RESEARCH ARTICLES CURRENT SCIENCE, VOL. 122, NO. 3, 10 FEBRUARY 2022 281 *For correspondence. (e-mail: mv@iisc.ac.in) † Contributed equally to the work. Structural variability of Mycobacterium tuberculosis SSB and susceptibility to inhibition Srikalaivani Raja 1,† , Anju Paul 1,† , Sriram Raghavan 1,4 , Sibi Narayanan 1,5 , Somnath Shee 2,3 , Amit Singh 2,3 , Umesh Varshney 2 , Balasubramanian Gopal 1 and Mamannamana Vijayan 1, * 1 Molecular Biophysics Unit, Indian Institute of Science, Bengaluru 560 012, India 2 Department of Microbiology and Cell Biology, Indian Institute of Science, Bengaluru 560 012, India 3 Centre for Infectious Disease Research, Indian Institute of Science, Bengaluru 560 012, India 4 Present address: RIKEN Center for Computational Science, Japan 5 Present address: Sitaram Ayurveda Private Limited, Thrissur 680 007, India Single-stranded DNA is formed at various stages of DNA metabolism. It is protected from degradation by single-stranded DNA-binding proteins (SSBs). Struc- tural 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 varia- bility. Docking studies carried out with inhibitors identified from DNA-binding assays allowed the char- acterization 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 tu- berculosis SSB (MtSSB) using DNA-binding and cellu- lar 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. MAJOR processes of DNA metabolism such as replica- tion, recombination and repair involve unwinding of the double-stranded DNA (dsDNA) to form transient, single- stranded DNA (ssDNA). In the cell, ssDNA is highly susceptible to attack by nucleases and other chemically reactive groups. They are also prone to form secondary structures. Single-stranded DNA-binding proteins (SSBs) are designed to protect vulnerable ssDNA from chemical attacks and aberrant secondary structure formation. SSBs are ubiquitous proteins found in viruses, archeae, bacteria and eukaryotes 1,2 . Though the overall structure and function of SSBs across all lifeforms are conserved, they share very low sequence similarity. They bind to DNA with high affi- nity and in a sequence-independent manner. In addition to maintaining the chemical integrity of DNA, SSBs are also involved in binding and controlling the function of other proteins involved in various stages of DNA metabolism. Most bacterial SSBs are homo-tetrameric, with each pro- tomer containing an N- and a C-domain. The N-domain folds into a highly conserved oligonucleotide-binding (OB) fold responsible for coating by ssDNA, whereas the C-terminal domain interacts with nearly a dozen other pro- teins that together constitute the SSB interactome 3,4 . The structure of the 164-residue long SSB from Myco- bacterium tuberculosis (MtSSB), determined as part of a concerted national and international effort 5–8 , showed the same tertiary structure as that seen in previously charac- terized SSBs from Escherichia coli (EcSSB) 9 and human mitochondria (HMtSSB) 10 . The MtSSB tetramer consists of an N-terminal DNA-binding domain and a C-terminal disordered tail. The N-terminal domain consists of three long -hairpin loops extending out of a globular core formed by a five-stranded -barrel capped by an α-helix, characteristic of the OB fold 11 . As shown in Figure 1, MtSSB tetramer is a dimer of dimers with molecular dy- ads along P, Q and R. While the quaternary structures of EcSSB and HMtSSB are comparable, the quaternary ar- rangement of MtSSB is different from the other homo- logues. One important feature that sets it apart from other well-characterized SSBs is the presence of a hook-like structure at the end of the N-domain. This peptide stretch is largely responsible for the unique quaternary structure adopted by the mycobacterial SSBs. In addition to the cano- nical SSB (SSBa), a paralogous protein (SSBb) has been found in several bacteria including M. tuberculosis. Structures of both paralogues of SSB from M. smegmatis and that of SSBa from M. leprae (MlSSB) have also been