Structural characterization of a transcriptional regulator of mycolic acid biosynthesis in Mycobacterium tuberculosis

JULIA LARA, LAUTARO DIACOVICH, GABRIELA GAGO, HUGO GRAMAJO

Santa Fe

Congreso; VIII Reunión Anual de la Asociación Argentina de Cristalografía; 2012

Asociación Argentina de Cristalografía (AACr)

Mycolic acids, the dominant feature of the Mycobacterium tuberculosis cell wall, are essential for the survival, virulence, and antibiotic exclusion of this human pathogen. Mycobacteria, unlike most bacteria, have two fatty acid synthases (FAS-I and FAS-II). Both of them are involved in the biosynthesis of mycolic acids, however, how these pathways were regulated at the transcriptional level was unknown until very recently. Our research group identified MabR (Mycolic acids biosynthesis regulator), a new transcriptional regulator that controls the expression of fasII genes, by binding specifically to the fasII operon of the promoter region. Pfam analysis of MabR and its orthologues showed that the C-terminal regions of these ~440-amino-acid proteins are relatively conserved and contain a helix–turn–helix (HTH) DNA-binding motif typically found in prokaryotic transcription factors [2]. However, a detailed analysis of the primary amino acid sequence and of the domains present in MabR using several software indicated that MabR is a member of a new family of HTH-containing transcription factors only found in actinomycetes. To screen for soluble expression of MabR, transformed E. coli (BL21, Rossetta, C41 and Origami) was processed following a fractional factorial approach combining 4 culture media (LB, SB, TB, 2YT), and 3 temperatures (37, 20, 17°C). As a result MabR was purified to homogeneity through Ni2+-NTA affinity chromatography. UV spectroscopy and near-UV CD spectroscopy analyses exhibited a highly soluble, non-denatured protein under low concentration conditions tested. Size exclusion chromatography coupled to an LC detector revealed a 179.2 KDa protein complex, which fits well to a tetrameric conformation. However, MabR presented a strong tendency to aggregate. Thus, in an attempt to find more appropriate conditions to analyze MabR through crystallographic studies, we decided to perform a solubility screening test by microdialysis in buffers containing different additives including EDTA, DTT, glutamine, arginine and low concentration of detergents. The best conditions found in which the protein was soluble up to 4 mg/ml concentration were Hepes 10 mM pH 7,5, NaCl 1M, DTT 1mM and Glycerol 30% v/v. The protein was initially screened using crystallization kits (AmSO4 Suite, ClassicsL Suite, PEGs Suite y pHClear Suite -Qiagen) based on sitting drop vapor diffusion at the Pasteur Institute of Montevideo where laminar crystals were obtained. Currently, we are optimizing the crystallization conditions in order to obtain a suitable crystal for structural function studies. The determination of the 3D structure of MabR will per se constitute a relevant and original objective since this protein belongs to a new family of transcriptional regulators. In the medically relevant genera Mycobacterium, MabR might become an excellent new target for the development of conceptually new antimycobacterium compounds for the design of inhibitors that prevents the interaction of MabR with its target promoters or its effector molecule.