifted to higher concentrations of denaturant and, vice versa. Denaturation profile of BmGK was also assessed with urea and GdnCl in presence of bound substrates GMP and ATP. It was observed from the results of fluorescence and CD spectroscopy that urea induced unfolding of BmGK was unaffected by binding of substrates (data not shown) while substrate binding stabilized the enzyme and protected its denaturation in presence of GdnCl (Fig. 6a & 6b). This suggests that indeed the electrostatic interactions are affected upon binding of substrate which play a role in stabilization of structure of BmGK.
The residual activity of BmGK as a function of temperature in the presence and absence of substrates is shown in Fig. 6c which also suggests that the enzyme was stabilized in presence of substrate. It was observed that activity of substrate bound BmGK was retained even at 50oC as compared to unbound form. The thermal unfolding of BmGK was also characterized by monitoring the loss of secondary structure of enzyme with rise in temperature [33]. Fig. 6d summarizes the changes in ellipticity at 222 nm of BmGK with increasing temperature, where two distinct transitions were observed between 30-85°C. These transitions were in the temperature regions from 45°C to 55°C and 65°C to 70°C and centered at about 50°C and 65°C, respectively. In presence of substrates i.e. GMP & ATP, the transition temperature shifted to higher temperature. Thus, thermal denaturation suggests that BmGK molecule contains two distinct structural domains of significantly different thermal stabilities which are influenced upon binding of substrates.
3.2.2. Tertiary and Secondary structural changes in BmGK upon binding of substrates A decrease in fluorescence intensity after binding of GMP was observed (Fig. 7a). The large reduction in the intrinsic fluorescence of BmGK is observed upon GMP binding which suggests that the nucleotide is in close juxtaposition to the single Trp51 residue in the protein [34].The lesser reduction during ATP binding suggests a slightly altered binding mode for the latter as compared to GMP (Fig. 7b). The fluorescence data were further analyzed according to the Chipman equation [35]. The Ka obtained was 2.5 X 104 M-1 for GMP binding (data not shown). Fig. 7c shows that addition of substrates also resulted in significant changes in both shape and intensity of the CD spectra, most strikingly in an increase of the CD signal around 222 nm. The significant increase in the secondary structure of BmGK upon binding of substrates provided further evidence of structural rearrangements, suggesting that the enzyme undergoes from a less ordered conformation to a more stable structured one. The susceptibility of BmGK under different conditions to trypsin proteolysis provided further insight into local conformational flexibility of the protein. The sequence analysis of BmGK revealed 11Arg and 17 Lys residues which depending on their accessibility in tertiary structure could serve as site for trypsin cleavage. The effect of substrates on the protection of BmGK from trypsin digestion is shown in Fig. 8. This data shows that binding of both substrates, GMP and ATP to the enzyme protected it to comparable extent suggesting that the proximity of binding in the active site of BmGK induces a conformational change and hence comparable level of protection from trypsin proteolysis has been observed. Based on the observations it could be speculated that GMP brings out a large conformational change upon binding as it protected the enzyme from tryptic digestion but complete protection occurs only when both GMP and ATP are bound to BmGK. 3.2.3. Binding site prediction and Molecular Docking of BmGK As currently high resolution X-ray crystallographic structure for BmGK is unavailable, 3D homology modeling may be an alternative to gain insights into potential substrate binding sites and the mechanisms of substrate interaction with the protein [36]. Equilibrated BmGK structure was used for the docking calculations with GMP and ATP. Initially, GMP was
docked based on the binding site information obtained from previous study. After docking, binding site for ATP was predicted through Sitemap and the best binding site was found based on the site score from the results provided in Table 2. Fig. S2 (a& b) highlights the predicted binding site region in Maroon colour surface with white dots. Further, ATP was docked with BmGK-GMP complex on the predicted binding site. Result of XP docking of both BmGK-GMP and BmGK-GMP-ATP complex were shown in Table 3. TwoDimensional interaction diagram of docked complexes were shown in Fig. 9, which exposes that the addition of ATP cause changes in interaction of GMP with BmGK. It was shown that GMP is interacting with residues like Arg38, Arg41, Tyr78, Asp98, Gln102, Arg134 and Arg145 in BmGK-GMP complex (Fig. 9a). In BmGK-GMP-ATP complex, the interacting residues for GMP have reduced to Arg38, Arg41, Glu69, Tyr78 and Arg145. Interacting residues of BmGK-GMP-ATP complex with ATP were found to be Ser10, Gly11, Gly12, Gly13, Lys14, Ser15, Tyr50, Arg134 and Arg145. GMP has lost its interaction with Asp98, Gln102 and Arg134 due to the addition of ATP in the binding site region whereas Arg134 was rather found to be interacting with ATP in BmGK-GMP-ATP complex. Interestingly, Arg145 was found to be interacting with both GMP and ATP in BmGK-GMP-ATP complex. Analogous Arg in mouse Guanylate kinase has been reported to act as a clamp by interacting with both phosphates of GMP and ATP [17]. Fig. S3 shows the closer view of GMP (Blue colour) and ATP (Brown colour) binding to the surface encoded binding site of BmGK. The interaction stability analysis of both BmGK-GMP and BmGK-GMP-ATP complex through MDS would bring clarity in substrate specificity of BmGK [37]. 3.2.4.Structural changes observed by molecular dynamics simulation Conformational changes and dynamics of proteins are monitored by molecular dynamic simulation. This method has been utilized to substantiate the structural changes in BmGK revealed by other probes and also to narrow down the substrate-induced changes to specific regions in the protein [38]. BmGK apoprotein was initially simulated for 100000ps to bring out well equilibrated structure for further analysis. After getting the equilibrated BmGK structure, docking calculations were performed with GMP and ATP. Two complexes of BmGK-GMP and BmGK-GMP-ATP were carried for further MDS for 100000ps of time period to procure a better understanding in structural changes of BmGK due to the interaction of GMP and ATP. The change in the structural integrity has been analyzed through calculating the root mean square deviation (RMSD) and root mean square fluctuations (RMSF) over backbone atoms. Fig. 10a shows the RMSD graph of all three MD simulations over 100000ps of time period. BmGK apoprotein is found to have more fluctuations in RMSD and it has attained stability around 6Å nearly at the end of simulation. The equilibrated BmGK structure was carried for further molecular docking and molecular dynamics simulation studies. RMSD of BmGK-GMP complex is found to attain stability at 3Å after 10000ps and maintained till the end, whereas RMSD of BmGK-GMP-ATP complex is found to have minor fluctuations progressively and after 60000ps the RMSD graph fluctuates around 4Å. RMSF graph shown in Fig. 10b clearly supports the result of RMSD of all three simulations. Difference in RMSF between the apoprotein and complex were seen around active site residues. It reveals that apoprotein gains stability due to addition of substrate GMP and ATP. Whereas the difference
in RMSF from 130 to 150 residue between BmGK-GMP and BmGK-GMP-ATP complex reveals that increased fluctuation in BmGK-GMP-ATP complex may be due to the change in interacting residues during the addition of ATP in the binding site. Further analysis of solvent accessible surface area (SASA) in bound receptor reveals that both complexes maintain stability during the simulation time period (Fig. 10c). Interaction of substrates with protein play major role in maintaining the stability of complex [37]. The time line of protein ligand contact is depicted in Fig. 11 which shows the interaction of GMP in BmGK-GMP (a) and BmGK-GMP-ATP (b) complex and also the interaction of ATP in BMGK-GMP-ATP complex (c). It uncovers the occurrence of interaction by each interacting residues in protein-ligand complex throughout the simulation. In Fig. 11a, residues like Arg41, Tyr78, Asp98, Arg134 and Arg145 are found to maintain the interaction throughout the simulation. Fig. 11b shows the interaction of Asp98, Arg134 were lost and the interaction of Arg38, Tyr50 and Phe73 were gained which clearly revealed the change in interaction of GMP in BmGK-GMP-ATP complex due to the addition of ATP. Fig. 11c, shows that Gly11, Gly12, Gly13, Lys14, Ser15 and Arg134 residues of BmGKGMP-ATP complex were interacting ATP throughout the simulation whereas Lys144 shows interaction after 17ns and Arg145 shows interaction after 40ns. It is clear from the Fig. 11c that Arg134 which has lost interaction with GMP is found to interact with ATP in BmGKGMP-ATP complex. It is also clear from Fig. 11, Arg145 which has stable interaction with GMP in BmGK-GMP complex (Fig. 11a) is found to change its interaction from GMP to ATP which is depicted in Fig. 11b and 11c. Percentage of interaction by interacting residues was represented through histogram in Fig. 12 which includes both hydrogen bond and hydrophobic bond interaction. Changes in the interaction and structural integrity clearly support the possible dynamic conformational changes in active site of BmGK protein upon substrate binding. Thus, our study supports the notion that binding of GMP alone induces the closure of the GMP domain and subsequent co binding of ATP results in the fully closed conformation [39]. 4. Conclusion Since protein exert their functional activity in different conformations in the cell passing through different states, present work represents the first report of detailed biophysical characterization of B. malayi Guanylate kinase illustrating different states during its folding pathway as well as the changes induced within the enzyme upon binding with its substrates. The study has provided comparative account of action of urea and GdnCl on structural, functional and stability properties of BmGK (Fig. 5). On the basis of our results, we propose that the unfolding of native BmGK proceeds with the initial formation of monomer molten globule like intermediate during GdnCl denaturation while urea denaturation proceeds with formation of expanded dimeric intermediate with loose tertiary contacts and disturbed structural rearrangements leading to final unfolded state. Various aspects of protein stability, flexibility & dynamics together in conjunction with each other have been studied which give a thorough picture of substrate (GMP and ATP) induced changes within BmGK. Different methods indicated that a conformational change takes place to reorganize the enzyme both
locally as well as globally for catalysis of reaction. It is evident from the results of molecular docking and molecular dynamic simulation studies that interaction of substrates like GMP and ATP brings high stability to BmGK. Thus, this study highlights the detailed unfolding pathway of BmGK and the conformational change in the protein upon binding of substrates probably leading to its transformation from open to closed state. Since detailed analysis of unfolding mechanism of any Guanylate kinase is not known till date, the overall results of the structural characterization of BmGK are noteworthy. This work could serve as a model to understand dynamic aspect of substrate binding for catalysis in absence of its crystal structure. Conflict of interest None declared.
Acknowledgement We gratefully acknowledge Council of Scientific and Industrial Research (CSIR), New Delhi, for offering a Senior Research fellowship to Smita Gupta to carry out this work. We would like to extend our gratitude to Director, CDRI for his invaluable support.
REFERENCES
[1] O.B. Ptitsyn, Adv Prot Chem, 47 (1995) 83-229. [2] C. Camilloni, A.G. Rocco, I. Eberini, E. Gianazza, R.A. Broglia, G. Tiana, Biophys J, 94 (2008) 4654-4661. [3] D.J. Fan, Y.W. Ding, J.M. Zhou, Biochim Biophys Acta, 6 (2009) 944-952. [4] V.K. Dubey, M. Jagannadham, Biochemistry, 42 (2003) 12287-12297. [5] Y. Kumar, S. Muzammil, S. Tayyab, J Biochem, 138 (2005) 335-341. [6] C.N. Pace, S. Trevino, E. Prabhakaran, J.M. Scholtz, Philos Trans R Soc Lond B Biol Sci, 359 (2004)1225-1234. [7] Y. Goto, N. Takahashi, A.L. Fink, Biochemistry, 29 (1990) 3480-3488. [8] D.A. Dolgikh, R.I. Gilmanshin, E.V. Brazhnikov, V.E. Bychkova, G.V. Semisotnov, S. Venyaminov,O.B. Ptitsyn, FEBS Lett, 136 (1981) 311-315. [9] Y. Goto, A.L. Fink, Biochemistry, 28 (1989) 945-952. [10] G.V. Semisotnov, N.A. Rodionova, O.I. Razgulyaev, V.N. Uversky, A.F. Gripas, R.I. Gilmanshin, Biopolymers, 31 (1991) 119-128. [11] F. Couthon, E. Clottes, C. Ebel, C. Vial, Eur J Biochem, 234 (1995) 160-170. [12] M.S. Celej, G.G. Montich, G.D. Fidelio, Protein Sci, 12 (2003) 1496-1506. [13] J.F. Brandts, L.N. Lin, Biochemistry, 29 (1990) 6927-6940. [14] A. Shrake, P.D. Ross, J Biol Chem, 265 (1990) 5055-5059. [15] A. Shrake, P.D. Ross, Biopolymers, 32 (1992) 925-940. [16] M. Kandeel, M. Nakanishi, T. Ando, K. El-Shazly, T. Yosef, Y. Ueno, Y. Kitade, Mol Biochem Parasitol, 159 (2008) 130-133. [17] N. Sekulic, L. Shuvalova, O. Spangenberg, M. Konrad, A. Lavie, J Bio Chem, 277 (2002)30236-30243. [18] J. Blaszczyk, Y. Li, H. Yan, X. Ji, J Mol Biol, 307 (2001) 247-257. [19] J. Ping, P. Hao, Y. X. Li, J.F. Wang, BioMed Res Int, 2013 (2013) 628536. [20] S. Gupta, S. Yadav, N. Singh, A. Verma, I. Siddiqi, J.K. Saxena, Parasitology, 141 (2014)1341-1352. [21] K. Agarwal, R. Miech, R. Parks, Methods Enzymol, 51 (1978) 483-490. [22] M.R. Eftink, C.A. Ghiron, Anal Biochem, 114 (1981) 199-227. [23] V. Suryanarayanan, S.K. Singh, J Recept Signal Transduct , 35 (2015) 370-80. [24] S.K. Tripathi, S.K. Singh, Mol BioSyst, 10 (2014) 2189-2201. [25] J.J. Plomer, A. Gafni, Biochim Biophys Acta, 1122 (1992) 234-242. [26] P. Alam, G. Rabbani, G. Badr, B.M. Badr, R.H. Khan, Cell Biochem Biophys, 71 (2015) 1199-1206. [27] S. Hayashi, S. Nakamura, Biochim Biophys Acta, 657 (1981) 40-51. [28] Gimenez-Gallego, Eur J Biochem, 246 (1997) 328-335. [29] S. Pawar, V. Deshpande, Eur J Biochem, 267 (2000) 6331-6338. [30] N. Silva, Jr., E. Gratton, G. Mei, N. Rosato, R. Rusch, A. Finazzi-Agro, Biophys Chem, 48(1993) 171-182. [31] P.R. Louzada, A. Sebollela, M.E. Scaramello, S.T. Ferreira, Biophys J, 85 (2003) 32553261. [32] A.N. Bhatt, M.Y. Khan, V. Bhakuni, Protein Sci, 13 (2004) 2184-2195. [33] B. Ahmad, G. Muteeb, P. Alam, A. Varshney, N. Zaidi, M. Ishtikhar, G. Badr, M.H. Mahmoud, R.H. Khan, Int J Biol Macromolec, 75 (2015) 447-452. [34] A.S. Abdelhameed, P. Alam, R.H. Khan, J Biomol Struct Dyn, 34 (2016) 2037-2044.
[35] D.M. Chipman, V. Grisaro, N. Sharon, J Biol Chem, 242 (1967) 4388-4394. [36] M.J. Hosen, A. Zubaer, S. Thapa, B. Khadka, A. De Paepe, O.M. Vanakker, PloS One, 9 (2014) e102779. [37] P. Alam, S.K. Chaturvedi, T. Anwar, M.K. Siddiqi, M.R. Ajmal, G. Badr, M.H. Mahmoud, R.H. Khan, J Lumin, 164 (2015) 123-130. [38] X. Du, Y. Li, Y.L. Xia, S.M. Ai, J. Liang, P. Sang, X. L. Ji, S.Q. Liu, Int J Mol Sci, 17 (2016) 144. [39] C. Vonrhein, G.J. Schlauderer, G.E. Schulz, Structure, 3 (1995) 483-490.
Legends to figures: Figure 1 Changes in BmGK activity. Effect of increasing concentrations of (a) urea, and (b) GdnCl on BmGK activity. Enzyme activity observed for BmGK in absence of urea and GdnCl is taken as 100%. Figure 2 Urea and GdnCl induced changes in tertiary and secondary structure of BmGK. (a) The fraction of unfolded BmGK (fi - fo)/(fd - fo) versus urea concentration; fi is wavelength for sample, fo is the wavelength in the absence of urea, and fd is the wavelength at urea concentrations <5M.(b) Fraction unfolded of BmGK (fi - fo)/ (fd - fo) versus GdnCl concentration; fi is wavelength for a particular sample, fo is the wavelength in the absence of GdnCl, and fd is the wavelength at GdnCl concentrations <2.5M. Inset shows the fluorescence curves of native and different urea and GdnCl denatured BmGK samples. (c) Urea and (d) GdnCl induced changes in secondary structure as monitored by following the changes in CD ellipticity at 222 nm from far-UV CD curves at increasing concentrations of urea and GdnCl. Data are represented as the percentages of ellipticity at 222 nm, taking the value observed for native protein in absence of urea and GdnCl as 100%. Inset shows the CD curves of native and BmGK denatured at different concentrations of urea or GdnCl. Near-UV CD spectra of BmGK under native condition and denatured at varying concentration of urea (e) and GdnCl (f). Figure 3 Fluorescence intensity of ANS. Binding of ANS to BmGK at various denaturing concentrations of (a) urea and (b) GdnCl. Quenching of intrinsic fluorescence of BmGK by acrylamide at different (c) urea and (d) GdnCl concentrations, fo: fluorescence intensity in the absence of acrylamide, f: observed fluorescence intensity in the presence of acrylamide. Figure 4 Size-exclusion chromatographic profile on Superdex 75 HR column and SDS-PAGE profiles of glutaraldehyde crosslinking. The chromatographic profiles of native BmGK on a Superdex 75 HR 10/300 column on AKTA FPLC (Amersham Pharmacia Biotech, Sweden) at different (a) Urea and (b) GdnCl concentrations. All the curves have been displaced along the Y-axis for display purposes. (c) Glutaraldehyde crosslinking of BmGK. Lane 1- glutaraldehyde crosslinked native BmGK, Lane 2- glutaraldehyde crosslinked 0.2 M GdnCl treated BmGK, Lane 3- glutaraldehyde crosslinked 0.8M GdnCl treated BmGK, Lane 4- glutaraldehyde crosslinked 1M GdnCl treated BmGK, Lane 5-uncrosslinked native BmGK (d) Lanes 1–3 represent native BmGK, glutaraldehyde crosslinked 1M urea treated BmGK and glutaraldehyde crosslinked 3M urea treated BmGK, respectively. (e) Calibration curve of standard molecular weight markers viz., Ribonuclease A (13.7KDa), Chymotrypsinogen (25KDa), Ova-albumin (43KDa) and albumin (67KDa). Figure 5 Representation of unfolding pathway of BmGK in presence of Urea and GdnCl
Figure 6 Effect of substrate binding on structural and functional properties of BmGK (a) GdnCl induced unfolding in absence of substrates (squares) and in presence of GMP+ATP (circles) (b) Denaturation of BmGK with 0.1M, 0.8M & 3M GdnCl in absence and presence of substrates, S (GMP+ATP) as revealed by far UV-CD spectroscopy (c) Activity of BmGK at different temperatures in absence and in presence of substrates i.e. GMP and ATP (d) Thermo stability of BmGK measured by far UV CD spectroscopy at 222 nm at different temperatures in absence and presence of substrates. Figure 7 Conformational changes in BmGK induced by substrate binding. (a) Fluorescence emission spectra of BmGK in the presence of different concentrations of GMP (b) and ATP (c) Changes in the ellipticity of BmGK upon binding of substrate by far-UV CD spectroscopy Figure 8 Effect of substrate binding on trypsin cleavage pattern of BmGK. Trypsin to BmGK (1: 500) was used in the experiment and the fragments separated on 15% SDS PAGE were visualised by staining with Coomassie brilliant blue. The digestion time (in minutes) is indicated at the top of the panel while substrates added are mentioned at the bottom of the panel. Figure 9 2D interaction diagram of (a) GMP with BmGK and (b) GMP (left) and ATP (right) with BmGK. Figure 10 (a) RMSD graph of BmGK Apoprotein, BmGK-GMP and BmGK-GMP-ATP complexes over 100000ps of simulation.(b) RMSF graph of BmGK Apoprotein, BmGK-GMP and BmGK-GMP-ATP complexes over 100000ps of simulation. (c) SASA of bound receptor of BmGK-GMP and BmGK-GMP-ATP complexes over 100000ps of simulation Figure 11 Timeline of Protein-substrate contacts of (a) GMP in BmGK-GMP complex, (b) GMP in BmGK-GMP-ATP complex and (c) ATP in BmGK-GMP-ATP complex. Figure 12 Histogram of Protein-substrate contacts of (a) GMP in BmGK-GMP complex, (b) GMP in BmGK-GMP-ATP complex and (c) ATP in BmGK-GMP-ATP complex.
Tables Table 1: Stern-Volmer constant (Ksv) values of BmGK under different denaturant concentrations
Urea (M) 0 0.5 1 2 4 7
Ksv(M-1) 7.8 8.7 8.9 9.2 11.5 13.9
GdnCl(M) 0 0.1 0.6 1 2
Ksv(M-1) 7.8 7.8 8.8 10.6 12.8
Table 2: Results of Binding site prediction through SiteMap S. No Entry Name
Site Score
volume
Dscore
Contact
phobic
philic
don/acc
1
Site_1
1.070
646.555
1.039
1.030
0.610
1.174
0.663
2
Site_2
0.737
146.118
0.749
0.644
0.483
0.687
1.830
3
Site_3
0.675
64.484
0.587
0.832
0.000
1.168
1.129
4
Site_4
0.669
100.499
0.505
0.887
0.017
1.362
0.355
5
Site_5
0.601
46.991
0.582
0.719
0.734
0.502
0.946
Table 3: Docking result of GMP and ATP with BmGK and BmGK-GMP complex S. No.
Compound
Protein
Docking score
Glide energy
Glide emodel
1.
GMP
BmGK
-10.561
-70.590
-101.032
2.
ATP
BmGK-GMP
-8.837
-62.627
-87.183
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11
Fig. 12
S0141-8130(16)31891-8 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.10.008 BIOMAC 6588
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
13-2-2016 1-10-2016 5-10-2016
Please cite this article as: Smita Gupta, Sunita Yadav, Venkatesan Suryanarayanan, Sanjeev K.Singh, Jitendra K.Saxena, Investigating the folding pathway and substrate induced conformational changes in B.malayi Guanylate kinase, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.10.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Investigating the folding pathway and substrate induced conformational changes in B. malayi Guanylate kinase Smita Gupta1, Sunita Yadav1, Venkatesan Suryanarayanan2, Sanjeev K. Singh2, Jitendra K. Saxena1* 1
Division of Biochemistry, CSIR- Central Drug Research Institute, Lucknow-226031, Uttar
Pradesh, India 2
Computer Aided Drug Design and Molecular Modeling Lab, Department of Bioinformatics, Alagappa University, Karaikudi, Tamilnadu, India
Abbreviations NMP kinase- Nucleoside monophosphate kinase BmGK - Brugia malayi Guanylate kinase SEC- Size Exclusion Chromatography GdnCl- Guanidine hydrochloride MS- Molecular Simulation
*
Corresponding author:
Jitendra K. Saxena Chief scientist Division of Biochemistry CSIR- Central Drug Research Institute BS10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow- 226031, Uttar Pradesh, India Telephone: +91 0522 2625932 Fax: +91 0522 2623405 E-mail: [email protected]
Highlights
Detailed biophysical characterization of BmGK carried out for the first time. Folding pathway of BmGK is altered upon binding of substrates (GMP & ATP). Binding of substrates stabilises BmGK. The binding interaction of Arg145 with GMP shows a switch over to ATP after 40ns simulation of BmGK-GMP-ATP ternary complex. Addition of ATP also results in increase in fluctuation in BmGK-GMP-ATP complex in the region from 130-150 residues.
ABSTRACT Guanylate kinase is one of the key enzymes in nucleotide biosynthesis. The study highlights the structural and functional properties of Brugia malayi Guanylate kinase (BmGK) in the presence of chemical denaturants. An inactive, partially unfolded, dimeric intermediate was observed at 1-2M urea while GdnCl unfolding showed monomer molten globule like intermediate at 0.8-1.0M. The results also illustrate the protective role of substrates in maintaining the integrity of the enzyme. The thermo stability of protein was found to be significantly enhanced in the presence of the substrates. Furthermore, binding of the substrates, GMP and ATP to BmGK changed its GdnCl induced unfolding pattern. Docking and molecular dynamic simulation performed for native BmGK, BmGK bound to GMP and GMP+ATP showed change in the fluctuation in the region between 130-150 residues. Arg134 lost its interaction with GMP and Arg145 interaction shifted to ATP after 40ns simulation upon binding of ATP to BmGK-GMP complex. We, thus, propose the importance of specific rearrangements contributed by binding of substrates which participate in the overall stability of the protein. The work here emphasizes on detailed biophysical characterization of BmGK along with the significant role of substrates in modulating the structural and functional properties of BmGK.
Keywords: B. malayi Guanylate Kinase; unfolding; Molten globule
1. Introduction Understanding the structure-function relationships of an enzyme under different conditions is fundamentally important for both theoretical and applicative aspects. Such studies may provide insight into the molecular basis of the stability of the enzyme. Denaturation is a useful means for studying the characteristics of protein folding and has given a better insight into the mechanism of the folding process [1-3]. Exposure of proteins to denaturing agents such as extremes of pH, temperature, high ionic strength, chemical denaturants and organic solvents has been utilized to study the unfolding/folding pathway [4-6]. Though partially folded forms of numerous proteins have been studied, it has yet not been possible to generalize the concept of „intermediate states‟ as universal equilibrium folding intermediates. However, characterization of protein folding intermediates is helpful in identifying and understanding various transition steps and forms [7]. A compact intermediate state, which possesses a high degree of secondary structure and a fluctuating tertiary structure, has been described in several recent studies under various denaturing conditions [8-11]. Ligand binding to proteins is a subject of interest to explain one of the most intriguing and perplexing question in structural and molecular biology i.e. the problem of protein folding into a unique, compact, highly ordered and functionally active form and especially the role of ligands in the structure and in the stabilization of proteins in their native states. The interaction of proteins with small ligands is associated with increase in protein thermostability due to the coupling of binding with unfolding process [12-15]. The binding of proteins to small molecules and protein– protein interactions are key processes in cellular biochemistry. Ligand binding results in change in the conformation of the target protein which in turn produces a given response. Since biological processes are carried out through binding events, therefore the induced structural changes upon ligand binding have been found to be a matter of extensive studies [12]. Guanylate kinase, GK (ATP:GMP phosphotransferases, EC 2.7.4.8) , a nucleoside monophosphate kinase (NMP kinase), catalyzes the reversible transfer of the terminal phosphoryl group from ATP to guanosine 5- monophosphate (GMP) substrate, subsequently releasing guanosine diphosphate (GDP) as product. This is a crucial intermediate step in RNA or DNA synthesis foregoing the formation of the key nucleotide guanosine triphosphate (GTP) or its deoxy form dGTP [16]. The structure of Guanylate kinase comprises of NMP binding region, CORE and the LID region. Crystal structures have been solved for enzymes from many organisms including Saccharomyces cerevisiae GK which is available in both nucleotide free form and in complex with GMP and also for mouse GK in an abortive complex with ADP and GMP (mGK-GMP-ADP) [17, 18]. This investigation describes the biophysical characterization of BmGK, as an endeavour of understanding the structure-function relationship, the basis and rationale of its distinct physicochemical properties and the folding unfolding mechanism. The study also reports the changes in the conformation and dynamic behaviour of BmGK on binding to substrates. Very little information about the folding aspects of Guanylate kinase is available; hence a detailed analysis of native, intermediate and denatured states of BmGK was carried out by measuring the intrinsic tryptophan fluorescence measurement, near & far-UV CD
analysis, 1- aniline-8- naphthalene sulphonic acid (ANS) binding and quenching of intrinsic tryptophan fluorescence. Large-scale conformational alterations are thought to mediate allosteric regulations, which are related with protein function in signal transduction, immune response, and enzymatic activity [19]. Thus, a fundamental problem is to understand the mechanism for the conformational transitions in BmGK upon binding of substrates which is not known. The relationship between protein stability upon substrate binding and the role of substrate in modulating the intrinsic flexibility of the enzyme using various spectroscopic techniques and bioinformatics tools such as molecular dynamic simulation have been carried out. Such a comprehensive and consistent description for BmGK is largely complementary to previous experimental studies carried out on this enzyme. 2. Materials and methods 2.1. Materials All the reagents were purchased from Sigma–Aldrich Chemical, USA unless specifically mentioned. Molecular weight markers for SDS–PAGE were purchased from MBI Fermentas, Maryland, USA. Ni–NTA agarose matrix was procured from Qiagen, Germany. 2.2. Protein expression, purification and activity assay Guanylate Kinase was expressed in E. coli BL21 (DE3) and purified to homogeneity by NiNTA affinity chromatography as described earlier [20]. The purity of the protein was checked on 12% SDS PAGE. Enzyme activity was measured according to the method of Agrawal et. al [21]. For studies involving denaturation with urea or GdnCl, the enzyme was incubated in assay buffer containing desired concentrations of the denaturants for 1 h. It was determined that the auxiliary enzymes (pyruvate kinase and lactate dehydrogenase) during assay remained active under the experimental conditions. The reversibility of the transitions was determined by incubating 20μM BmGK for 1 h in the presence of the different concentrations of denaturants. The samples were then diluted with 50mM Tris buffer pH 7.5 to attain the final concentration of 10mM urea and 2.5mM GdnCl respectively. The diluted enzyme mixture was then immediately used for the activity. The loss of enzyme activity as a function of temperature was followed, in the presence and absence of substrates in 50mM Tris-Cl buffer (pH 7.5). The enzyme samples were incubated for 15 min at different temperatures 25°C to 55°C and after cooling to 4°C, the residual activity was measured on spectrophotometer at 25°C. 2.3. Fluorescence spectroscopy Fluorescence spectra of BmGK were recorded in Perkin-Elmer LS 5B luminescence spectrometer with a 5mm path-length quartz cell. Samples containing different concentrations of urea or GdnCl were equilibrated for 5h in 50mM sodium phosphate buffer (pH 7.0) at 25°C. The spectra were normalized for the solvent contribution to fluorescence. Titrations for binding of nucleotides (GMP and ATP) with BmGK were carried out by adding small aliquots (0-2mM) to 5μM of BmGK in buffer containing 50mM phosphate buffer (pH 7.0)
after an equilibration period of 2 h. For tryptophan fluorescence measurements, an excitation wavelength of 290 nm was used and the emission was recorded between 300-400 nm. ANS binding was estimated by incubating BmGK previously denatured at different concentrations of urea or GdnCl with optimised concentration (100μM) of ANS at 25°C. The excitation wavelength was 380 nm and emission was recorded between 400 nm- 600 nm. 2.4. Fluorescence quenching experiment Acrylamide quenching experiments were performed by adding acrylamide to BmGK denatured at different concentrations of urea or GdnCl for 5h and the fluorescence quenching data were analyzed according to the Stern–Volmer equation [22]. 2.5. Circular dichroism measurements CD experiments were performed with a JASCO J810 spectropolarimeter (equipped with peltier temperature controller system) in a 1cm path length cell at 25oC. The far UV CD spectra were measured for 5μM BmGK treated with desired concentration of denaturants for 5h in 50mM sodium phosphate buffer (pH 7.0) at 25°C. Readings obtained were normalized by subtracting the baseline observed for the buffer with similar denaturant concentration under similar conditions. The tertiary structure of the enzyme (20μM) was monitored in the near UV region in wavelength range 250–350 nm using a cell of path length of 0.5 cm. Each spectrum was recorded as an average of three scans. For binding experiments, the CD spectra of 5μM BmGK were measured in buffer containing 50mM phosphate buffer (pH 7.0) with 0.25mM GMP and (0.25mM GMP+0.25mM ATP). The values obtained were normalized by subtracting the baseline recorded for the buffer having same concentration of substrate under similar conditions. Thermal denaturation was studied by monitoring changes in molar ellipticity at 222 nm as a function of temperature from 30oC to 85oC. 2.6. Limited proteolysis BmGK (0.5 mg/ml) was subjected to limited proteolysis with trypsin for 1 h at 37oC with protease to protein ratio at 1:500 (w/w). Samples were preincubated with 0.25mM substrates for 1hr before trypsin digestion. The reaction was stopped by adding PMSF (1mM) in the reaction mixture and samples were analyzed on 15 % SDS-PAGE. 2.7. Size Exclusion chromatography Gel filtration experiments were carried out on Superdex TM 75, 10/300GL column on AKTA FPLC (GE Health care) pre-calibrated with standard molecular weight markers. The column was pre-equilibrated with 50mM sodium phosphate buffer (pH 7.0) and protein was eluted with or without the desired denaturant concentration at 25oC at a flow rate of 0.3 ml/min with detection at 280 nm. 2.8. Crosslinking using glutaraldehyde 100μg of protein denatured with desired concentration of urea or GdnCl were incubated with 5μl of 1% glutaraldehyde in a 100μl reaction volume in 50mM sodium phosphate buffer
(pH7.0) for 30 min at 37◦C. The reactions were stopped by adding 5μl of 1M Tris–HCl pH 8.0 in the reaction mixture and the samples were analysed on 10% SDS-PAGE. 2.9. Binding site prediction and Molecular Docking BmGK protein docked with GMP was taken from the previous studies [20] and the protein was prepared through Protein Preparation Wizard (Schrodinger, LLC, New York, NY, 2014), implemented in Maestro 9.6. GMP and ATP were downloaded from Pubchem and they were prepared through LigPrep (Suite 2014: LigPrep, version 2.9, Schrodinger, LLC, New York, NY, 2014) module from Schrodinger. Binding site of GMP was noted from the previous studies and it was docked in the same site after getting the equilibrated structure of BmGK from Molecular dynamics simulation using „extra precision‟ glide docking (Glide XP) method (Suite 2014: Glide, version 6.2, Schrodinger, LLC, New York, NY, 2014). Further, the binding site for ATP was investigated through SiteMap (Suite 2014: SiteMap, version 3.0, Schrodinger, LLC, New York, NY, 2014), which generates binding site‟s characteristic information using novel search and analytical facilities. ATP molecule was also docked in the predicted binding site of BmGK protein using Glide XP docking method. In docking calculations, to soften the potential for non polar parts of the receptor, we scaled the van der Waals radii of receptor atoms by 1.00 with a partial atomic charge of 0.25. The default grid size was adopted from the Glide program, and no constraints were applied for all the docking studies. Glide generates conformations internally and passes these through a series of filters. The lowest-energy docked complex was found in the majority of similar docking conformations, which was selected for further study. 2.10. Molecular Dynamics Simulation BmGK Apoprotein and its complexes with GMP and GMP-ATP were carried for computing MD simulation by using Desmond (Suite 2014: Desmond, version 3.7, Schrodinger, LLC, New York, NY, 2014) with Optimized Potentials for Liquid Simulations (OPLS) 2005 force field. All the three systems were imported in Desmond‟s System Builder panel and solvated in an orthorhombic box of TIP3P water molecules and neutralized using appropriate number of counter ions and 0.15M of salt concentration. The system was subjected to the local energy minimization using a hybrid method of the steepest decent and the limited-memory Broyden– Fletcher– Goldfarb–Shanno (LBFGS) algorithms with a maximum of 5000 steps until a gradient threshold (25kcal/mol/Å) was reached. The simulation system was relaxed by constant NPT (number of atoms N, pressure P, temperature T) ensemble condition to generate simulation data for post-simulation analyses. The temperature value was defined as 300 K for the whole simulation process using Nose–Hoover thermostats and stable atmospheric pressure (1atm) carried out by Martina– Tobias–Klein barostat method. The multi-time step RESPA integrator algorithm was used to investigate the equation of motion in dynamics. The time step for bonded, “near” non bonded, and “far” non bonded interactions were 2, 2, and 6fs, respectively. SHAKE algorithm was employed to constrain the atoms which are involved in hydrogen bond interaction. The short range electrostatic and Lennard– Jones interactions were estimated by setting up the cutoff value as 9- Å radius. The longrange electrostatic interactions were evaluated by using particle mesh Ewald (PME) method with the simulation process using periodic boundary conditions (PBC). Energy and trajectory
analysis data were documented at 1.2- and 4.8-ps intervals respectively for statistical analysis. The Final production MD was carried for 100000ps for all three systems and the results were analyzed using Simulation event analysis and Simulation Interaction diagram available in Desmond module [23, 24]. 3. Results & Discussion 3.1. Unfolding of BmGK with urea and GdnCl 3.1.1. Activity is lost prior to structural denaturation Activity of the enzyme is considered the most sensitive probe to study the changes in an enzyme‟s conformation as it reflects subtle readjustments at the active site, allowing very small conformational variations in structure to be detected [25,26]. Fig. 1 shows the activity of BmGK with increasing concentrations of denaturants (urea/GdnCl). The activity was lost at much lower concentration of urea or GdnCl at which no detectable changes in either secondary structure or intrinsic fluorescence occurred. An early loss of enzymatic activity (<0.1M GdnCl) has been detected prior to other changes which may be due to binding of guanidinium ions to BmGK disturbing the electrostatic interactions involved in the stabilization of the active-site conformation, as observed in several cases [11]. It has been reported that restored enzymatic activity is the real marker for assessing the refolding not only the structural properties. Fig. S1 summarizes the refolding efficiency of GdnCl or urea treated BmGK. The observations demonstrated that complete refolding to native BmGK can be achieved after denaturation of BmGK with low denaturant concentrations, whereas denaturation with high denaturant concentration is irreversible. 3.1.2. Changes in tertiary and secondary structure of BmGK upon unfolding Fluorescence spectra provide a sensitive means of characterizing proteins and their conformation. The spectrum is determined by the polarity of the tryptophan and tyrosine residues‟ environment and by their specific interactions [27]. The tryptophan fluorescence emission maxima of BmGK showed red shift in wavelength with increasing concentration of urea or GdnCl (Fig. 2a & b). Two separate transitions were observed for BmGK unfolding in presence of urea or GdnCl. An intermediate state was found to be populated at 1-2M urea. Above 4M urea, the complete unfolding marked by a prominent red shift in wavelength was observed. The results indicated that the single tryptophan residue of BmGK is significantly exposed to the solvent. Similarly, a two state transition was observed during GdnCl unfolding of BmGK. An initial transition between 0 and 0.8M GdnCl and the plateau region at 0.8- 1M GdnCl followed by sharp transition ending into complete unfolding of BmGK at 3M GdnCl was observed. BmGK showed a typical α/β type secondary structure composition as observed by the far-UV CD spectrum of the protein. Changes in the secondary structure of BmGK induced by urea or GdnCl were examined by far UV- CD measurements (Fig. 2c & 2d). Addition of 2M urea and 0.5M GdnCl caused only a minor decrease in ellipticity at 222 nm. However, a significant drop in ellipticity was observed at 4M urea and 2.5M GdnCl. The near-UV CD spectrum of BmGK was characterized by several positive peaks and shoulders suggesting that
the aromatic residues are incorporated into a rigid tertiary structure giving rise to aromatic CD signals (Fig. 2e & 2f). The CD signal in the near-UV region at 1M GdnCl is significantly lower than native state of BmGK suggesting that the addition of 1M GdnCl induces a looser and more flexible environment nearby aromatic residues. Similarly at 1-2M urea there was change in the tertiary structure of BmGK as evident in near UV region of CD spectra. Addition of 5M urea and 3M GdnCl abolished the far and near UV CD signals, indicating extensive or complete unfolding of BmGK. During unfolding, the non-coincidence of the ellipticity curve with those monitoring changes in enzymatic activity and tertiary structure also suggest the existence of intermediate states during urea or GdnCl denaturation. Although at 1-2 M urea, a 40-50% loss of tertiary structure was observed in tryptophan fluorescence spectra but it still retained 75-85% secondary contents as can be seen in far UV CD spectra. All the denaturation experiments were also performed as a function of enzyme concentration (1μM, 2μM 4μM, 6μM) and there was no change in the denaturation profile of BmGK either with urea or GdnCl (data not shown). 3.1.3. ANS binding to BmGK ANS is a fluorescent dye that binds to hydrophobic regions within the proteins. Moltenglobule (MG) intermediate states expose a significant hydrophobic core to the solvent [10, 28]. Hence, ANS binds strongly to the MG state of proteins and shows increased fluorescence intensity. To gain an insight into the unfolding intermediate state, the binding of ANS to BmGK was studied as a function of increasing concentration of urea or GdnCl (Fig. 3a & b). It was observed that ANS binding did not show any increase in fluorescence intensity at lower concentration of urea. This further strengthened the absence of MG like properties for intermediate at 1-2M urea while an increase in ANS fluorescence intensity could be observed at 1M GdnCl as shown in Fig. 3b. Thus, the presence of an intermediate state possessing the characteristics of molten globule could be speculated during the unfolding of BmGK with GdnCl showing compact secondary as well as loose tertiary structure as observed by CD spectroscopy and enhanced ANS fluorescence intensity. 3.1.4. Fluorescence quenching of tryptophan residue of BmGK Fluorescence quenching of the tryptophan residues by quenchers has been shown to be useful in getting information about the solvent accessibility of these residues in proteins and the polarity of their microenvironment [29]. The quenching of fluorescence intensity of tryptophan residue under varying concentrations of urea or GdnCl was monitored in order to probe accessibility of acrylamide to the protein core at different conditions (Fig. 3c & d). It was observed that urea below 4M concentration did not bring any prominent change in fluorescence intensity while at 7M concentration significant quenching was monitored. BmGK treated with 0.6 and 1M GdnCl showed increase in fluorescence quenching as compared to native protein indicating an increased exposure of aromatic hydrophobic residues. The presence of molten globule state at 1.0 M GdnCl with characteristics compact structure and exposed hydrophobic residues as indicated by enhanced ANS fluorescence intensity further supports the quenching result. The Stern–Volmer quenching constants for the urea and GdnCl denatured states are shown in Table 1.
3.1.5. Changes in the quaternary structure of BmGK upon urea and GdnCl induced unfolding Size Exclusion chromatography (SEC) experiments were performed to analyze changes in the quaternary structure of the enzyme in the presence of increasing concentration of urea or GdnCl as shown in Fig. 4. For native BmGK, single peak at 12.27 ml was observed. This is similar to the peak observed for ovalbumin corresponding to retention volume of 12.3 ml with molecular weight of 43KDa. This indicates BmGK is dimeric in nature. For 0.2M GdnCl treated BmGK, single peak with retention volume of 12.8 ml was observed. Two peaks have been observed for 0.8M GdnCl corresponding to retention volume of 9.1ml and 13.0ml for aggregated protein species and compact monomer species of BmGK, respectively. However at 1M GdnCl single peak with retention volume 13.5 ml has been observed corresponding to monomer BmGK. Molten globule monomers have been reported as intermediates in the dissociation of dimeric proteins such as human superoxide dismutase [30], rabbit muscle creatine kinase [11], yeast glutathione reductase [31]. Fig. 4b shows urea induced changes in BmGK. For 1M urea denatured BmGK, two peaks at 12.27 ml and 10.9 ml were observed designated as peak A and B, respectively. The peak A corresponds to native dimeric form of enzyme while peak B represents the slight expansion of the enzyme structure forming highly solvated structure, thus enhanced hydrodynamic radii as determined by size exclusion chromatography. Such a situation occurs only when the dimeric enzyme undergoes progressive unfolding without dissociation into monomers [32]. The preferential interactions at the external regions of BmGK might be the principal reason for a local unfolding of the protein structure, far from the dimeric interface [31]. Further increases in urea concentration to 2M resulted in single peak at 10.5ml with loss or complete vanishing of dimeric form peak of BmGK at 12.27 ml. However, at 4 M urea and 3M GdnCl the protein eluted in void volume at retention volume of 7.8 ml, which was due to complete unfolding of the enzyme resulting in significant enhancement in the hydrodynamic radii of the enzyme. The results were supported by glutaraldehyde crosslinking data as evident in Fig. 4c&d. 3.2. Substrate induced changes within BmGK 3.2.1. Binding of substrate to BmGK alters the folding pathway of BmGK The protein having several domains and each contributing to the parameter being followed shows a multi transitional denaturation curve depending on the method of following the unfolding process, reflecting the variable stabilities of the different domains. If a ligand is found to stabilize the overall structure of the protein, the denaturation curve will be shifted to higher concentrations of denaturant and, vice versa. Denaturation profile of BmGK was also assessed with urea and GdnCl in presence of bound substrates GMP and ATP. It was observed from the results of fluorescence and CD spectroscopy that urea induced unfolding of BmGK was unaffected by binding of substrates (data not shown) while substrate binding stabilized the enzyme and protected its denaturation in presence of GdnCl (Fig. 6a & 6b). This suggests that indeed the electrostatic interactions are affected upon binding of substrate which play a role in stabilization of structure of BmGK.
The residual activity of BmGK as a function of temperature in the presence and absence of substrates is shown in Fig. 6c which also suggests that the enzyme was stabilized in presence of substrate. It was observed that activity of substrate bound BmGK was retained even at 50oC as compared to unbound form. The thermal unfolding of BmGK was also characterized by monitoring the loss of secondary structure of enzyme with rise in temperature [33]. Fig. 6d summarizes the changes in ellipticity at 222 nm of BmGK with increasing temperature, where two distinct transitions were observed between 30-85°C. These transitions were in the temperature regions from 45°C to 55°C and 65°C to 70°C and centered at about 50°C and 65°C, respectively. In presence of substrates i.e. GMP & ATP, the transition temperature shifted to higher temperature. Thus, thermal denaturation suggests that BmGK molecule contains two distinct structural domains of significantly different thermal stabilities which are influenced upon binding of substrates.
3.2.2. Tertiary and Secondary structural changes in BmGK upon binding of substrates A decrease in fluorescence intensity after binding of GMP was observed (Fig. 7a). The large reduction in the intrinsic fluorescence of BmGK is observed upon GMP binding which suggests that the nucleotide is in close juxtaposition to the single Trp51 residue in the protein [34].The lesser reduction during ATP binding suggests a slightly altered binding mode for the latter as compared to GMP (Fig. 7b). The fluorescence data were further analyzed according to the Chipman equation [35]. The Ka obtained was 2.5 X 104 M-1 for GMP binding (data not shown). Fig. 7c shows that addition of substrates also resulted in significant changes in both shape and intensity of the CD spectra, most strikingly in an increase of the CD signal around 222 nm. The significant increase in the secondary structure of BmGK upon binding of substrates provided further evidence of structural rearrangements, suggesting that the enzyme undergoes from a less ordered conformation to a more stable structured one. The susceptibility of BmGK under different conditions to trypsin proteolysis provided further insight into local conformational flexibility of the protein. The sequence analysis of BmGK revealed 11Arg and 17 Lys residues which depending on their accessibility in tertiary structure could serve as site for trypsin cleavage. The effect of substrates on the protection of BmGK from trypsin digestion is shown in Fig. 8. This data shows that binding of both substrates, GMP and ATP to the enzyme protected it to comparable extent suggesting that the proximity of binding in the active site of BmGK induces a conformational change and hence comparable level of protection from trypsin proteolysis has been observed. Based on the observations it could be speculated that GMP brings out a large conformational change upon binding as it protected the enzyme from tryptic digestion but complete protection occurs only when both GMP and ATP are bound to BmGK. 3.2.3. Binding site prediction and Molecular Docking of BmGK As currently high resolution X-ray crystallographic structure for BmGK is unavailable, 3D homology modeling may be an alternative to gain insights into potential substrate binding sites and the mechanisms of substrate interaction with the protein [36]. Equilibrated BmGK structure was used for the docking calculations with GMP and ATP. Initially, GMP was
docked based on the binding site information obtained from previous study. After docking, binding site for ATP was predicted through Sitemap and the best binding site was found based on the site score from the results provided in Table 2. Fig. S2 (a& b) highlights the predicted binding site region in Maroon colour surface with white dots. Further, ATP was docked with BmGK-GMP complex on the predicted binding site. Result of XP docking of both BmGK-GMP and BmGK-GMP-ATP complex were shown in Table 3. TwoDimensional interaction diagram of docked complexes were shown in Fig. 9, which exposes that the addition of ATP cause changes in interaction of GMP with BmGK. It was shown that GMP is interacting with residues like Arg38, Arg41, Tyr78, Asp98, Gln102, Arg134 and Arg145 in BmGK-GMP complex (Fig. 9a). In BmGK-GMP-ATP complex, the interacting residues for GMP have reduced to Arg38, Arg41, Glu69, Tyr78 and Arg145. Interacting residues of BmGK-GMP-ATP complex with ATP were found to be Ser10, Gly11, Gly12, Gly13, Lys14, Ser15, Tyr50, Arg134 and Arg145. GMP has lost its interaction with Asp98, Gln102 and Arg134 due to the addition of ATP in the binding site region whereas Arg134 was rather found to be interacting with ATP in BmGK-GMP-ATP complex. Interestingly, Arg145 was found to be interacting with both GMP and ATP in BmGK-GMP-ATP complex. Analogous Arg in mouse Guanylate kinase has been reported to act as a clamp by interacting with both phosphates of GMP and ATP [17]. Fig. S3 shows the closer view of GMP (Blue colour) and ATP (Brown colour) binding to the surface encoded binding site of BmGK. The interaction stability analysis of both BmGK-GMP and BmGK-GMP-ATP complex through MDS would bring clarity in substrate specificity of BmGK [37]. 3.2.4.Structural changes observed by molecular dynamics simulation Conformational changes and dynamics of proteins are monitored by molecular dynamic simulation. This method has been utilized to substantiate the structural changes in BmGK revealed by other probes and also to narrow down the substrate-induced changes to specific regions in the protein [38]. BmGK apoprotein was initially simulated for 100000ps to bring out well equilibrated structure for further analysis. After getting the equilibrated BmGK structure, docking calculations were performed with GMP and ATP. Two complexes of BmGK-GMP and BmGK-GMP-ATP were carried for further MDS for 100000ps of time period to procure a better understanding in structural changes of BmGK due to the interaction of GMP and ATP. The change in the structural integrity has been analyzed through calculating the root mean square deviation (RMSD) and root mean square fluctuations (RMSF) over backbone atoms. Fig. 10a shows the RMSD graph of all three MD simulations over 100000ps of time period. BmGK apoprotein is found to have more fluctuations in RMSD and it has attained stability around 6Å nearly at the end of simulation. The equilibrated BmGK structure was carried for further molecular docking and molecular dynamics simulation studies. RMSD of BmGK-GMP complex is found to attain stability at 3Å after 10000ps and maintained till the end, whereas RMSD of BmGK-GMP-ATP complex is found to have minor fluctuations progressively and after 60000ps the RMSD graph fluctuates around 4Å. RMSF graph shown in Fig. 10b clearly supports the result of RMSD of all three simulations. Difference in RMSF between the apoprotein and complex were seen around active site residues. It reveals that apoprotein gains stability due to addition of substrate GMP and ATP. Whereas the difference
in RMSF from 130 to 150 residue between BmGK-GMP and BmGK-GMP-ATP complex reveals that increased fluctuation in BmGK-GMP-ATP complex may be due to the change in interacting residues during the addition of ATP in the binding site. Further analysis of solvent accessible surface area (SASA) in bound receptor reveals that both complexes maintain stability during the simulation time period (Fig. 10c). Interaction of substrates with protein play major role in maintaining the stability of complex [37]. The time line of protein ligand contact is depicted in Fig. 11 which shows the interaction of GMP in BmGK-GMP (a) and BmGK-GMP-ATP (b) complex and also the interaction of ATP in BMGK-GMP-ATP complex (c). It uncovers the occurrence of interaction by each interacting residues in protein-ligand complex throughout the simulation. In Fig. 11a, residues like Arg41, Tyr78, Asp98, Arg134 and Arg145 are found to maintain the interaction throughout the simulation. Fig. 11b shows the interaction of Asp98, Arg134 were lost and the interaction of Arg38, Tyr50 and Phe73 were gained which clearly revealed the change in interaction of GMP in BmGK-GMP-ATP complex due to the addition of ATP. Fig. 11c, shows that Gly11, Gly12, Gly13, Lys14, Ser15 and Arg134 residues of BmGKGMP-ATP complex were interacting ATP throughout the simulation whereas Lys144 shows interaction after 17ns and Arg145 shows interaction after 40ns. It is clear from the Fig. 11c that Arg134 which has lost interaction with GMP is found to interact with ATP in BmGKGMP-ATP complex. It is also clear from Fig. 11, Arg145 which has stable interaction with GMP in BmGK-GMP complex (Fig. 11a) is found to change its interaction from GMP to ATP which is depicted in Fig. 11b and 11c. Percentage of interaction by interacting residues was represented through histogram in Fig. 12 which includes both hydrogen bond and hydrophobic bond interaction. Changes in the interaction and structural integrity clearly support the possible dynamic conformational changes in active site of BmGK protein upon substrate binding. Thus, our study supports the notion that binding of GMP alone induces the closure of the GMP domain and subsequent co binding of ATP results in the fully closed conformation [39]. 4. Conclusion Since protein exert their functional activity in different conformations in the cell passing through different states, present work represents the first report of detailed biophysical characterization of B. malayi Guanylate kinase illustrating different states during its folding pathway as well as the changes induced within the enzyme upon binding with its substrates. The study has provided comparative account of action of urea and GdnCl on structural, functional and stability properties of BmGK (Fig. 5). On the basis of our results, we propose that the unfolding of native BmGK proceeds with the initial formation of monomer molten globule like intermediate during GdnCl denaturation while urea denaturation proceeds with formation of expanded dimeric intermediate with loose tertiary contacts and disturbed structural rearrangements leading to final unfolded state. Various aspects of protein stability, flexibility & dynamics together in conjunction with each other have been studied which give a thorough picture of substrate (GMP and ATP) induced changes within BmGK. Different methods indicated that a conformational change takes place to reorganize the enzyme both
locally as well as globally for catalysis of reaction. It is evident from the results of molecular docking and molecular dynamic simulation studies that interaction of substrates like GMP and ATP brings high stability to BmGK. Thus, this study highlights the detailed unfolding pathway of BmGK and the conformational change in the protein upon binding of substrates probably leading to its transformation from open to closed state. Since detailed analysis of unfolding mechanism of any Guanylate kinase is not known till date, the overall results of the structural characterization of BmGK are noteworthy. This work could serve as a model to understand dynamic aspect of substrate binding for catalysis in absence of its crystal structure. Conflict of interest None declared.
Acknowledgement We gratefully acknowledge Council of Scientific and Industrial Research (CSIR), New Delhi, for offering a Senior Research fellowship to Smita Gupta to carry out this work. We would like to extend our gratitude to Director, CDRI for his invaluable support.
REFERENCES
[1] O.B. Ptitsyn, Adv Prot Chem, 47 (1995) 83-229. [2] C. Camilloni, A.G. Rocco, I. Eberini, E. Gianazza, R.A. Broglia, G. Tiana, Biophys J, 94 (2008) 4654-4661. [3] D.J. Fan, Y.W. Ding, J.M. Zhou, Biochim Biophys Acta, 6 (2009) 944-952. [4] V.K. Dubey, M. Jagannadham, Biochemistry, 42 (2003) 12287-12297. [5] Y. Kumar, S. Muzammil, S. Tayyab, J Biochem, 138 (2005) 335-341. [6] C.N. Pace, S. Trevino, E. Prabhakaran, J.M. Scholtz, Philos Trans R Soc Lond B Biol Sci, 359 (2004)1225-1234. [7] Y. Goto, N. Takahashi, A.L. Fink, Biochemistry, 29 (1990) 3480-3488. [8] D.A. Dolgikh, R.I. Gilmanshin, E.V. Brazhnikov, V.E. Bychkova, G.V. Semisotnov, S. Venyaminov,O.B. Ptitsyn, FEBS Lett, 136 (1981) 311-315. [9] Y. Goto, A.L. Fink, Biochemistry, 28 (1989) 945-952. [10] G.V. Semisotnov, N.A. Rodionova, O.I. Razgulyaev, V.N. Uversky, A.F. Gripas, R.I. Gilmanshin, Biopolymers, 31 (1991) 119-128. [11] F. Couthon, E. Clottes, C. Ebel, C. Vial, Eur J Biochem, 234 (1995) 160-170. [12] M.S. Celej, G.G. Montich, G.D. Fidelio, Protein Sci, 12 (2003) 1496-1506. [13] J.F. Brandts, L.N. Lin, Biochemistry, 29 (1990) 6927-6940. [14] A. Shrake, P.D. Ross, J Biol Chem, 265 (1990) 5055-5059. [15] A. Shrake, P.D. Ross, Biopolymers, 32 (1992) 925-940. [16] M. Kandeel, M. Nakanishi, T. Ando, K. El-Shazly, T. Yosef, Y. Ueno, Y. Kitade, Mol Biochem Parasitol, 159 (2008) 130-133. [17] N. Sekulic, L. Shuvalova, O. Spangenberg, M. Konrad, A. Lavie, J Bio Chem, 277 (2002)30236-30243. [18] J. Blaszczyk, Y. Li, H. Yan, X. Ji, J Mol Biol, 307 (2001) 247-257. [19] J. Ping, P. Hao, Y. X. Li, J.F. Wang, BioMed Res Int, 2013 (2013) 628536. [20] S. Gupta, S. Yadav, N. Singh, A. Verma, I. Siddiqi, J.K. Saxena, Parasitology, 141 (2014)1341-1352. [21] K. Agarwal, R. Miech, R. Parks, Methods Enzymol, 51 (1978) 483-490. [22] M.R. Eftink, C.A. Ghiron, Anal Biochem, 114 (1981) 199-227. [23] V. Suryanarayanan, S.K. Singh, J Recept Signal Transduct , 35 (2015) 370-80. [24] S.K. Tripathi, S.K. Singh, Mol BioSyst, 10 (2014) 2189-2201. [25] J.J. Plomer, A. Gafni, Biochim Biophys Acta, 1122 (1992) 234-242. [26] P. Alam, G. Rabbani, G. Badr, B.M. Badr, R.H. Khan, Cell Biochem Biophys, 71 (2015) 1199-1206. [27] S. Hayashi, S. Nakamura, Biochim Biophys Acta, 657 (1981) 40-51. [28] Gimenez-Gallego, Eur J Biochem, 246 (1997) 328-335. [29] S. Pawar, V. Deshpande, Eur J Biochem, 267 (2000) 6331-6338. [30] N. Silva, Jr., E. Gratton, G. Mei, N. Rosato, R. Rusch, A. Finazzi-Agro, Biophys Chem, 48(1993) 171-182. [31] P.R. Louzada, A. Sebollela, M.E. Scaramello, S.T. Ferreira, Biophys J, 85 (2003) 32553261. [32] A.N. Bhatt, M.Y. Khan, V. Bhakuni, Protein Sci, 13 (2004) 2184-2195. [33] B. Ahmad, G. Muteeb, P. Alam, A. Varshney, N. Zaidi, M. Ishtikhar, G. Badr, M.H. Mahmoud, R.H. Khan, Int J Biol Macromolec, 75 (2015) 447-452. [34] A.S. Abdelhameed, P. Alam, R.H. Khan, J Biomol Struct Dyn, 34 (2016) 2037-2044.
[35] D.M. Chipman, V. Grisaro, N. Sharon, J Biol Chem, 242 (1967) 4388-4394. [36] M.J. Hosen, A. Zubaer, S. Thapa, B. Khadka, A. De Paepe, O.M. Vanakker, PloS One, 9 (2014) e102779. [37] P. Alam, S.K. Chaturvedi, T. Anwar, M.K. Siddiqi, M.R. Ajmal, G. Badr, M.H. Mahmoud, R.H. Khan, J Lumin, 164 (2015) 123-130. [38] X. Du, Y. Li, Y.L. Xia, S.M. Ai, J. Liang, P. Sang, X. L. Ji, S.Q. Liu, Int J Mol Sci, 17 (2016) 144. [39] C. Vonrhein, G.J. Schlauderer, G.E. Schulz, Structure, 3 (1995) 483-490.
Legends to figures: Figure 1 Changes in BmGK activity. Effect of increasing concentrations of (a) urea, and (b) GdnCl on BmGK activity. Enzyme activity observed for BmGK in absence of urea and GdnCl is taken as 100%. Figure 2 Urea and GdnCl induced changes in tertiary and secondary structure of BmGK. (a) The fraction of unfolded BmGK (fi - fo)/(fd - fo) versus urea concentration; fi is wavelength for sample, fo is the wavelength in the absence of urea, and fd is the wavelength at urea concentrations <5M.(b) Fraction unfolded of BmGK (fi - fo)/ (fd - fo) versus GdnCl concentration; fi is wavelength for a particular sample, fo is the wavelength in the absence of GdnCl, and fd is the wavelength at GdnCl concentrations <2.5M. Inset shows the fluorescence curves of native and different urea and GdnCl denatured BmGK samples. (c) Urea and (d) GdnCl induced changes in secondary structure as monitored by following the changes in CD ellipticity at 222 nm from far-UV CD curves at increasing concentrations of urea and GdnCl. Data are represented as the percentages of ellipticity at 222 nm, taking the value observed for native protein in absence of urea and GdnCl as 100%. Inset shows the CD curves of native and BmGK denatured at different concentrations of urea or GdnCl. Near-UV CD spectra of BmGK under native condition and denatured at varying concentration of urea (e) and GdnCl (f). Figure 3 Fluorescence intensity of ANS. Binding of ANS to BmGK at various denaturing concentrations of (a) urea and (b) GdnCl. Quenching of intrinsic fluorescence of BmGK by acrylamide at different (c) urea and (d) GdnCl concentrations, fo: fluorescence intensity in the absence of acrylamide, f: observed fluorescence intensity in the presence of acrylamide. Figure 4 Size-exclusion chromatographic profile on Superdex 75 HR column and SDS-PAGE profiles of glutaraldehyde crosslinking. The chromatographic profiles of native BmGK on a Superdex 75 HR 10/300 column on AKTA FPLC (Amersham Pharmacia Biotech, Sweden) at different (a) Urea and (b) GdnCl concentrations. All the curves have been displaced along the Y-axis for display purposes. (c) Glutaraldehyde crosslinking of BmGK. Lane 1- glutaraldehyde crosslinked native BmGK, Lane 2- glutaraldehyde crosslinked 0.2 M GdnCl treated BmGK, Lane 3- glutaraldehyde crosslinked 0.8M GdnCl treated BmGK, Lane 4- glutaraldehyde crosslinked 1M GdnCl treated BmGK, Lane 5-uncrosslinked native BmGK (d) Lanes 1–3 represent native BmGK, glutaraldehyde crosslinked 1M urea treated BmGK and glutaraldehyde crosslinked 3M urea treated BmGK, respectively. (e) Calibration curve of standard molecular weight markers viz., Ribonuclease A (13.7KDa), Chymotrypsinogen (25KDa), Ova-albumin (43KDa) and albumin (67KDa). Figure 5 Representation of unfolding pathway of BmGK in presence of Urea and GdnCl
Figure 6 Effect of substrate binding on structural and functional properties of BmGK (a) GdnCl induced unfolding in absence of substrates (squares) and in presence of GMP+ATP (circles) (b) Denaturation of BmGK with 0.1M, 0.8M & 3M GdnCl in absence and presence of substrates, S (GMP+ATP) as revealed by far UV-CD spectroscopy (c) Activity of BmGK at different temperatures in absence and in presence of substrates i.e. GMP and ATP (d) Thermo stability of BmGK measured by far UV CD spectroscopy at 222 nm at different temperatures in absence and presence of substrates. Figure 7 Conformational changes in BmGK induced by substrate binding. (a) Fluorescence emission spectra of BmGK in the presence of different concentrations of GMP (b) and ATP (c) Changes in the ellipticity of BmGK upon binding of substrate by far-UV CD spectroscopy Figure 8 Effect of substrate binding on trypsin cleavage pattern of BmGK. Trypsin to BmGK (1: 500) was used in the experiment and the fragments separated on 15% SDS PAGE were visualised by staining with Coomassie brilliant blue. The digestion time (in minutes) is indicated at the top of the panel while substrates added are mentioned at the bottom of the panel. Figure 9 2D interaction diagram of (a) GMP with BmGK and (b) GMP (left) and ATP (right) with BmGK. Figure 10 (a) RMSD graph of BmGK Apoprotein, BmGK-GMP and BmGK-GMP-ATP complexes over 100000ps of simulation.(b) RMSF graph of BmGK Apoprotein, BmGK-GMP and BmGK-GMP-ATP complexes over 100000ps of simulation. (c) SASA of bound receptor of BmGK-GMP and BmGK-GMP-ATP complexes over 100000ps of simulation Figure 11 Timeline of Protein-substrate contacts of (a) GMP in BmGK-GMP complex, (b) GMP in BmGK-GMP-ATP complex and (c) ATP in BmGK-GMP-ATP complex. Figure 12 Histogram of Protein-substrate contacts of (a) GMP in BmGK-GMP complex, (b) GMP in BmGK-GMP-ATP complex and (c) ATP in BmGK-GMP-ATP complex.
Tables Table 1: Stern-Volmer constant (Ksv) values of BmGK under different denaturant concentrations
Urea (M) 0 0.5 1 2 4 7
Ksv(M-1) 7.8 8.7 8.9 9.2 11.5 13.9
GdnCl(M) 0 0.1 0.6 1 2
Ksv(M-1) 7.8 7.8 8.8 10.6 12.8
Table 2: Results of Binding site prediction through SiteMap S. No Entry Name
Site Score
volume
Dscore
Contact
phobic
philic
don/acc
1
Site_1
1.070
646.555
1.039
1.030
0.610
1.174
0.663
2
Site_2
0.737
146.118
0.749
0.644
0.483
0.687
1.830
3
Site_3
0.675
64.484
0.587
0.832
0.000
1.168
1.129
4
Site_4
0.669
100.499
0.505
0.887
0.017
1.362
0.355
5
Site_5
0.601
46.991
0.582
0.719
0.734
0.502
0.946
Table 3: Docking result of GMP and ATP with BmGK and BmGK-GMP complex S. No.
Compound
Protein
Docking score
Glide energy
Glide emodel
1.
GMP
BmGK
-10.561
-70.590
-101.032
2.
ATP
BmGK-GMP
-8.837
-62.627
-87.183
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11
Fig. 12