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Dissection of Heme Binding to Plasmodium Falciparum Glyceraldehyde-3-Phosphate Dehydrogenase Using Spectroscopic Methods and Molecular Docking

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Dissection of Heme Binding to Plasmodium Falciparum Glyceraldehyde-3-Phosphate Dehydrogenase Using Spectroscopic Methods and Molecular Docking Indian Journal of Biochemistry & Biophysics Vol. 54, February-April 2017, pp. 24-31 Dissection of heme 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 hemoglobin, 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 enzyme, 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-histidine 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).
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