Spectroscopic Studies of Asparaginyl-tRNA Synthetase from Entamoeba histolytica

Author(s): Priyanka Biswas*, Dillip K. Sahu, Kalyanasis Sahu, Rajat Banerjee*.

Journal Name: Protein & Peptide Letters

Volume 26 , Issue 6 , 2019

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Abstract:

Background: Aminoacyl-tRNA synthetases play an important role in catalyzing the first step in protein synthesis by attaching the appropriate amino acid to its cognate tRNA which then transported to the growing polypeptide chain. Asparaginyl-tRNA Synthetase (AsnRS) from Brugia malayi, Leishmania major, Thermus thermophilus, Trypanosoma brucei have been shown to play an important role in survival and pathogenesis. Entamoeba histolytica (Ehis) is an anaerobic eukaryotic pathogen that infects the large intestines of humans. It is a major cause of dysentery and has the potential to cause life-threatening abscesses in the liver and other organs making it the second leading cause of parasitic death after malaria. Ehis-AsnRS has not been studied in detail, except the crystal structure determined at 3 Å resolution showing that it is primarily α-helical and dimeric. It is a homodimer, with each 52 kDa monomer consisting of 451 amino acids. It has a relatively short N-terminal as compared to its human and yeast counterparts.

Objective: Our study focusses to understand certain structural characteristics of Ehis-AsnRS using biophysical tools to decipher the thermodynamics of unfolding and its binding properties.

Methods: Ehis-AsnRS was cloned and expressed in E. coli BL21DE3 cells. Protein purification was performed using Ni-NTA affinity chromatography, following which the protein was used for biophysical studies. Various techniques such as steady-state fluorescence, quenching, circular dichroism, differential scanning fluorimetry, isothermal calorimetry and fluorescence lifetime studies were employed for the conformational characterization of Ehis-AsnRS. Protein concentration for far-UV and near-UV circular dichroism experiments was 8 µM and 20 µM respectively, while 4 µM protein was used for the rest of the experiments.

Results: The present study revealed that Ehis-AsnRS undergoes unfolding when subjected to increasing concentration of GdnHCl and the process is reversible. With increasing temperature, it retains its structural compactness up to 45ºC before it unfolds. Steady-state fluorescence, circular dichroism and hydrophobic dye binding experiments cumulatively suggest that Ehis-AsnRS undergoes a two-state transition during unfolding. Shifting of the transition mid-point with increasing protein concentration further illustrate that dissociation and unfolding processes are coupled indicating the absence of any detectable folded monomer.

Conclusion: This article indicates that GdnHCl induced denaturation of Ehis-AsnRS is a two – state process and does not involve any intermediate; unfolding occurs directly from native dimer to unfolded monomer. The solvent exposure of the tryptophan residues is biphasic, indicating selective quenching. Ehis-AsnRS also exhibits a structural as well as functional stability over a wide range of pH.

Keywords: Ehis-AsnRS, fluorescence, circular dichroism, two-state transition, acrylamide quenching, ITC.

[1]
Rumfeldt, J.A.O.; Galvagnion, C.; Vassall, K.A.; Meiering, E.M. Conformational stability and folding mechanisms of dimeric proteins. Prog. Biophys. Mol. Biol., 2008, 98(1), 61-84.
[2]
Eftink, M.R.; Helton, K.J.; Beavers, A.; Ramsay, G.D. The unfolding of trp aporepressor as a function of pH: Evidence for an unfolding intermediate. Biochemistry, 1994, 33(34), 10220-10228.
[3]
Hornby, J.A.T.; Luo, J.K.; Stevens, J.M.; Wallace, L.A.; Kaplan, W.; Armstrong, R.N.; Dirr, H.W. Equilibrium folding of dimeric class µ glutathione transferases involves a stable monomeric intermediate. Biochemistry, 2000, 39, 12336-12344.
[4]
Park, Y.C.; Bedouelle, H. Dimeric tyrosyl-tRNA synthetase from Bacillus stearothermophilus unfolds through a monomeric intermediate a quantitative analysis under equilibrium conditions. J. Bio. Chem., 1998, 273(29), 18052-18059.
[5]
Clark, A.C.; Sinclair, J.F.; Baldwin, T.O. Folding of bacterial luciferase involves a non-native heterodimeric intermediate in equilibrium with the native enzyme and the unfolded subunits. J. Bio. Chem., 1993, 268(15), 10773-10779.
[6]
Grimsley, J.K.; Scholtz, J.M.; Pace, C.N.; Wild, J.R. Organophosphorus hydrolase is a remarkably stable enzyme that unfolds through a homodimeric interrmediate. Biochemistry, 1997, 36(47), 14366-14374.
[7]
Neet, K.E.; Timm, D.E. Conformational stability of dimeric proteins: Quantitative studies by equilibrium denaturation. Protein Sci., 1994, 3, 2167-2174.
[8]
Kaplan, W.; Husler, P.; Klump, H.; Erhardt, J.; Cremer, N.S.; Dirr, H. Conformational stability of pGEX-expressed Schistosoma japonicum glutathione S-transferase: A detoxification enzyme and fusion-protein affinity tag. Protein Sci., 1997, 6, 399-406.
[9]
Berg, J.M.; Tymoczko, J.L.; Stryer, L. Biochemistry, 5th ed; W.H. Freeman: New York, USA, 2002.
[10]
O’Donoghue, P.; Luthey-Schulten, Z. On the evolution of structure in aminoacyl-tRNA synthetases. Microbiol. Mol. Biol. Rev., 2003, 67(4), 550-573.
[11]
Eriani, G.; Delarue, M.; Poch, O.; Gangloff, J.; Moras, D. Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs. Nature, 1990, 347(6289), 203-206.
[12]
Cusack, S. Aminoacyl-tRNA synthetases. Curr. Opin. Struct. Biol., 1997, 7(6), 881-889.
[13]
Cusack, S.; Härtlein, M.; Leberman, R. Sequence, structural and evolutionary relationships between class II aminoacyl-tRNA synthetases. Nucleic Acids Res., 1991, 19(13), 3489-3498.
[14]
Perona, J.J., and ; Hadd, A. Structural diversity and protein engineering of the aminoacyl-tRNA synthetases. Biochemistry, 2012, 51, 8705-8729.
[15]
Ibba, M., and ; Soll, D. Aminoacyl-tRNA synthesis. Annu. Rev. Biochem., 2000, 69, 617-650.
[16]
Bullwinkle, T.J.; Ibba, M. Emergence and evolution. Top. Curr. Chem., 2014, 344, 43-87.
[17]
Walter, F.; Putz, J.; Giege, R.; Westhof, E. Binding of tobramycin leads to conformational changes in yeast tRNAAsp and inhibition of aminoacylation. EMBO J., 2002, 21, 760-768.
[18]
Mikkelsen, N.E.; Johansson, K.; Virtanen, A.; Kirsebom, L.A. Aminoglycoside binding displaces a divalent metal ion in a tRNA-neomycin B complex. Nat. Struct. Biol., 2001, 8, 510-514.
[19]
Pham, J.S.; Dawson, K.L.; Jackson, K.E.; Lim, E.E.; Pasaje, C.F.; Turner, K.E.; Ralph, S.A. Aminoacyl-tRNA synthetases as drug targets in eukaryotic parasites. Int. J. Parasitol. Drugs Drug Resist., 2013, 4(1), 1-13.
[20]
Hurdle, J.G.; O’Neill, A.J.; Chopra, I. Prospects for aminoacyl-tRNA synthetase inhibitors as new antimicrobial agents. Antimicrob. Agents Chemother., 2005, 49(12), 4821-4833.
[21]
Hoen, R.; Novoa, E.M.; López, A.; Camacho, N.; Cubells, L.; Vieira, P.; Santos, M.; Marin-Garcia, P.; Bautista, J.M.; Cortés, A.; Ribas de Pouplana, L.; Royo, M. Selective inhibition of an apicoplastic aminoacyl-tRNA synthetase from Plasmodium falciparum. ChemBioChem, 2013, 14(4), 499-509.
[22]
Khan, S. Recent advances in the biology and drug targeting of malaria parasite aminoacyl-tRNA synthetases. Malar. J., 2016, 15, 203.
[23]
Jothi, D.J.; Dhanraj, M.; Solaiappan, S.; Sivanesan, S.; Kron, M.; Dhanasekaran, A. Brugia malayi Asparaginyl-tRNA synthetase stimulates endothelial cell proliferation, vasodilation and angiogenesis. PLoS One, 2016, 12(2), e0171402.
[24]
Gowri, V.S.; Ghosh, I.; Sharma, A.; Madhubala, R. Unusual domain architecture of aminoacyl tRNA synthetases and their paralogs from Leishmania major. BMC Genomics, 2012, 13, 621-636.
[25]
Berthet-Colominas, C.; Seignovert, L.; Härtlein, M.; Grotli, M.; Cusack, S.; Leberman, R. The crystal structure of asparaginyl-tRNA synthetase from Thermus thermophilus and its complexes with ATP and asparaginyl-adenylate: The mechanism of discrimination between asparagine and aspartic acid. EMBO J., 1998, 17(10), 2947-2960.
[26]
Kalidas, S.; Cestari, I.; Monnerat, S.; Li, Q.; Regmi, S.; Hasle, N.; Labaied, M.; Parsons, M.; Stuart, K.; Phillips, M.A. Genetic validation of aminoacyl-tRNA synthetases as drug targets in Trypanosoma brucei. Eukaryotic Cell J., 2014, 13(4), 504-516.
[27]
Ryan, K.J.; Ray, C.G. Sherris Medical Microbiology, 4th ed; McGraw Hill: New York City, USA, 2004, pp. 733-738.
[28]
Tanyuksel, M.; Petri, W.A., Jr Laboratory diagnosis of amebiasis. Clin. Microbiol. Rev., 2003, 16(4), 713-729.
[29]
Rodríguez, L.; Cervantes, E.; Ortiz, R. Malnutrition and gastrointestinal and respiratory infections in children: A public health problem. Int. J. Environ. Res. Public Health, 2011, 8(4), 1174-1205.
[30]
Larson, E.T.; Merritt, E.A. Medical Structural Genomics of Pathogenic Protozoa 2010. PDB ID: 3M4Q
[31]
Park, Y.C.; Bedouelle, H. Dimeric tyrosyl-tRNA synthetase from Bacillus stearothermophilus unfolds through a monomeric intermediate. J. Bio. Chem., 1998, 273(17), 18052-18059.
[32]
Dignam, J.D.; Qu, X.; Chaires, J.B. Equilibrium unfolding of Bombyx mori glycyl-tRNA synthetase. J. Bio. Chem., 2001, 276(6), 4028-4037.
[33]
Banerjee, B.; Banerjee, R. Guanidine hydrochloride mediated denaturation of E. coli alanyl-tRNA synthetase: Identification of an inactive dimeric intermediate. Protein J., 2014, 33, 119-127.
[34]
Alexandrov, A.; Vignali, M.; LaCount, D.J.; Quartley, E.; de Vries, C.; De Rosa, D.; Babulski, J.; Mitchell, S.F.; Schoenfeld, L.W.; Fields, S.; Hol, W.G.; Dumont, M.E.; Phizicky, E.M.; Grayhack, E.J. A facile method for high-throughput co-expression of protein pairs. Mol. Cell. Proteomics, 2004, 3(9), 934-938.
[35]
Guild, K.; Zhang, Y.; Stacy, R.; Mundt, E.; Benbow, S.; Green, A.; Myler, P.J. Wheat germ cell-free expression system as a pathway to improve protein yield and solubility for the SSGCID pipeline. Struc. Biol. Cryst. Comm., 2011, 67(9), 1027-1031.
[36]
Studier, F.W. Protein production by auto-induction in high-density shaking cultures. Protein Exp. Pur., 2005, 41, 207-234.
[37]
Kenoth, R.; Swami, M.J. Steady-state and time resolved fluorescence studies on Trichosanthes cucumerina seed lectin. J. Photochem. Photobiol. B Biol, 2003, 69, 193-201.
[38]
Sreejith, R.K.; Yadav, V.N.; Varshney, N.K.; Berwal, S.K.; Suresh, C.G.; Gaikwad, S.M.; Pal, J.K. Conformational characterization of human eukaryotic initiation factor 2α: A single tryptophan protein. J. Biochem. Biophy. Res. Comm., 2009, 390, 273-279.
[39]
Kishore, D.; Kundu, S.; Kayastha, A.M. Thermal, chemical and pH induced denaturation of a multimeric β-galactosidase reveals multiple unfolding pathways. PLoS One, 2012, 7(11), 1-9.
[40]
Lavinder, J.J.; Hari, S.B.; Sullivan, B.J.; Magliery, T.J. High-throughput thermal scanning: A general, rapid dye-binding thermal shift screen for protein engineering. J. Am. Chem. Soc., 2009, 131(11), 3794-3795.
[41]
Chatterjee, A.; Mandal, D.K. Denaturant-induced equilibrium unfolding of concanavalin A is expressed by a three-state mechanism and provides an estimate of its protein stability. Biochem. Biophys. Acta, 2003, 1648(2), 174-183.
[42]
Baez, M.; Cabrera, R.; Guixé, V.; Babul, J. Unfolding pathway of the dimeric and tetrameric forms of phosphofructokinase-2 from Escherichia coli. Biochemistry, 2007, 46(20), 6141-6148.
[43]
Mukherjee, S.; Saha, B.; Das, A.K. Differential chemical and thermal unfolding pattern of Rv3588c and Rv1284 of Mycobacterium tuberculosis - A comparison by fluorescence and circular dichroism spectroscopy. Biophys. Chem., 2009, 141, 94-104.
[44]
Eftink, M.R.; Ghiron, C.A. Exposure of tryptophanyl residues in proteins quantitative determination by fluorescence quenching studies. Biochemistry, 1976, 15(3), 672-680.
[45]
Cestari, I.; Stuart, K. A Spectrophotometric Assay for quantitative measurement of aminoacyl-tRNA synthetase activity. J. Biomol. Screening., 2012, 18(4), 490-497.
[46]
Danel, F.; Caspers, P.; Nuoffer, C.; Härtlein, M.; Kron, M.A. Page, M.G. Asparaginyl-tRNA synthetase pre-transfer editing Assay. Curr. Drug Disc. Tech., 2011, 8, 66-75.
[47]
Grove, A.; Kushwaha, A.K.; Nguyen, K.H. Protein Cages: Methods and Protocols, Ch-9 Determining the role of metal binding in protein cage assembly. Methods in Molecular Biology. In: Thermal Stability Assay; Orner, B.P., Ed.; Humana Press: New York City, USA, 2014; Vol. 1252, pp. 91-100.
[48]
Eftink, M.R. Fluorescence techniques for studying protein structure. Methods Biochem. Anal., 1991, 35, 127-205.
[49]
Tripathi, P.; Hofmann, H.; Kayastha, A.M.; Ulbrich-Hofmann, R. Conformational stability and integrity of a-amylase from mung beans: Evidence of kinetic intermediate in GdmCl-induced unfolding. Biophys. Chem., 2008, 137, 95-99.
[50]
Harder, M.; Deinzer, M.L.; Leid, M.E.; Schimerlik, M.I. Global analysis of three state protein unfolding data. Protein Sci., 2004, 13(8), 2207-2222.
[51]
Riley, W.P.; Cheng, H.; Samuel, D.; Roder, H.; Walsh, P.N. Dimer dissociation and unfolding mechanism of coagulation factor XI Apple 4 domain: Spectroscopic and mutational analysis. J. Mol. Bio., 2007, 367(2), 558-573.
[52]
Telley, K.; Alexov, E. On the pH-optimum of activity and stability of proteins. Proteins, 2010, 78(12), 2699-2706.
[53]
Semisotnov, G.V.; Rodionova, N.A.; Razgulyaev, O.I.; Uversky, V.N.; Gripas, A.F.; Gilmanshin, R.I. Study of the “molten globule” intermediate state in protein folding by a hydrophobic fluorescent probe. Biopolymers, 1991, 31(1), 119-128.
[54]
Diamandis, E.P.; Christopoulos, T.K. Nonradioactive analysis of biomolecules. Time-Resolved Fluorescence, Ch-23, Springer Lab Manual, , 289-294.
[55]
Phillips, S.R.; Wilson, L.J.; Borkman, R.F. Acrylamide and iodide fluorescence quenching as a structural probe of tryptophan microenvironment in bovine lens crystallins. Curr. Eye Res., 1986, 5(8), 611-619.
[56]
Strambini, G.B.; Gonnelli, M. Fluorescence quenching of buried trp residues by acrylamide does not require penetration of the protein fold. J. Phys. Chem. B, 2010, 114(2), 1089-1093.
[57]
Xing, D.; Yi, L.; Yuan-Ling, X.; Shi-Meng, A.; Jing, L.; Peng, S.; Xing-Lai, J.; Shu-Qun, L. Insights into protein – ligand interactions: Mechanisms, models, and methods. Int. J. Of Mol. Sci., 2016, 17(2), 144.
[58]
Ghai, R.; Falconer, R.J.; Collins, B.M. Applications of isothermal titration calorimetry in pure and applied research--survey of the literature from 2010. J. Mol. Recognit., 2012, 25(1), 32-52.
[59]
Grolier, J.P.E.; del Rio, J.M. Isothermal titration calorimetry: A thermodynamic interpretation of measurements. J. Chem. Therm., 2012, 55, 193-202.
[60]
Chuawong, P.; Hendrickson, T.L. The nondiscriminating aspartyl-tRNA synthetase from Helicobacter pylori: Anticodon-binding domain mutations that impact tRNA specificity and heterologous toxicity. Biochemistry, 2006, 45(26), 8079-8087.
[61]
Choi, H.; Gabriel, K.; Schneider, J.; Otten, S.; McClain, W.H. Recognition of acceptor-stem structure of tRNA(Asp) by Escherichia coli aspartyl-tRNA synthetase. RNA, 2003, 9(4), 386-393.
[62]
Briand, C.; Poterszman, A.; Eiler, S.; Webster, G.; Thierry, J.C.; Moras, D. An intermediate step in the recognition of tRNA(Asp) by aspartyl-tRNA synthetase. J. Bio. Mol., 2000, 299(4), 1051-1060.
[63]
Francin, M.; Kaminska, M.; Kerjan, P.; Mirande, M. The N-terminal domain of mammalian lysyl-tRNA synthetase is a functional tRNA-binding domain. J. Bio Chem., 2002, 277(3), 1762-1769.
[64]
Guo, M.; Ignatov, M.; Musier-Forsyth, K.; Schimmel, P.; Yang, X.L. Crystal structure of tetrameric form of human lysyl-tRNA synthetase: Implications for multisynthetase complex formation. PNAS, 2008, 105(7), 2331-2336.
[65]
Dams, T.; Jaenicke, R. Stability and folding of dihydrofolate reductase from the hyperthermophilic bacterium Thermotoga maritima. Biochemistry, 1999, 38(28), 9169-9178.
[66]
Andersen, N.H. Protein structure, stability and folding. In: Methods in Molecular Biology; Humana Press: New York City, USA, 2010; Vol. 168, .
[67]
Sundd, M.; Kundu, S.; Jagannadham, M.V. Acid and chemical induced conformational changes of Ervatamin B. Presence of partially structured multiple intermediates. J. Biochem. Mol. Biol., 2002, 35, 143-154.
[68]
Mok, Y.K.; Gay, G.; Butler, P.J.; Bycroft, M. Equilibrium dissociation and unfolding of the dimeric human papillomavirus strain-16 E2 DNA-binding domain. Protein Sci., 1996, 5, 310-319.
[69]
Zwanzig, R. Two-state models of protein folding kinetics. PNAS, 1997, 94(1), 148-150.
[70]
Frimurer, T.M.; Peters, G.H.; Iversen, L.F.; Andersen, H.S.; Moller, N.P.H.; Olsen, O.H. Ligand-induced conformational changes: Improved predictions of ligand binding conformations and affinities. Biophys. J., 2003, 84(4), 2273-2281.
[71]
Koike, R.; Amemiya, T.; Ota, M.; Kidera, A. Protein structural change upon ligand binding correlates with enzymatic reaction mechanism. J. Mol. Bio., 2008, 379(3), 397-401.
[72]
Möller, M.; Denicola, A. Protein tryptophan accessibility studied by fluorescence quenching. Biochem. Mo. Bio. Edu., 2002, 30(3), 175-178.
[73]
Lakowicz, J.R. Principles of Fluorescence Spectroscopy, 3rd edition; Springer Publishers: Berlin, Germany, 2007.


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VOLUME: 26
ISSUE: 6
Year: 2019
Page: [435 - 448]
Pages: 14
DOI: 10.2174/0929866526666190327122419
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