Indian Journal of Biochemistry & Biophysics Vol. 54, February-April 2017, pp. 24-31

Dissection of binding to Plasmodium falciparum glyceraldehyde-3-phosphate dehydrogenase using spectroscopic methods and molecular docking

Biswajit Pal* CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad–500 007, India

Received 20 July 2016; revised 06 March 2017

Heme binds to a large group of proteins and controls their functions. However, quantitative estimation of heme binding to a protein using spectroscopic methods is challenging as heme molecules assume different forms depending on solvent compositions. Therefore, spectroscopic methods used for binding studies need to distinguish between free and bound heme for quantification of bound ligands and curve fitting is useful for the purpose. Malarial GAPDH (PfGAPDH) has been reported to bind heme, and heme binding inhibits its enzymatic activity. Heme binding to PfGAPDH was studied, and dissociation constant was estimated using curve fitted Soret bands. Dissociation constant was also estimated using Isothermal Titration Calorimetry in the presence of DMSO. Intrinsic tryptophan fluorescence indicated that heme binding resulted in a dynamic change in PfGAPDH. Docking studies indicated that heme binding pocket overlaps with NAD+ binding pocket. However, the orientation of heme is different in the case of PfGAPDH compared to that of human GAPDH, resulting in higher levels of steric hindrance between PfGAPDH-bound heme and NAD+. This possibly results in inhibition of enzymatic activity in PfGAPDH upon heme binding. Results show that PfGAPDH has weak heme binding compared to the mammalian GAPDHs. This may have implications as PfGAPDH may be involved in the storage or transport of cellular heme pool, produced by degradation of , which is toxic to the parasite.

Keywords: Activity, Binding constant, GAPDH, Heme binding, Plasmodium falciparum

Heme (iron protoporphyrin IX) modulates diverse the binding constant in the case of heme binding to functions in living organisms, and these functions are GAPDHs4. However, heme dimerises in aqueous controlled mostly by proteins in heme bound forms. solutions depending on the pH and components of the One such example is binding of heme to a glycolytic buffer and, therefore, heme binding studies of proteins , glyceraldehyde-3-phosphate dehydrogenase are in general difficult5. In most of these studies, Soret (GAPDH, EC 1.2.1.12) and related modulation of peak of free heme, which normally forms hematin in its activity1-4. The main physiological function of aqueous solution, has been characterized at 385 nm4. GAPDH is the conversion of glyceraldehyde 3- However, it is well established that the free hematin phosphate (GAP) into glycerate 1, 3-bisphosphate in has a Soret peak around 365 nm and the peak around the presence of nicotinamide adenine dinucleotide 385 nm corresponds to µ-oxobisheme6,7. Therefore, (NAD+) and phosphate, although many calculations of binding constants using Soret peak at “moonlighting” functions of it have been discovered 385 nm might give erroneous values, and these over the years1,2. Heme binding to different GAPDHs studies need to be verified independently using other has mainly been studied using spectrophotometric methods. If these binding studies are complemented methods3,4. In these studies, differences of absorbance with functional assays, it will provide an insight in to at two different wavelengths were used to calculate the mechanism. —————— It was reported earlier that heme binds to the *Correspondence: GAPDHs tested but does not alter the enzymatic Phone: +91-40-2719-2831; Fax: + 91-40-2716-0591 activity of most of these GAPDHs3,4. The only E-mail: [email protected] exception known till date is Plasmodium falciparum Abbreviations: DMSO; Dimethyl sulfoxide, GAP; Glyceraldehyde GAPDH (PfGAPDH) and its activity is totally 3-phosphate, GAPDH; Glyceraldehyde-3-phosphate dehydrogenase, 3 ITC; Isothermal Titration Calorimetry, LB; Luria-Bertani, NAD+; inhibited upon heme binding . Enzymatic inhibition Nicotinamide adenine dinucleotide studies were used for heme binding by measuring PAL: INTERACTIONS OF HEME AND PfGAPDH 25

NADH production and inhibitor constant, Ki, was and human GAPDH (HsGAPDH). Based on all these calculated for PfGAPDH3. However, it had been studies, I proposed the mechanism of inhibition of proposed that the absorbance of heme at higher PfGAPDH by heme. concentrations could interfere with the measurement of NADH production during the assay3. Therefore, Materials and Methods precise estimation of binding parameters along with Cloning, expression, and purification enzymatic activity is extremely important to understand the mechanism of inhibition of GAPDH Cloning, expression, purification, and characterization of rPfGAPDH were carried out by heme. It is particularly important, as, in one hand, 14 it has been proposed that heme is essential for according to the reported literature with some malarial growth8, and, on the other hand, free heme is modifications. In brief, the coding sequence for toxic to the parasite, and this has been explored to PfGAPDH (PF3D7_1462800) was amplified from design drugs against malaria9,10. Structure of cDNA and cloned into E. coli expression vector, PfGAPDH has been solved by two independent pET15b with an N-terminal hexa- tag groups11,12. In one of the studies, heme was docked on (Novagen). The recombinant protein was expressed in the structure and was proposed that one of the two E. coli BL21 (DE3) pLysS. Bacterial culture was propionate groups of heme interferes with NAD+ grown at 37°C in a LB medium containing suitable 12 antibiotics, and 2 mM lactose was added to the mid- binding . However, no comparative mechanism has 15 been proposed yet. Despite several studies about log phase . The culture was further grown at 23°C heme binding to GAPDHs are available, none of these overnight. Recombinant proteins were purified from studies properly correlated binding parameters with the soluble fraction of the bacterial lysate using functional studies. immobilized metal affinity chromatography on Ni- To eliminate the ambiguity, it’s extremely NTA column, followed by size exclusion important to estimate the amount of bound and chromatography on Superdex 200 column. Purified unbound components of heme as precisely as protein was used for further studies with or without possible. To quantify the bound heme from free further concentration. moieties, curve fitted bands of bound components could give better estimation and could be used for the Enzymatic Assay calculation of binding constant. Binding constant thus Enzymatic activity of rPfGAPDH was measured by 16 calculated should be validated using other the modified method of Ferdinand . Assay buffer independent methods. One of the widely used contained 40 mM Triethanolamine, 50 mM Na2HPO4, + methods to calculate binding constant is isothermal pH 7.4, containing, 0.2 mM EDTA, 2 mM NAD and titration calorimetry (ITC). This method is label free 2 mM DL-GAP. The reaction was started by the and directly calculates the parameters. However, addition of about 40 ng of enzyme. Total assay binding studies using heme is difficult using ITC, volume was 100 µL and was carried out at room since DMSO, used to keep heme in monomeric form, temperature of 22°C. For the assay, a blank solution interferes as is has a high heat of dilution. As a result, containing reaction mixture without enzyme was reports of heme binding using ITC are scanty, placed in the reference cell. Formation of NADH was although, there are reports of heme binding studies measured at 340 nm for 5 min. However, data of using ITC in the absence of DMSO13. initial 30-60 sec were used for calculations as above In this work, I cloned, expressed and purified One min the production of NADH deviated from enzymatically active PfGAPDH and assayed the linearity. One unit of enzyme activity was defined as activity in the presence or absence of heme. The the amount of enzyme causing 1 µmol of NAD+ binding constant was calculated using the intensity for converted to NADH per minute at 22°C. For heme the curve fitted band of bound heme. Then heme was bound GAPDH assay, GAPDH was incubated with titrated with protein using ITC in reverse mode to 1 µM hemin solution for 10 min before the assay. To calculate the binding constant in the presence of cross check the activity of the heme-bound enzyme, DMSO and validated the values obtained from 50 µM (monomeric) enzyme was incubated with spectrophotometric titrations. Further, using steady 250 µM hemin and excess hemin was removed using state fluorescence, binding of heme to PfGAPDH was a desalting column. An equivalent amount of 0.1 M confirmed. Finally, heme was docked on PfGAPDH NaOH containing 5% DMSO was also used as a 26 INDIAN J. BIOCHEM. BIOPHYS., VOL. 54, FEBRUARY-APRIL 2017

control. These were used to compare specific reaction. Eadie-Hofstee plot of ΔA vs. ΔA/[P] was 18 activities. used to calculate Kd and ΔAmax .

Heme binding studies Isothermal calorimetric titration Electronic absorption spectroscopy Stock solution (2.5 mM) of hemin was prepared Heme–rPfGAPDH interactions were studied using dissolving the measured amount of hemin in 100 mM titration method17. Two sets of experiments were NaOH containing 5% DMSO. This was further performed in each case: one without protein, which diluted to 1 mM using 100 mM NaOH containing 5% serves as a control and one containing protein. DMSO, if required. To prepare a working solution, All spectra were recorded in a Shimadzu UV-VIS this solution was diluted in required concentration in Spectrophotometer, UV-2600. A fixed concentration working buffer, immediately before the experiments. of hemin solution was titrated with a varied amount of Heme binding experiments were carried out in a protein each time in 100 µL total reaction. The Microcal VP-ITC 200 instrument. Since the ligand experiments were repeated and found to be solution contained DMSO, reverse titrations were reproducible within experimental errors. All the carried out keeping ligand (25 µM) in the cell and the absorption spectra were recorded at room temperature protein in the syringe (125 µM, tetramer). Both of about 22°C. protein and heme solutions were equilibrated at 25°C The molar extinction coefficient (ε) of the protein and extensively degassed before loading to the cell or was determined theoretically using the expassy tool syringe prior to the titrations. Binding was carried out and was used to calculate the unknown concentration at 25°C with 2 µL of injection volume for each using absorption at 280 nm. The molar extinction titration. Blanks were obtained by titrating the buffer coefficient of free hemin was calculated using with the same concentration of hemin. Curve fitting Lambert-Beer law. The extinction coefficient of the was performed for a single site using the software bound heme was calculated by mixing protein and Origin supplied by MICROCAL. heme at 100:1 ratio and then incubating at room temp Steady state fluorescence studies for 10 min before scanning the absorption spectra. Fluorescence measurements were carried out using To calculate the stoichiometry and binding a Hitachi F-7000 spectrofluorometer using a quartz constant, titration was carried out. The binding of a cuvette of 1 cm path length. Excitation wavelength ligand to a single site of a protein molecule could be used was 295 nm. Blank spectra of buffer (or ligands represented as P + L PL, and, apparent dissociation in the buffer) were recorded and subtracted from each constant, K , could be represented as d sample spectrum to minimize contribution due to the K = [P][L]/[PL], where [P] and [L] were concentrations d solvent Raman peak and other scattering artifacts. of unbound protein and ligand, respectively, and [PL] 6 µM enzyme was treated with either 125 µM NAD+ was the concentration of the complex18. Since the free or 685 µM GAP. Either 1.25 µM or 6 µM hemin and bound had Soret maxima at different solutions were used for fluorescence studies. wavelengths, the changes in the absorbance of bound heme could be directly proportional to the amount of Docking studies bound complex produced. In this case, a series of To identify the binding site of heme on PfGAPDH, experiments along with control sets were carried out, heme was docked on PfGAPDH using SwissDock, a keeping the heme concentration constant (10 µM) and web based molecular interactions prediction titrating with increasing concentration of protein, server20,21. Coordinates of the rPfGAPDH structure at from 0-35 µM, and absorption spectra were recorded. 2.25 Å (2B4R) and HsGAPDHat 1.75 Å (1U8F) were Free and bound heme spectra were curve-fitted using used as templates. Coordinates of heme without iron IgorPro software and used for the calculation of the was used as the ligand. Among several docked heme amount of bound complex19. Amount of bound structures, minimum energy one was used for complex was directly proportional to the change in representation using PyMOL22. absorbance, ΔA, and could be expressed as ΔA = ΔAmax− Kd (ΔA/[P]). Results and Discussion In a modified titration, when heme concentration Characterization of rPfGAPDH was kept constant, and protein concentration was The gene was amplified from cDNA and cloned varied, as this reaction followed a pseudo first order into pET15b and sequencing confirmed the desired PAL: INTERACTIONS OF HEME AND PfGAPDH 27

gene. This rPfGAPDH was overexpressed in which exists as hematin in aqueous solution, appears E. coli using lactose as an inducer15 and resulted in around 365 nm6,7. Soret peak of freshly prepared soluble protein. Two steps protocol was used for hemin solution shifted at 385 nm over the period of purification, and the purified protein appeared as a time. In protein bound state, Soret maximum showed major band of about 35 kDa on SDS-PAGE. The a bathochromic shift and appeared at 424 nm with protein was nearly homogeneous. The yield was more increase in absorption in fully bound state. Extinction than 50 mg/L culture indicating excellent induction by coefficients of free and bound heme were calculated lactose. Orbitrap LC-MS was used to confirm it as to be 19.8 and 132.4 mM-1.cm-1 at 367 nm and malarial GAPDH (data not shown). The typical 424 nm, respectively. specific activity of the purified enzyme was To calculate the binding constant, a fixed 20 units/mg protein. Specific activity was in the range concentration of heme (10 µM) was titrated with an of other known GAPDHs, although low compared increasing concentration of protein (Fig. 1). In an to the rPfGAPDH reported earlier. The specific earlier study, GAPDH was titrated with increasing activity of rPfGAPDH was reported to be between concentrations of heme and found that at higher heme 90-126 unit/mg at 25°C3,14. However, for the second concentration, the non-specific complex was formed4. report14, the assay was carried out at pH 8.9 by the To check the formation of GAPDH complex at higher method of Ferdinand where it had been shown that heme concentration, titration was carried out in a nearly 50% of the specific activity of GAPDH was reverse way. Further, in this way, the concentration of lost when the pH of the assay was reduced to 7.5 from DMSO was kept constant during binding. The key in 8.716. In this case, pH 7.4 was selected for assay as this method was to use fresh heme solution. Since NAD+ has been reported to be very labile in alkaline there would be both free and bound heme in solutions, especially in the presence of phosphate23. equilibrium in the solution; each spectrum would In the presence of 1 µM heme, the specific activity of represent the mixture of both24. Earlier, curve fitting the enzyme was reduced to nearly 30% of its original was used to successfully quantify different heme value. When 2.5 µM heme was used, the inhibition of moieties in solution19. In these types of titrations, an enzymatic activity was near total. However, it had isosbestic point is expected. However, no clear been reported that the heme had no effect on the isosbestic point was observed in this case. A possible activity of other GAPDHs4. To check it further, the reason could be that the higher absorbances of specific activity of heme bound enzyme was proteins at increasing concentrations mask the calculated after desalting the excess heme. In this case individual components of the heme moieties. also, 85% of the activity was abolished. Therefore, each spectrum was curve fitted to calculate the components of free and bound heme. A point of Binding of heme

Estimation of dissociation constant: Electronic absorption spectroscopy To calculate the binding constant of heme binding to rPfGAPDH and to compare it with reported results, spectrophotometric titration was used. To investigate the occurrence of binding, absorption spectra for free heme and heme–protein mixture with an excess amount of protein were studied. Presuming that binding was taking place, two spectra in each case were compared. Shifting of Soret maximum of heme was considered as binding and absorptions were used to calculate extinction coefficients for free and bound heme. In this case, the Soret peak of free heme appeared at 367 nm, which shifted to higher Fig. 1 — Titration of heme by increasing concentrations of wavelength over the period of time. In a recent study, rPfGAPDH. Concentration of hemin was 10 µM and the Soret maximum of heme was reported at 385 nm4. concentrations of protein were 0 (hemin only), 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 9.5, 14.5, 19.5, 24.5, 29.5, 34.5 µM. However, a Soret peak with a maxima at 385 nm The inset shows the change in absorbance against the protein indicates a µ-oxobisheme, whereas, heme monomer, concentration used. 28 INDIAN J. BIOCHEM. BIOPHYS., VOL. 54, FEBRUARY-APRIL 2017

intersection around 400 nm was observed for free and Steady state fluorescence studies bound components for all the fitted spectra (not Thermodynamic parameters indicate that the shown). From the intensity of curve fitted peaks, binding of heme is driven by both enthalpy and fractions of bound heme were calculated. The binding entropy. In ITC, change in enthalpy is measured constant was estimated using the Eadie-Hofstee plot. directly, whereas entropy is calculated. Therefore, to Kd value thus calculated was 3.5 µM with ΔAmax of study the dynamic changes of proteins upon ligand 0.4. From the direct plot (not shown) also it’s evident binding, tryptophan fluorescence could be utilized25. that at half of ΔAmax, the concentration of rPfGAPDH To check the effects of binding of heme and would be around 3.5 µM. It has to be noted that this substrates to rPfGAPDH, which had four tryptophans, calculated value is very sensitive to the composition intrinsic tryptophan fluorescence was exploited, and of buffer and heme state and thus can not be measured rPfGAPDH was titrated against different ligands. accurately. In an earlier report, Ki value has been Binding of GAP did not result in an appreciable shown as 0.2 µM3. It could be noted that at higher change in the spectrum (Fig. 3). Structures of E. coli concentration of heme, the absorbance of heme at GAPDH in apo and holo forms (GAP and NAD+ 340 nm could interfere with that of NADH, the bound) are available26. Comparisons of these product of the enzymatic reaction3. structures show that rmsd between apo and GAP bound form is much less compared to the apo and Isothermal Titration Calorimetry NAD+ bound form26. A similar observation was noted The binding constant of heme to rPfGAPDH in rPfGAPDH also, and the binding of NAD+ calculated in the present studydiffers from the values quenched the fluorescence. Interestingly, upon the reported for other GAPDHs4. Therefore, ITC was addition of heme, quenching of fluorescence was used to directly calculate it. However, it’s difficult to measure heme binding to a protein using ITC as heme needs DMSO (or another suitable solvent) to prevent dimer formation4 and high heat of dilution of DMSO interferes with the heat change of binding of heme to proteins. As a result, reports of these types of binding studies using ITC in the literature are scanty. To overcome this problem, ITC binding studies were carried out using reverse titration. In this case, stock heme solution was freshly diluted using the buffer to minimize DMSO concentration and used in the cell, and concentrated protein in appropriate buffer was taken in the syringe. This was done to minimize the effect of heat of dilution of DMSO present in the heme solution. Binding isotherm fitted using the single site and is shown in Fig. 2. Titrations confirmed the binding of heme to the rPfGAPDH and showed 4 binding sites per tetramer (Fig. 1). The dissociation constant, Kd, calculated from ITC studies was 2.1 µM, which is close to the Kd value calculated using the spectrophotometric method, which is

3.5 µM. ITC results suggest that the heme binding to rPfGAPDH is driven by negative standard molar Fig. 2 — Isothermal titration calorimetric study of heme binding enthalpy as well as positive standard molar entropy to rPfGAPDH. 25 µM freshly prepared heme was taken in the cell changes. To the best of my knowledge, this is the first and titrated with 125 µM of tetramericrPfGAPDH. The molar ratio shows heme per rPfGAPDH tetramer. report of heme binding study using ITC in the presence of DMSO. This sets a platform for these types of studies in the presence of non-aqueous Ligand n ΔH TΔS G=ΔH–TΔS Kd solvents and could be further fine-tuned for ligand kcal/mole kcal/mole kcal/mole µM binding in the presence of such solvents. Hemin 4.15 ± .08 −35.7 ± 0.6 -27.9 −63.7 2.1 PAL: INTERACTIONS OF HEME AND PfGAPDH 29

Fig. 3 — Fluorescence emission spectra of 6 µM rPfGAPDH in the absence or presence of lignads. (A) protein only; (B) with 685 µM GAP; (C) with 125 µM NAD+; (D) with 1.25 µM heme and (E) with 6 µM heme. observed along with the slight blue shift of about 3 nm, indicating binding and related structural reorientation of surface exposed tryptophan residues. + Thus, upon NAD and heme binding, rPfGAPDH exhibited similar dynamic changes, whereas, binding of GAP did not perturb the structure much, despite 5 Fig. 4 — Docking of heme in GAPDHs: (A)PfGAPDH (cyan); and (B) HsGAPDH (magenta) have been represented as ribbons, heme fold excess of the concentration of GAP compared to + + has been represented as semi-transparent surface, and NAD has that of NAD . These studies clearly demonstrated that been represented as sticks. Red represents , blue represents the tryptophan residues located on the protein surface nitrogen whereas grey represents carbon for the ligands. of rPfGAPDH were strongly perturbed upon NAD+ or However, the orientations of heme were different for heme binding. HsGAPDH and PfGAPDH (Fig. 4). In the case of Docking PfGAPDH, one of the propionate side chains of the It has been demonstrated earlier that PfGAPDH heme occupied the NAD+ binding site in. In an earlier binds heme and as a result, enzymatic activity is study also, a similar observation was reported11. reduced3. On the other hand, it has been shown that However, in the case of HsGAPDH, both the GAPDHs from several organisms including human propionate groups were directed towards the GAP erythrocyte GAPDH bind heme with no change in binding pocket. Superimposition of docked heme on enzymatic activity4. In this study, from the the NAD+ bound structure indicated that the heme and spectroscopic titrations and ITC binding studies, we the NAD+ had two independent, but close binding could confirm that heme bound to rPfGAPDH with sites in both the cases (Fig. 4).The orientation of heme micromolar affinity and bound heme inhibited the towards the NAD+ binding pockets in PfGAPDH enzymatic activity of rPfGAPDH as reported earlier. indicated a possible steric clash of NAD+ with the However, no mechanism of inhibition has been heme (Fig. 4). In the presence of bound heme, NAD+ proposed so far, although in a docking study earlier, it would possibly be not able to occupy the pocket was proposed that heme might interfere with the properly, and this could explain the mechanism of NAD+ binding11. To understand the mechanism of inhibition of enzymatic activity of rPfGAPDH. In inhibition, heme was docked on PfGAPDH as well as fact, the Ki value of heme for PfGAPDH was nearly on HsGAPDH. Docking resulted in many similar 50% less in the presence of substrates3. In the case of conformations for both the cases. Among these, the HsGAPDH, the clash is less compared to the minimum energy conformer was used for further PfGAPDH, and that probably accounts for the demonstration. Heme moiety docked in the cleft difference in activities. Also, heme binding may affect between the N-terminal NAD+-binding domain and the cooperative nature of these , which are the C-terminal catalytic domain in the both the cases. known to show cooperativity, resulting in a difference 30 INDIAN J. BIOCHEM. BIOPHYS., VOL. 54, FEBRUARY-APRIL 2017

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