Investigating the mechanism through which amino acids activate a conserved serine/threonine protein kinase in Actinobacteria

Bacteria have evolved sensory pathways which allow the detection and response to changes in the environment which is essential for survival. Some of these pathways rely on eukaryotic-like serine/threonine protein kinases (STPKs).

One STPK conserved within Actinobacteria is PknG which is found within ecologically, industrially and medically important bacteria. One such bacteria is Mycobacterium tuberculosis, which causes Tuberculosis in humans. Research has highlighted that PknG, and its phosphorylation target GarA, respond to amino acid availability and are essential for the regulation of glutamate metabolism. Currently, there is no proven mechanism which could explain how cytoplasmic PknG could be activated by external amino acids. A model has been proposed, involving two proteins conserved in the pknG operon: GlnH, a predicted glutamine-binding protein, and GlnX, a membrane protein with unknown function.

The main aim of this project was to provide evidence supporting the hypothesis that both GlnH and GlnX are involved in the activation of PknG in response to the presence of amino acids. Recombinant GlnH from M. tuberculosis and Corynebacterium glutamicum was characterised using ligand screens and Kd measurements. This highlighted that GlnH binds, with a high affinity, the amino acids known to trigger GarA phosphorylation in bacteria: aspartate and glutamate. Furthermore, weak ligands were identified for each protein: asparagine, histidine, cysteine and isocitrate. Structure determination of histidine-bound and aspartate-bound M. tuberculosis GlnH allows further understanding of the binding specificity.

To test for a putative protein-protein interaction between GlnH and GlnX, attempts were made to produce a soluble form of GlnX for interaction assays. The understanding of the activation mechanism of the PknG/GarA pathway could allow therapeutics to be developed which could successfully target this essential pathway.