Functional studies of serine/threonine protein kinase signalling in Mycobacterium tuberculosis
Fork-head associated (FHA) domains function in the assembly of signalling complexes through specific interactions with phospho-threonine motifs. FHA domains are found in eukaryotes, and intriguingly in a subset of bacterial species including Mycobacteria tuberculosis. In addition, the M. tuberculosis genome encodes 11 eukaryotic-like serine/threonine protein kinases (STPKs). In M. tuberculosis, the co-expression of FHA domain-containing proteins and STPKs strongly suggest that these bacterial FHA domain-containing proteins engage in phospho-dependent protein-protein interaction, and FHA dependent processes in bacteria are controlled by STPK-dependent phosphorylation. This study describes a body of biophysical and biochemical experiments which characterises the interactions between two STPKs, PknA and PknB, and three FHA domain-containing proteins, Rv0019, Rv0020 and Rv1827. Isothermal titration calorimetry and surface plasmon resonance combined with site-directed mutagenesis are used to investigate interactions of kinase domains with FHA domains. These experiments reveal that the FHA domains interact with specific phospho-threonine residues located within the kinase domain activation loop. Additionally, in vitro kinase assays demonstrated that the interactions also involve phosphorylation of the FHA domains and/or adjacent segments of the protein. These data suggest that STPK-mediated signalling in M. tuberculosis involves a complex series of interactions and suggests a 'molecular docking' model for the phosphorylation of FHA domain-containing proteins. Finally, high-throughput assays have been used to identify several inhibitors of PknB which will serve as the basis for development of novel antimicrobial therapies.