Current Science

Open Access Open Access  Restricted Access Subscription Access

Structural variability of Mycobacterium tuberculosis SSB and susceptibility to inhibition


Affiliations
1 Molecular Biophysics Unit, Indian Institute of Science, Bengaluru 560 012, India
2 Molecular Biophysics Unit, Indian Institute of Science, Bengaluru 560 012, India; Present address: RIKEN Center for Computational Science, Japan
3 Molecular Biophysics Unit, Indian Institute of Science, Bengaluru 560 012, India; Present address: Sitaram Ayurveda Private Limited, Thrissur 680 007, India
4 Department of Microbiology and Cell Biology, Indian Institute of Science, Bengaluru 560 012, India; Centre for Infectious Disease Research, Indian Institute of Science, Bengaluru 560 012, India
5 Department of Microbiology and Cell Biology, Indian Institute of Science, Bengaluru 560 012, India
 

Single-stranded DNA is formed at various stages of DNA metabolism. It is protected from degradation by single-stranded DNA-binding proteins (SSBs). Structural variability has been observed in the quaternary arrangement of tetrameric SSBs from mycobacteria and other sources. Here we describe two novel crystal forms which illustrate the extent of structural variability. Docking studies carried out with inhibitors identified from DNA-binding assays allowed the characterization of eight distinct potential binding regions or grooves on each tetramer that circumvent structurally variable regions. Compounds known to inhibit certain bacterial SSBs were tested against Mycobacterium tuberculosis SSB (MtSSB) using DNA-binding and cellular assays. We report two compounds that inhibit MtSSB and growth of the bacterium. Together, this structural analysis reveals a strategy to exploit the variability of MtSSB for the design of inhibitors to this protein. The variability in structure of MtSSB could contribute to its susceptibility to inhibition

Keywords

Binding regions, crystal structure, docking, inhibitor development, Mycobacterium tuberculosis, structural plasticity.
User
Notifications
Font Size

  • Chase, J. W. and Williams, K. R., Single-stranded DNA binding proteins required for DNA replication. Annu. Rev. Biochem., 1986, 55, 103–136.

  • Lohman, T. M. and Ferrari, M. E., Escherichia coli singlestranded DNA-binding protein: multiple DNA-binding modes and cooperativities. Annu. Rev. Biochem., 1994, 63, 527–570.

  • Shereda, R. D., Kozlov, A. G., Lohman, T. M., Cox, M. M. and Keck, J. L., SSB as an organizer/mobilizer of genome maintenance complexes. Crit. Rev. Biochem. Mol. Biol., 2008, 43(5), 289–318.

  • Costes, A., Lecointe, F., McGovern, S., Quevillon-Cheruel, S. and Polard, P., The C-terminal domain of the bacterial SSB protein acts as a DNA maintenance hub at active chromosome replication forks. PLOS Genet., 2010, 6(12), e1001238.

  • Terwilliger, T. C. et al., The TB structural genomics consortium: a resource for Mycobacterium tuberculosis biology. Tuberculosis, 2003, 83(4), 223–249.

  • Vijayan, M., Structural biology of mycobacterial proteins: the Bangalore effort. Tuberculosis, 2005, 85(5), 357–366.

  • Arora, A. et al., Structural biology of Mycobacterium tuberculosis proteins: the Indian efforts. Tuberculosis, 2011, 91(5), 456–468.

  • Arcus, V. L., Lott, J. S., Johnston, J. M. and Baker, E. N., The potential impact of structural genomics on tuberculosis drug discovery. Drug Discov. Today, 2006, 11(1–2), 28–34.

  • Raghunathan, S., Ricard, C. S., Lohman, T. M. and Waksman, G., Crystal structure of the homo-tetrameric DNA binding domain of Escherichia coli single-stranded DNA-binding protein determined by multiwavelength X-ray diffraction on the selenomethionyl protein at 2.9-A resolution. Proc. Natl. Acad. Sci. USA, 1997, 94(13), 6652–6657.

  • Yang, C., Curth, U., Urbanke, C. and Kang, C., Crystal structure of human mitochondrial single-stranded DNA binding protein at 2.4 A resolution. Nature Struct. Biol., 1997, 4(2), 153–157.

  • Saikrishnan, K. et al., Crystallization and preliminary X-ray studies of the single-stranded DNA-binding protein from Mycobacterium tuberculosis. Acta Crystallogr., Sect. D, 2002, 58(2), 327–329.

  • Saikrishnan. K. et al., Structure of Mycobacterium smegmatis single-stranded DNA-binding protein and a comparative study involving homologus SSBs: biological implications of structural plasticity and variability in quaternary association. Acta Crystallogr. Sect. D, 2005, 61(8), 1140–1148.

  • Kaushal, P. S., Singh, P., Sharma, A., Muniyappa, K. and Vijayan, M., X-ray and molecular-dynamics studies on Mycobacterium leprae single-stranded DNA-binding protein and comparison with other eubacterial SSB structures. Acta Crystallogr., Sect. D, 2010, 66(10), 1048–1058.

  • Singh, A., Vijayan, M. and Varshney, U., Distinct properties of a hypoxia specific paralog of single stranded DNA binding (SSB) protein in mycobacteria. Tuberculosis, 2018, 108, 16–25.

  • Arif, S. M. and Vijayan, M., Structural diversity based on variability in quaternary association. A case study involving eubacterial and related SSBs. Methods Mol. Biol., 2012, 922, 23–35.

  • Voter, A. F., Killoran, M. P., Ananiev, G. E., Wildman, S. A., Hoffmann, F. M. and Keck, J. L., A high-throughput screening strategy to identify inhibitors of SSB protein–protein interactions in an academic screening facility. SLAS Discov., 2018, 23(1), 94– 101.

  • Glanzer, J. G., Endres, J. L., Byrne, B. M., Liu, S., Bayles, K. W. and Oakley, G. G., Identification of inhibitors for single-stranded DNA-binding proteins in eubacteria. J. Antimicrob. Chemother., 2016, 71(12), 3432–3340.

  • Huang, C. Y., Crystal structure of SSB complexed with inhibitor myricetin. Biochem. Biophys. Res. Commun., 2018, 504(4), 704– 708.

  • Battye, T. G. G., Kontogiannis, L., Johnson, O., Powell, H. R. and Leslie, A. G. W., iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. Sect. D, 2011, 67(4), 271–281.

  • Winn, M. D. et al., Overview of the CCP4 suite and current developments. Acta Crystallogr. Sect. D, 2011, 67(4), 235–242.

  • Matthews, B. W., Solvent content of protein crystals. J. Mol. Biol., 1968, 33(2), 491–497.

  • McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. and Read, R. J., Phaser crystallographic software. J. Appl. Crystallogr., 2007, 40(4), 658–674.

  • Murshudov, G. N. et al., REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. Sect. D, 2011, 67(4), 355–367.

  • Emsley, P. and Cowtan, K., Coot: model-building tools for molecular graphics. Acta Crystallogr. Sect. D, 2004, 60(12 and 1), 2126–2132.

  • Sastry, G. M., Adzhigirey, M., Day, T., Annabhimoju, R. and Sherman, W., Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments. J. Comput. Aided Mol. Des., 2013, 27(3), 221–234.

  • Halgren, T. A., Identifying and characterizing binding sites and assessing druggability. J. Chem. Inf. Model., 2009, 49(2), 377–389.

  • Friesner, R. A. et al., Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. J. Med. Chem., 2006, 49(21), 6177–6196.

  • Greenwood, J. R., Calkins, D., Sullivan, A. P. and Shelley, J. C., Towards the comprehensive, rapid, and accurate prediction of the favorable tautomeric states of drug-like molecules in aqueous solution. J. Comput. Aided Mol. Des., 2010, 24(6–7), 591–604.

  • Cohen, G., ALIGN: a program to superimpose protein coordinates, accounting for insertions and deletions. J. Appl. Crystallogr., 1997, 30(6), 1160–1161.

  • Hubbard, S. J. and Thornton, J. M., ‘NACCESS’, computer program. Department of Biochemistry and Molecular Biology, University College, London, UK, 1993.

  • Pettersen, E. F. et al., UCSF Chimera – a visualization system for exploratory research and analysis. J. Comput. Chem., 2004, 25(13), 1605–1612.

  • DeLano, W. L., PyMOL, 2002, http://www.pymol.org.

  • Madeira, F. et al., The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res., 2019, 47(W1), W636–W641.

  • Robert, X. and Gouet, P., Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res., 2014, 42(W1), W320–W324.

  • Kuriata, A. et al., CABS-flex 2.0: a web server for fast simulations of flexibility of protein structures. Nucleic Acids Res., 2018, 46(W1), W338–W343.

  • Saikrishnan, K. et al., Structure of Mycobacterium tuberculosis single-stranded DNA-binding protein. Variability in quaternary structure and its implications. J. Mol. Biol., 2003, 331(2), 385–393.

  • Dubiel, K. et al., Structural mechanisms of cooperative DNA binding by bacterial single-stranded DNA-binding proteins. J. Mol. Biol., 2019, 431(2), 178–195.

  • Waldman, V. M., Weiland, E., Kozlov, A. G. and Lohman, T. M., Is a fully wrapped SSB-DNA complex essential for Escherichia coli survival? Nucleic Acids Res., 2016, 44(9), 4317–4329.


Abstract Views: 385

PDF Views: 141




  • Structural variability of Mycobacterium tuberculosis SSB and susceptibility to inhibition

Abstract Views: 385  |  PDF Views: 141

Authors

Srikalaivani Raja
Molecular Biophysics Unit, Indian Institute of Science, Bengaluru 560 012, India
Anju Paul
Molecular Biophysics Unit, Indian Institute of Science, Bengaluru 560 012, India
Sriram Raghavan
Molecular Biophysics Unit, Indian Institute of Science, Bengaluru 560 012, India; Present address: RIKEN Center for Computational Science, Japan
Sibi Narayanan
Molecular Biophysics Unit, Indian Institute of Science, Bengaluru 560 012, India; Present address: Sitaram Ayurveda Private Limited, Thrissur 680 007, India
Somnath Shee
Department of Microbiology and Cell Biology, Indian Institute of Science, Bengaluru 560 012, India; Centre for Infectious Disease Research, Indian Institute of Science, Bengaluru 560 012, India
Amit Singh
Department of Microbiology and Cell Biology, Indian Institute of Science, Bengaluru 560 012, India; Centre for Infectious Disease Research, Indian Institute of Science, Bengaluru 560 012, India
Umesh Varshney
Department of Microbiology and Cell Biology, Indian Institute of Science, Bengaluru 560 012, India
Balasubramanian Gopal
Molecular Biophysics Unit, Indian Institute of Science, Bengaluru 560 012, India
Mamannamana Vijayan
Molecular Biophysics Unit, Indian Institute of Science, Bengaluru 560 012, India

Abstract


Single-stranded DNA is formed at various stages of DNA metabolism. It is protected from degradation by single-stranded DNA-binding proteins (SSBs). Structural variability has been observed in the quaternary arrangement of tetrameric SSBs from mycobacteria and other sources. Here we describe two novel crystal forms which illustrate the extent of structural variability. Docking studies carried out with inhibitors identified from DNA-binding assays allowed the characterization of eight distinct potential binding regions or grooves on each tetramer that circumvent structurally variable regions. Compounds known to inhibit certain bacterial SSBs were tested against Mycobacterium tuberculosis SSB (MtSSB) using DNA-binding and cellular assays. We report two compounds that inhibit MtSSB and growth of the bacterium. Together, this structural analysis reveals a strategy to exploit the variability of MtSSB for the design of inhibitors to this protein. The variability in structure of MtSSB could contribute to its susceptibility to inhibition

Keywords


Binding regions, crystal structure, docking, inhibitor development, Mycobacterium tuberculosis, structural plasticity.

References





DOI: https://doi.org/10.18520/cs%2Fv122%2Fi3%2F281-289