High-throughput strategy for identification of Mycobacterium tuberculosis membrane protein expression conditions using folding reporter GFP.

Mycobacterium tuberculosis membrane protein biochemistry and structural biology studies are often hampered by challenges in protein expression and selection for well-expressing protein candidates, suitable for further investigation. Here we present a folding reporter GFP (frGFP) assay, adapted for M. tuberculosis membrane protein screening in Escherichia coli Rosetta 2 (DE3) and Mycobacterium smegmatis mc24517. This method allows protein expression condition screening for multiple protein targets simultaneously by monitoring frGFP fluorescence in growing cells. We discuss the impact of common protein expression conditions on 42 essential M. tuberculosis H37Rv helical transmembrane proteins and establish the grounds for their further analysis. We have found that the basal expression of the lac operon in the T7-promoter expression system generally leads to high recombinant protein yield in M. smegmatis, and we suggest that a screening condition without the inducer is included in routine protein expression tests. In addition to the general observations, we describe conditions allowing high-level expression of more than 25 essential M. tuberculosis membrane proteins, containing 2 to 13 transmembrane helices. We hope that these findings will stimulate M. tuberculosis membrane protein research and aid the efforts in drug development against tuberculosis.

The Bacillus anthracis class Ib ribonucleotide reductase subunit NrdF intrinsically selects manganese over iron.

Correct protein metallation in the complex mixture of the cell is a prerequisite for metalloprotein function. While some metals, such as Cu, are commonly chaperoned, specificity towards metals earlier in the Irving-Williams series is achieved through other means, the determinants of which are poorly understood. The dimetal carboxylate family of proteins provides an intriguing example, as different proteins, while sharing a common fold and the same 4-carboxylate 2-histidine coordination sphere, are known to require either a Fe/Fe, Mn/Fe or Mn/Mn cofactor for function. We previously showed that the R2lox proteins from this family spontaneously assemble the heterodinuclear Mn/Fe cofactor. Here we show that the class Ib ribonucleotide reductase R2 protein from Bacillus anthracis spontaneously assembles a Mn/Mn cofactor in vitro, under both aerobic and anoxic conditions, when the metal-free protein is subjected to incubation with MnII and FeII in equal concentrations. This observation provides an example of a protein scaffold intrinsically predisposed to defy the Irving-Williams series and supports the assumption that the Mn/Mn cofactor is the biologically relevant cofactor in vivo. Substitution of a second coordination sphere residue changes the spontaneous metallation of the protein to predominantly form a heterodinuclear Mn/Fe cofactor under aerobic conditions and a Mn/Mn metal center under anoxic conditions. Together, the results describe the intrinsic metal specificity of class Ib RNR and provide insight into control mechanisms for protein metallation.

Redox-induced structural changes in the di-iron and di-manganese forms of Bacillus anthracis ribonucleotide reductase subunit NrdF suggest a mechanism for gating of radical access.

Class Ib ribonucleotide reductases (RNR) utilize a di-nuclear manganese or iron cofactor for reduction of superoxide or molecular oxygen, respectively. This generates a stable tyrosyl radical (Y·) in the R2 subunit (NrdF), which is further used for ribonucleotide reduction in the R1 subunit of RNR. Here, we report high-resolution crystal structures of Bacillus anthracis NrdF in the metal-free form (1.51 Å) and in complex with manganese (MnII/MnII, 1.30 Å). We also report three structures of the protein in complex with iron, either prepared anaerobically (FeII/FeII form, 1.32 Å), or prepared aerobically in the photo-reduced FeII/FeII form (1.63 Å) and with the partially oxidized metallo-cofactor (1.46 Å). The structures reveal significant conformational dynamics, likely to be associated with the generation, stabilization, and transfer of the radical to the R1 subunit. Based on observed redox-dependent structural changes, we propose that the passage for the superoxide, linking the FMN cofactor of NrdI and the metal site in NrdF, is closed upon metal oxidation, blocking access to the metal and radical sites. In addition, we describe the structural mechanics likely to be involved in this process.

Structure of Mycobacterium tuberculosis phosphatidylinositol phosphate synthase reveals mechanism of substrate binding and metal catalysis.

Tuberculosis causes over one million yearly deaths, and drug resistance is rapidly developing. Mycobacterium tuberculosis phosphatidylinositol phosphate synthase (PgsA1) is an integral membrane enzyme involved in biosynthesis of inositol-derived phospholipids required for formation of the mycobacterial cell wall, and a potential drug target. Here we present three crystal structures of M. tuberculosis PgsA1: in absence of substrates (2.9 Å), in complex with Mn2+ and citrate (1.9 Å), and with the CDP-DAG substrate (1.8 Å). The structures reveal atomic details of substrate binding as well as coordination and dynamics of the catalytic metal site. In addition, molecular docking supported by mutagenesis indicate a binding mode for the second substrate, D-myo-inositol-3-phosphate. Together, the data describe the structural basis for M. tuberculosis phosphatidylinositol phosphate synthesis and suggest a refined general catalytic mechanism-including a substrate-induced carboxylate shift-for Class I CDP-alcohol phosphotransferases, enzymes essential for phospholipid biosynthesis in all domains of life.