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Molecular Cloning, Purification and Characterization of Mce1R of Mycobacterium tuberculosis

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Abstract

The mce1 operon of Mycobacterium tuberculosis, important for lipid metabolism/transport, host cell invasion, modulation of host immune response and pathogenicity, is under the transcriptional control of Mce1R. Hence characterizing Mce1R is an important step for novel anti-tuberculosis drug discovery. The present study reports functional and in silico characterization of Mce1R. In this work, we have computationally modeled the structure of Mce1R and have validated the structure by computational and experimental methods. Mce1R has been shown to harbor the canonical VanR-like structure with a flexible N-terminal 'arm', carrying conserved positively charged residues, most likely involved in the operator DNA binding. The mce1R gene has been cloned, expressed, purified and its DNA-binding activity has been measured in vitro. The Kd value for Mce1R-operator DNA interaction has been determined to be 0.35 ± 0.02 µM which implies that Mce1R binds to DNA with moderate affinity compared to the other FCD family of regulators. So far, this is the first report for measuring the DNA-binding affinity of any VanR-type protein. Despite significant sequence similarity at the N-terminal domain, the wHTH motif of Mce1R exhibits poor conservancy of amino acid residues, critical for DNA-binding, thus results in moderate DNA-binding affinity. The N-terminal DNA-binding domain is structurally dynamic while the C-terminal domain showed significant stability and such profile of structural dynamics is most likely to be preserved in the structural orthologs of Mce1R. In addition to this, a cavity has been detected in the C-terminal domain of Mce1R which contains a few conserved residues. Comparison with other FCD family of regulators suggests that most of the conserved residues might be critical for binding to specific ligand. The max pKd value and drug score for the cavity are estimated to be 9.04 and 109 respectively suggesting that the cavity represents a suitable target site for novel anti-tuberculosis drug discovery approaches.

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References

  1. World Health Organization, Global Tuberculosis Report, 2019, WHO, Geneva, Switzerland. (www.who.int/tb/publications/global_report/en/).

  2. Sturgill-Koszycki, S., Schlesinger, P. H., Chakraborty, P., Haddix, P. L., Collins, H. L., Fok, A. K., et al. (1994). Lack of acidification in mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase. Science, 263, 678–681.

    Article  CAS  PubMed  Google Scholar 

  3. Arruda, S., Bonfim, G., Knights, R., Huima-Byron, T., & Riley, L. W. (1993). Cloning of an M. tuberculosis DNA fragment associated with entry and survival inside cells. Science, 261, 1454–1457.

    Article  CAS  PubMed  Google Scholar 

  4. Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., et al. (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature, 393, 537–544.

    Article  CAS  PubMed  Google Scholar 

  5. Kumar, A., Chandolia, A., Chaudhry, U., Brahmachari, V., & Bose, M. (2005). Comparison of mammalian cell entry operons of mycobacteria: in silico analysis and expression profiling. FEMS Immunology and Medical Microbiology, 43, 185–195.

    Article  CAS  PubMed  Google Scholar 

  6. Casali, N., & Riley, L. W. (2007). A phylogenomic analysis of the Actinomycetales mce operons. BMC Genomics, 8, 60.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Chitale, S., Ehrt, S., Kawamura, I., Fujimura, T., Shimono, N., Anand, N., et al. (2001). Recombinant Mycobacterium tuberculosis protein associated with mammalian cell entry. Cellular Microbiology, 4, 247–254.

    Article  Google Scholar 

  8. Shimono, N., Morici, L., Casali, N., Cantrell, S., Sidders, B., Ehrt, S., & Riley, L. W. (2003). Hypervirulent mutant of Mycobacterium tuberculosis resulting from disruption of the mce1 operon. Proceedings of the National Academy of Sciences of the United States of America, 100, 15918–15923.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Tekaia, F., Gordon, S. V., Garnier, T., Brosch, R., Barrell, B. G., & Cole, S. T. (1999). Analysis of the proteome of Mycobacterium tuberculosis in silico. Tubercle and Lung Disease, 79, 329–342.

    Article  CAS  PubMed  Google Scholar 

  10. Kumar, A., Bose, M., & Brahmachari, V. (2003). Analysis of expression profile of mammalian cell entry (mce) operons of Mycobacterium tuberculosis. Infection and Immunity, 71, 6083–6087.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Cantrell, S. A., Leavell, M. D., Marjanovic, O., Iavarone, A. T., Leary, J. A., & Riley, L. W. (2013). Free mycolic acid accumulation in the cell wall of the mce1 operon mutant strain of Mycobacterium tuberculosis. Journal Microbiology, 51, 619–626.

    Article  CAS  Google Scholar 

  12. Forrellad, M. A., McNeil, M., Santangelo Mde, L., Blanco, F. C., Garcia, E., Klepp, L. I., et al. (2014). Role of the Mce1 transporter in the lipid homeostasis of Mycobacterium tuberculosis. Tuberculosis (Edinb)., 94, 170–177.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Marjanovic, O., Iavarone, A. T., & Riley, L. W. (2011). Sulfolipid accumulation in Mycobacterium tuberculosis disrupted in the mce2 operon. Journal of Microbiology, 49, 441–447.

    Article  CAS  Google Scholar 

  14. de la Paz Santangelo, M., Klepp, L., Nuñez-García, J., Blanco, F. C., Soria, M., García-Pelayo, M. C., et al. (2009). Mce3R, a TetR-type transcriptional repressor, controls the expression of a regulon involved in lipid metabolism in Mycobacterium tuberculosis. Microbiology, 155, 2245–2255.

    Article  Google Scholar 

  15. Pandey, A. K., & Sassetti, C. M. (2008). Mycobacterial persistence requires the utilization of host cholesterol. Proceedings of the National Academy of Sciences of the United States of America, 105, 4376–4380.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Khan, S., Khan, F. I., Mohammad, T., Khan, P., Hasan, G. M., Lobb, K. A., et al. (2018). Exploring molecular insights into the interaction mechanism of cholesterol derivatives with the Mce4A: a combined spectroscopic and molecular dynamic simulation studies. International Journal of Biological Macromolecules, 111, 548–560.

    Article  CAS  PubMed  Google Scholar 

  17. Checkley, A. M., Wyllie, D. H., Scriba, T. J., Golubchik, T., Hill, A. V., Hanekom, W. A., & McShane, H. (2011). Identification of antigens specific to non-tuberculous mycobacteria: the Mce family of proteins as a target of T cell immune responses. PLoS One, 6(10), e26434.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Stavrum, R., Stavrum, A. K., Valvatne, H., Riley, L. W., Ulvestad, E., Jonassen, I., et al. (2011). Modulation of transcriptional and inflammatory responses in murine macrophages by the Mycobacterium tuberculosis mammalian cell entry (Mce) 1 complex. PLoS One, 6(10), e26295.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Xu, G., Li, Y., Yang, J., Zhou, X., Yin, X., Liu, M., & Zhao, D. (2007). Effect of recombinant Mce4A protein of Mycobacterium bovis on expression of TNF-alpha, iNOS, IL-6, and IL-12 in bovine alveolar macrophages. Molecular and Cellular Biochemistry, 302, 1–7.

    Article  CAS  PubMed  Google Scholar 

  20. Uchida, Y., Casali, N., White, A., Morici, L., Kendall, L. V., & Riley, L. W. (2007). Accelerated immunopathological response of mice infected with Mycobacterium tuberculosis disrupted in the mce1 operon negative transcriptional regulator. Cellular Microbiology, 9, 1275–1283.

    Article  CAS  PubMed  Google Scholar 

  21. Senaratne, R. H., Sidders, B., Sequeira, P., Saunders, G., Dunphy, K., Marjanovic, O., et al. (2008). Mycobacterium tuberculosis strains disrupted in mce3 and mce4 operons are attenuated in mice. Journal of Medical Microbiology, 57, 164–170.

    Article  CAS  PubMed  Google Scholar 

  22. Marjanovic, O., Miyata, T., Goodridge, A., Kendall, L. V., & Riley, L. W. (2010). Mce2 operon mutant strain of Mycobacterium tuberculosis is attenuated in C57BL/6 mice. Tuberculosis, 90, 50–56.

    Article  CAS  PubMed  Google Scholar 

  23. Li, J., Chai, Q. Y., Zhang, Y., Li, B. X., Wang, J., Qiu, X. B., & Liu, C. H. (2015). Mycobacterium tuberculosis Mce3E suppresses host innate immune responses by targeting ERK1/2 signaling. Journal of Immunology, 194, 3756–3767.

    Article  CAS  Google Scholar 

  24. Zhang, F., & Xie, J. P. (2011). Mammalian cell entry gene family of Mycobacterium tuberculosis. Molecular and Cellular Biochemistry, 352, 1–10.

    Article  CAS  PubMed  Google Scholar 

  25. Qiang, L., Wang, J., Zhang, Y., Ge, P., Chai, Q., Li, B., et al. (2019). Mycobacterium tuberculosis Mce2E suppresses the macrophage innate immune response and promotes epithelial cell proliferation. Cellular & Molecular Immunology, 16, 380–391.

    Article  CAS  Google Scholar 

  26. Zhang, Y., Li, J., Li, B., Wang, J., & Liu, C. H. (2018). Mycobacterium tuberculosis Mce3C promotes mycobacteria entry into macrophages through activation of β2 integrin-mediated signaling pathway. Cellular Microbiology, 20, e12800.

    Article  Google Scholar 

  27. Shazly, S. E., Ahmad, S., Mustafa, A. S., Attiyah, R. A., & Krajci, D. (2007). Internalization by HeLa cells of latex beads coated with mammalian cell entry (Mce) proteins encoded by the mce3 operon of Mycobacterium tuberculosis. Journal of Medical Microbiology, 56, 1145–1151.

    Article  PubMed  Google Scholar 

  28. Saini, N. K., Sharma, M., Chandolia, A., Pasricha, R., Brahmachari, V., & Bose, M. (2008). Characterization of Mce4A protein of Mycobacterium tuberculosis: role in invasion and survival. BMC Microbiology, 8, 200.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Fenn, K., Wong, C. T., & Darbari, V. C. (2020). Mycobacterium tuberculosis uses Mce proteins to interfere with host cell signaling. Frontiers in Molecular Biosciences, 6, 149.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Gioffré, A., Infante, E., Aguilar, D., Santangelo, M. P., Klepp, L., Amadio, A., et al. (2005). Mutation in mce operons attenuates Mycobacterium tuberculosis virulence. Microbes and Infection, 7, 325–334.

    Article  PubMed  Google Scholar 

  31. Lima, P., Sidders, B., Morici, L., Reader, R., Senaratne, R., Casali, N., & Riley, L. W. (2007). Enhanced mortality despite control of lung infection in mice aerogenically infected with a Mycobacterium tuberculosis mce1 operon mutant. Microbes and Infection, 9, 1285–1290.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. McCann, J. R., McDonough, J. A., Sullivan, J. T., Feltcher, M. E., & Braunstein, M. (2011). Genome-wide identification of Mycobacterium tuberculosis exported proteins with roles in intracellular growth. Journal of Bacteriology, 193, 854–861.

    Article  CAS  PubMed  Google Scholar 

  33. Wiker, H. G., Spierings, E., Kolkman, M. A., Ottenho, T. H., & Harboe, M. (1999). The mammalian cell entry operon 1 (mce1) of Mycobacterium leprae and Mycobacterium tuberculosis. Microbial Pathogenesis, 27, 173–177.

    Article  CAS  PubMed  Google Scholar 

  34. Haile, Y., Caugant, D. A., Bjune, G., & Wiker, H. G. (2002). Mycobacterium tuberculosis mammalian cell entry operon (mce1) homologs in Mycobacterium other than tuberculosis (MOTT). FEMS Immunology and Medical Microbiology, 33, 125–132.

    Article  CAS  PubMed  Google Scholar 

  35. Singh, P., Katoch, V. M., Mohanty, K. K., & Chauhan, D. S. (2016). Analysis of expression profile of mce operon genes (mce1, mce2, mce3 operon) in different Mycobacterium tuberculosis isolates at different growth phases. Indian Journal of Medical Research, 143, 487–494.

    Article  CAS  Google Scholar 

  36. Rathor, N., Garima, K., Sharma, N. K., Narang, A., Varma-Basil, M., & Bose, M. (2016). Expression profile of mce4 operon of Mycobacterium tuberculosis following environmental stress. International Journal of Mycobacteriology, 5, 328–332.

    Article  PubMed  Google Scholar 

  37. Rathor, N., Chandolia, A., Saini, N. K., Sinha, R., Pathak, R., Garima, K., et al. (2013). An insight into the regulation of mce4 operon of Mycobacterium tuberculosis. Tuberculosis, 93, 389–397.

    Article  CAS  PubMed  Google Scholar 

  38. Casali, N., White, A. M., & Riley, L. W. (2006). Regulation of the Mycobacterium tuberculosis mce1 operon. Journal of Bacteriology, 188, 441–449.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. de la Paz Santangelo, M., Blanco, F., Campos, E., Soria, M., Bianco, M. V., Klepp, L., et al. (2009). Mce2R from Mycobacterium tuberculosis represses the expression of the mce2 operon. Tuberculosis, 89, 22–28.

    Article  Google Scholar 

  40. Kendall, S. L., Withers, M., Soffair, C. N., Moreland, N. J., Gurcha, S., Sidders, B., et al. (2007). A highly conserved transcriptional repressor controls a large regulon involved in lipid degradation in Mycobacterium smegmatis and Mycobacterium tuberculosis. Molecular Microbiology, 65, 684–699.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Zeng, J., Cui, T., & He, Z. G. (2012). A genome-wide regulator-DNA interaction network in the human pathogen Mycobacterium tuberculosis H37Rv. Journal of Proteome Research, 11, 4682–4692.

    Article  CAS  PubMed  Google Scholar 

  42. Vindal, V., Ranjan, S., & Ranjan, A. (2007). In silico analysis and characterization of GntR family of regulators from Mycobacterium tuberculosis. Tuberculosis, 87, 242–247.

    Article  CAS  PubMed  Google Scholar 

  43. Rigali, S., Derouaux, A., Giannotta, F., & Dusart, J. (2002). Subdivision of the helix-turn-helix GntR family of bacterial regulators in the FadR, HutC, MocR, and YtrA subfamilies. Journal of Biological Chemistry, 277, 12507–12515.

    Article  CAS  Google Scholar 

  44. Rigali, S., Schlicht, M., Hoskisson, P., Nothaft, H., Merzbacher, M., Joris, B., & Titgemeyer, F. (2004). Extending the classification of bacterial transcription factors beyond the helix-turn-helix motif as an alternative approach to discover new cis/trans relationships. Nucleic Acids Research, 32, 3418–3426.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Dirusso, C. C., & Black, P. N. (2004). Bacterial long chain fatty acid transport: gateway to a fatty acid-responsive signaling system. Journal of Biological Chemistry, 279, 49563–49566.

    Article  CAS  Google Scholar 

  46. Sambrook, J., & Russell, D. W. (2014). Molecular cloning: a laboratory manual (4th ed.). Cold Spring Harbor: Cold Spring Harbor Laboratory Press.

    Google Scholar 

  47. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.

    Article  CAS  PubMed  Google Scholar 

  48. Ganguly, T., Bandhu, A., Chattoraj, P., Chanda, P. K., Das, M., Mandal, N. C., & Sau, S. (2007). Repressor of temperate mycobacteriophage L1 harbors a stable C-terminal domain and binds to different asymmetric operator DNAs with variable affinity. Virology Journal, 4, 64.

    Article  PubMed  PubMed Central  Google Scholar 

  49. Bandhu, A., Ganguly, T., Jana, B., Mondal, R., & Sau, S. (2010). Regions and residues of an asymmetric operator DNA interacting with the monomeric repressor of temperate mycobacteriophage L1. Biochemistry, 49, 4235–4243.

    Article  CAS  PubMed  Google Scholar 

  50. Larouche, K., Bergeron, M.-J., Leclere, S., & Guerin, S. L. (1996). Optimization of competitor poly (dI-dC)•poly (dI-dC) levels is advised in DNA-protein interaction studies involving enriched nuclear proteins. Biotechniques, 20, 439–444.

    Article  CAS  PubMed  Google Scholar 

  51. Chen, X., Farmer, G., Zhu, H., Prywes, R., & Prives, C. (1993). Cooperative DNA binding of p53 with TFIID (TBP): a possible mechanism for transcriptional activation. Genes & Development, 7, 1837–1849.

    Article  CAS  Google Scholar 

  52. Kelly, L. A., Mezulis, S., Yates, C. M., Wass, M. N., & Sternberg, M. J. E. (2015). The Phyre2 web portal for protein modeling, prediction and analysis. Nature Protocols, 10, 845–858.

    Article  Google Scholar 

  53. Guex, N., & Peitsch, M. C. (1997). SWISS-MODEL and the Swiss-Pdb viewer: an environment for comparative protein modeling. Electrophoresis, 18, 2714–2723.

    Article  CAS  PubMed  Google Scholar 

  54. Wiederstein, M., & Sippl, M. J. (2007). ProSA-web: Interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Reserach, 35, W407–W410.

    Article  Google Scholar 

  55. Zargarian, L., Tilly, V. L., Jamin, N., Chaffotte, A., Gabrielsen, O. S., Toma, F., & Alpert, B. (1999). Myb-DNA recognition: role of tryptophan residues and structural changes of the minimal DNA binding domain of c-Myb. Biochemistry, 38, 1921–1929.

    Article  CAS  PubMed  Google Scholar 

  56. Larsson, T., Wedborg, M., & Turner, D. (2007). Correction of inner-filter effect in fluorescence excitation-emission matrix spectrometry using Raman scatter. Analytica Chimica Acta, 583, 357–363.

    Article  CAS  PubMed  Google Scholar 

  57. Lehrer, S. S. (1971). Solute perturbation of protein fluorescence. The quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion. Biochemistry, 10, 3254–3263.

    Article  CAS  PubMed  Google Scholar 

  58. Kuriata, A., Gierut, A. M., Oleniecki, T., Ciemny, M. P., Kolinski, A., Kurcinski, M., & Kmiecik, S. (2018). CABS-flex 2.0: A web server for fast simulations of flexibility of protein structures. Nucleic Acids Research, 46, W338–W343.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Jamroz, M., Orozco, M., Kolinski, A., & Kmiecik, S. (2013). Consistent view of protein fluctuations from all-atom molecular dynamics and coarse-grained dynamics with knowledge-based force-field. Journal of Chemical Theory and Computation, 9, 119–125.

    Article  CAS  PubMed  Google Scholar 

  60. Koliński, A. (2004). Protein modeling and structure prediction with a reduced representation. Acta Biochimica Polonica, 51, 349–371.

    Article  PubMed  Google Scholar 

  61. Holm, L. (2019). Benchmarking fold detection by DaliLite vol 5. Bioinformatics, 35, 5326–5327.

    Article  CAS  PubMed  Google Scholar 

  62. Holm, L., Kaariainen, S., Rosenstrom, P., & Schenkel, A. (2008). Searching protein structure databases with DaliLite vol 3. Bioinformatics, 24, 2780–2781.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Pei, J., Kim, B. H., & Grishim, N. V. (2008). PROMALS3D: a tool for multiple sequence and structure alignment. Nucleic Acids Research, 36, 2295–2300.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Kumar, S., Stecher, G., Li, M., Knyaz, C., & Tamura, K. (2018). MEGA X: Molecular evolutionary genetic analysis across computing platforms. Molecular Biology and Evolution, 35, 1547–1549.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Xu, Y., Wang, S., Hu, Q., Gao, S., Ma, X., Zhang, W., et al. (2018). CavityPlus: a web server for protein cavity detection with pharmacophore modelling, allosteric site identification and covalent ligand binding ability prediction. Nucleic Acids Research, 46, W374–W379.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Zheng, H., Lu, L., Wang, B., Pu, S., Zhang, X., Zhu, G., et al. (2008). Genetic basis of virulence attenuation revealed by comparative genomic analysis of Mycobacterium tuberculosis strain H37Ra versus H37Rv. PLoS One, 3(6), e2375.

    Article  PubMed  PubMed Central  Google Scholar 

  67. Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel, R. D. and Bairoch, A. (2005) Protein Identification and Analysis Tools on the ExPASy Server; (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press, pp. 571–607.

  68. Bowie, J. U., Luthy, R., & Eisenberg, D. (1991). A method to identify protein sequences that fold into a known three-dimensional structure. Science, 253, 164–170.

    Article  CAS  PubMed  Google Scholar 

  69. Colovos, C., & Yeates, T. O. (1993). Verification of protein structures: patterns of nonbonded atomic interactions. Protein Science, 2, 151–1519.

    Article  Google Scholar 

  70. Laskowski, R. A., MacArthur, M. W., Moss, D. S., & Thornton, J. M. (1993). PROCHECK - a program to check the stereochemical quality of protein structures. Journal of Applied Crystallography, 26, 283–291.

    Article  CAS  Google Scholar 

  71. Roy, S., Maheshwari, N., Chauhan, R., Sen, N. K., & Sharma, A. (2011). Structure prediction and functional characterization of secondary metabolite proteins of Ocimum. Bioinformation, 6, 315–319.

    Article  PubMed  PubMed Central  Google Scholar 

  72. Eftink, M. R., & Ghiron, C. A. (1981). Fluorescence quenching studies with proteins. Analytical Biochemistry, 114, 199–227.

    Article  CAS  PubMed  Google Scholar 

  73. Vivian, J. T., & Callis, P. R. (2001). Mechanisms of tryptophan fluorescence shifts in proteins. Biophysical Journal, 80, 2093–2109.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Xu, Y., Heath, R. J., Li, Z., Rock, C. O., & White, S. W. (2001). The FadR•DNA complex: Transcriptional control of fatty acid metabolism in Escherichia coli. Journal of Biological Chemistry, 276, 17373–17379.

    Article  CAS  Google Scholar 

  75. Boehr, D. D., D’Amico, R. N., & O’Rourke, K. F. (2018). Engineered control of enzyme structural dynamics and function. Protein. Sci., 27, 825–838.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Clarke, N. D., Beamer, K. J., Goldberg, H. F., Berkower, C., & Pabo, C. O. (1991). The DNA binding arm of lambda repressor: critical contacts form a flexible region. Science`, 254, 267–270.

    CAS  Google Scholar 

  77. Das, A. K., Kumar, V. A., Sevalkar, R. R., Bansal, R., & Sarkar, D. (2013). Unique N-terminal arm of Mycobacterium tuberculosis PhoP protein plays an unusual role in its regulatory function. Journal of Biological Chemistry, 288, 29182–29192.

    Article  CAS  Google Scholar 

  78. Palena, C. M., Tron, A. E., Bertoncini, C. W., Gonzalez, D. H., & Chan, R. L. (2001). Positively charged residues at the N–terminal arm of the homeodomain are required for efficient DNA binding by homeodomain–leucine zipper proteins. Journal of Molecular Biology, 308, 39–47.

    Article  CAS  PubMed  Google Scholar 

  79. Miyazono, K., Zhi, Y., Takamura, Y., Nagata, K., Saigo, K., Kojima, T., & Tanokura, M. (2010). Cooperative DNA–binding and sequence–recognition mechanism of aristaless and clawless. EMBO Journal, 29, 1613–1623.

    Article  CAS  Google Scholar 

  80. Domsic, J. F., Chen, H.-S., Lu, F., Marmorstein, R., & Lieberman, P. M. (2013). Molecular basis for oligomeric–DNA binding and episome maintenance by KSHV LANA. PLoS Pathogens, 9(10), e1003672.

    Article  PubMed  PubMed Central  Google Scholar 

  81. Tioni, M. F., Viola, I. L., Chan, R. L., & Gonzalez, D. H. (2005). Site–directed mutagenesis and footprinting analysis of the interaction of the sunflower KNOX protein HAKN1 with DNA. FEBS Journal, 272, 190–202.

    Article  CAS  Google Scholar 

  82. Hart, B. R., Mishra, P. K., Lintner, R. E., Hinerman, J. M., Herr, A. B., & Blumenthal, R. M. (2011). Recognition of DNA by the helix–turn–helix global regulatory protein Lrp is modulated by the amino terminus. Journal Bacteriology, 193, 3794–3803.

    Article  CAS  Google Scholar 

  83. Zheng, M., Cooper, D. R., Grossoehme, N. E., Yu, M., Hung, L. W., Cieslik, M., et al. (2009). Structure of Thermotoga maritima TM0439: implications for the mechanism of bacterial GntR transcriptional regulators with Zn2+–binding FCD domains. Acta Crystallographica D, 65, 356–365.

    Article  CAS  Google Scholar 

  84. Lord, D. M., Baran, A. U., Soo, V. W. C., Wood, T. K., Peti, W., & Page, R. (2014). McbR/YncC: Implications for the mechanism of ligand and DNA binding by a bacterial GntR transcriptional regulator involved in biofilm formation. Biochemistry, 53, 7223–7231.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Pinheiro, J., Lisboa, J., Pombinho, R., Carvalho, F., Carreaux, A., Brito, C., et al. (2018). MouR controls the expression of the Listeria monocytogenes Agr system and mediates virulence. Nucleic Acids Research, 46, 9338–9352.

    CAS  PubMed  PubMed Central  Google Scholar 

  86. Van Aalten, D. M. F., Dirusso, C. C., & Knudsen, J. (2001). The structural basis of acyl Coenzyme A–dependent regulation of the transcription factor FadR. EMBO Journal, 20, 2041–2050.

    Article  Google Scholar 

  87. Gräslund, S., Nordlund, P., Weigelt, J., Hallberg, B. M., Bray, J., Gileadi, O., et al. (2008). Protein production and purification. Nature Methods., 5, 135–146.

    Article  PubMed  Google Scholar 

  88. Goldstone, R. M., Moreland, N. J., Bashiri, G., Baker, E. N., & Shaun Lott, J. (2008). A new Gateway vector and expression protocol for fast and efficient recombinant protein expression in Mycobacterium smegmatis. Protein Expression and Purification, 57, 81–87.

    Article  CAS  PubMed  Google Scholar 

  89. Yamaguchi, H., & Miyazaki, M. (2014). Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies. Biomolecules, 4, 235–251.

    Article  PubMed  PubMed Central  Google Scholar 

  90. DiRusso, C. C., Heimert, T. L., & Metzger, A. K. (1992). Characterization of FadR, a global transcriptional regulator of fatty acid metabolism in Escherichia coli: Interaction with the fadB promoter by long chain fatty acyl Coenzyme As. Journal of Biological Chemistry, 267, 8685–8691.

    Article  CAS  Google Scholar 

  91. Rao, M., Liu, H., Yang, M., Zhao, C., & He, Z. G. (2012). A copper-responsive global repressor regulates expression of diverse membrane-associated transporters and bacterial drug resistance in mycobacteria. Journal of Biological Chemistry, 287, 39721–39731.

    Article  CAS  Google Scholar 

  92. Gao, Y. G., Suzuki, H., Itou, H., Zhou, Y., Tanaka, Y., Wachi, M., et al. (2008). Structural and functional characterization of the LldR from Corynebacterium glutamicum: a transcriptional repressor involved in L–lactate and sugar utilization. Nucleic Acids Research, 36, 7110–7123.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Suvorova, I. A., Korostelev, Y. D., & Gelfand, M. S. (2015). GntR family of bacterial transcription factors and their DNA binding motifs: structure, positioning and co-evolution. PLoS One, 10, e0132618.

    Article  PubMed  PubMed Central  Google Scholar 

  94. Aravind, L., Anantharaman, V., Balaji, S., Babu, M. M., & Iyer, L. M. (2005). The many faces of the helix–turn–helix domain: Transcription regulation and beyond. FEMS Microbiological Review, 29, 231–262.

    Article  CAS  Google Scholar 

  95. Luscombe, N. M., Laskowski, R. A., & Thornton, J. M. (2001). Amino acid–base interactions: a three–dimensional analysis of protein–DNA interactions at an atomic level. Nucleic Acids Research, 29, 2860–2874.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Bromberg, Y., & Rost, B. (2009). Correlating protein function and stability through the analysis of single amino acid substitutions. BMC Bioinformatics, 10, S8.

    Article  PubMed  PubMed Central  Google Scholar 

  97. Teng, S., Wang, L., Srivastava, A. K., Schwartz, C. E., & Alexov, E. (2011). Structural assessment of the effects of amino acid substitutions on protein stability and protein–protein interaction. International Journal of Computational Biology and Drug Design, 3, 334–349.

    Article  PubMed Central  Google Scholar 

  98. Miller, S., Janin, J., Lesk, A., & Chothia, C. (1987). Interior and surface of monomeric proteins. Journal of Molecular Biology, 196, 641–656.

    Article  CAS  PubMed  Google Scholar 

  99. Jana, B., Bandhu, A., Mondal, R., Biswas, A., Sau, K., & Sau, S. (2012). Domain structure and denaturation of a dimeric Mip–like peptidyl–prolyl cis–trans isomerase from Escherichia coli. Biochemistry, 51, 1223–1237.

    Article  CAS  PubMed  Google Scholar 

  100. Tisi, L. C., & Evans, P. A. (1995). Conserved structural features on protein surfaces: Small exterior hydrophobic clusters. Journal of Molecular Biology, 249, 251–258.

    Article  CAS  PubMed  Google Scholar 

  101. Frigerio, F., Margarit, I., Nogarotto, R., de Filippis, V., & Grandi, G. (1996). Cumulative stabilizing effects of hydrophobic interactions on the surface of the neutral protease from Bacillus subtilis. Protein Engineering, 9, 439–445.

    Article  CAS  PubMed  Google Scholar 

  102. Kannan, N., & Vishveshwara, S. (2000). Aromatic clusters: A determinant of thermal stability of thermophilic proteins. Protein Engineering, 13, 753–761.

    Article  CAS  PubMed  Google Scholar 

  103. Karplus, M., & Petsko, G. A. (1990). Molecular dynamics simulations in biology. Nature, 347, 631–639.

    Article  CAS  PubMed  Google Scholar 

  104. Childers, M. C., & Daggett, V. (2017). Insights from molecular dynamics simulations for computational protein design. Molecular Systems Design & Engineering, 2, 9–33.

    Article  CAS  Google Scholar 

  105. Kmiecik, S., Kouza, M., Badaczewska-Dawid, A. E., Kloczkowski, A., & Kolinski, A. (2018). Modeling of protein structural flexibility and large–scale dynamics: Coarse-Grained simulations and elastic network models. International Journal of Molecular Science, 19, 3496.

    Article  Google Scholar 

  106. Teichmann, M., Dumay-Odelot, H., & Fribourg, S. (2012). Structural and functional aspects of winged-helix domains at the core of transcription complexes. Transcription, 3, 2–7.

    Article  PubMed  Google Scholar 

  107. Fernández-Tornero, C., Böttcher, B., Rashid, U. J., Steuerwald, U., Flörchinger, B., Devos, D. P., et al. (2010). Conformational flexibility of RNA polymerase III during transcriptional elongation. EMBO Journal, 29, 3762–3772.

    Article  Google Scholar 

  108. Clubb, R. T., Mizuuchi, M., Huth, J. R., Omichinski, J. G., Savilahti, H., Mizuuchi, K., et al. (1996). The wing of the enhancer-binding domain of Mu phage transposase is flexible and is essential for efficient transposition. Proceedings of the National Academy of Sciences of the United States of America, 93, 1146–1150.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  109. Milano, T., Gulzar, A., Narzi, D., Guidoni, L., & Pascarella, S. (2017). Molecular dynamics simulation unveils the conformational flexibility of the interdomain linker in the bacterial transcriptional regulator GabR from Bacillus subtilis bound to pyridoxal 5’–phosphate. PLoS One, 12, e0189270.

    Article  PubMed  PubMed Central  Google Scholar 

  110. Komeiji, Y., & Uebayasi, M. (1999). Change in fonformation by DNA–peptide association: molecular dynamics of the Hin–recombinase–hixl complex. Biophysical Journal, 77, 123–138.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Ghosh, S., & Bagchi, A. (2019). Structural study to analyze the DNA–binding properties of DsrC protein from the dsr operon of sulfur–oxidizing bacterium Allochromatium vinosum. Journal of Molecular Modeling, 25, 74.

    Article  PubMed  Google Scholar 

  112. Kachhap, S. and Singh, N. Role of DNA conformation & energetic insights in Msx–1–DNA recognition as revealed by molecular dynamics studies on specific and nonspecific complexesc. Journal of Biomolecular Structure and Dynamics 33, 2069–2082.

  113. Fujihashi, M., Nakatani, T., Hirooka, K., Matsuka, H., Fujita, Y., & Miki, K. (2014). Structural characterization of a ligand–bound form of Bacillus subtilis FadR involved in the regulation of fatty acid degradation. Proteins, 82, 1301–1310.

    Article  CAS  PubMed  Google Scholar 

  114. Shi, W., Kovacikova, G., Lin, W., Taylor, R. K., Skorupski, K., & Kull, F. J. (2015). The 40–residue insertion in Vibrio cholerae FadR facilitates binding of an additional fatty acyl–CoA ligand. Nature Communiation, 6, 6032.

    Article  CAS  Google Scholar 

  115. Nazarova, E. V., Montague, C. R., La, T., Wilburn, K. M., Sukumar, N., Lee, W., Caldwell, S., Russell, D. G. and VanderVen, B. C. (2017). Rv3723/LucA coordinates fatty acid and cholesterol uptake in Mycobacterium tuberculosis. eLIFE 6, e26969.

  116. Dunphy, K. Y., Senaratne, R. H., Masuzawa, M., Kendall, L. V., & Riley, L. W. (2010). Attenuation of Mycobacterium tuberculosis functionally disrupted in a fatty acyl–CoA synthetase gene fadD5. Journal of Infectious Diseases, 201, 1232–1239.

    Article  CAS  Google Scholar 

  117. Queiroz, A., Medina–Cleghorn, D., Marjanovic, O., Nomura, D. K. and Riley, L. W. (2015) Comparative metabolic profiling of mce1 operon mutant vs wild–type Mycobacterium tuberculosis strains. FEMS. Pathogens Disease 73, ftv066.

  118. Trivedi, O. A., Arora, P., Sridharan, V., Tickoo, R., Mohanty, D., & Gokhale, R. S. (2004). Enzymatic activation and transfer of fatty acids as acyl–adenylates in mycobacteria. Nature, 428, 441–445.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the Grants from SERB and CSIR (Govt. of India) to Dr. Amitava Bandhu (Grant Nos: SB/YS/LS-184/2014 and 27/(0327)/17/EMR-II dated: 12.04.2017). Mrs. Dipanwita Maity and Dr. Rajasekhara Reddy Katreddy are the recipients of fellowships from SERB and CSIR (Govt. of India), respectively. The authors also acknowledge Mrs. M. Nirupama for her assistance during the work.

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  1. Dipanwita Maity and Rajasekhara Reddy Katreddy have contributed equally to this work.

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  1. Department of Biotechnology, National Institute of Technology Warangal, Warangal, Telangana, 506004, India

    Dipanwita Maity, Rajasekhara Reddy Katreddy & Amitava Bandhu

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  1. Dipanwita Maity
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Maity, D., Katreddy, R.R. & Bandhu, A. Molecular Cloning, Purification and Characterization of Mce1R of Mycobacterium tuberculosis. Mol Biotechnol 63, 200–220 (2021). https://doi.org/10.1007/s12033-020-00293-5

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