The DNA Binding Protein Lsr2 from Mycobacterium tuberculosis
Lsr2 is a small, basic DNA binding protein that is highly conserved in mycobacteria and related actinomycetes. Lsr2 is essential for growth in Mycobacterium tuberculosis and previous studies have shown that Lsr2 is involved in down-regulating a wide range of genes involved in cell wall synthesis and metabolic functions. This regulatory function is likely to influence bacterial growth and survival. This research investigated the biochemistry and 3D structure of Lsr2 from M. tuberculosis. Transmission electron microscopy (TEM) analysis of Lsr2 in complex with DNA revealed a regular fibril-like arrangement of protein coating double-stranded DNA. In addition, it was shown that Lsr2 physically protected DNA from DNase activity. The structure of the C-terminal DNA binding domain of Lsr2, determined by others part way through this research, prompted site directed mutagenesis of residues proposed to interact with DNA. Modification of arginine residues significantly reduced the binding of Lsr2 to DNA and fibril-like structures were not observed using TEM, for arginine mutants. The first crystal structure of the N-terminal domain of Lsr2 is reported here. Two high resolution structures in monoclinic and hexagonal space groups were solved using X-ray crystallography and ab initio phasing. Proteolytic processing of the N-terminus of Lsr2 was revealed by the structure in P2₁ and this process was recreated using the protease trypsin which resulted in crystal formation in a P3₁21 space group. Both structures show linear chains of dimeric N-terminal Lsr2, as shown by crystallographic symmetry, linked by overlapping anti-parallel β-sheets, revealing a mechanism of protein oligomerisation. Oligomerisation only occurs after the removal of the first three residues M1, A2 and K3. In solution, protein oligomerisation was recreated with trypsin, resulting in the formation of large protein complexes. A change in DNA topology after the addition of trypsin to full-length Lsr2/DNA complexes was observed using TEM. This mechanism is likely to be important to M. tuberculosis under “stress” conditions where proteases are known to be upregulated and where cross-linking and condensation of DNA is critical.
DNA binding protein, Mycobacterium tuberculosis, Tuberculosis, Lsr2
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