Structure, function, and regulation of eukaryotic-like Serine/Threonine protein kinases in Mycobacterium tuberculosis

The M. tuberculosis genome contains 11 genes with significant homology to eukaryotic Ser/Thr protein kinases (STPKs). Crystallographic studies showed conservation of the eukaryotic kinase fold and elucidated a novel dimer interface. Sequence comparisons revealed that the analogous interface is conserved in orthologs from diverse bacterial species. To analyze the roles of dimerization, we constructed Mtb Protein Kinase D (PknD) kinase domain (KD) fusion proteins that formed dimers upon addition of rapamycin. Dimerization of unphosphorylated Mtb PknD KD fusions stimulated phosphorylation activity. Mutations in the dimer interface reduced this activation, limited autophosphorylation and altered substrate specificity. In contrast, an inactive, catalytic-site mutant retained the ability to stimulate the wild-type KD by dimerization. These results support the idea that dimer formation allosterically activates unphosphorylated PknD. The conservation of analogous dimers in diverse prokaryotic and eukaryotic STPKs implies that this mechanism of protein kinase regulation is ancient and broadly distributed.;The M. tuberculosis STPKs are candidates for sensors that govern developmental changes and disease progression in tuberculosis (TB), but the functions of these kinases are not established. We showed that Mtb Protein Kinase D (PknD) overexpression alters transcription of numerous bacterial genes, including Rv0516c, a putative anti-anti-sigma-factor, and genes regulated by sigma factor F. The PknD kinase domain directly phosphorylated Rv0516c on Thr2, but no other sigma-factor regulator, in vitro. This phosphorylation inhibited Rv0516c binding in vitro to a homologous anti-anti-sigma-factor, Rv2638. These results support a model in which signals transmitted through PknD alter the transcriptional program of Mtb by stimulating phosphorylation of a sigma-factor regulator at an unprecedented regulatory site.