Interdomain interaction reconstitutes the functionality of PknA, a eukaryotic type Ser/Thr kinase from Mycobacterium tuberculosis.

Eukaryotic type Ser/Thr protein kinases have recently been shown to regulate a variety of cellular functions in bacteria. PknA, a transmembrane Ser/Thr protein kinase from Mycobacterium tuberculosis, when constitutively expressed in Escherichia coli resulted in cell elongation and therefore has been thought to be regulating morphological changes associated with cell division. Bioinformatic analysis revealed that PknA has N-terminal catalytic, juxtamembrane, transmembrane, and C-terminal extracellular domains, like known eukaryotic type Ser/Thr protein kinases from other bacteria. To identify the minimum region capable of exhibiting phosphorylation activity of PknA, we created several deletion mutants. Surprisingly, we found that the catalytic domain itself was not sufficient for exhibiting phosphorylation ability of PknA. However, the juxtamembrane region together with the kinase domain was necessary for the enzymatic activity and thus constitutes the catalytic core of PknA. Utilizing this core, we deduce that the autophosphorylation of PknA is an intermolecular event. Interestingly, the core itself was unable to restore the cell elongation phenotype as manifested by the full-length protein in E. coli; however, its co-expression along with the C-terminal region of PknA can associate them in trans to reconstitute a functional protein in vivo. Therefore, these findings argue that the transmembrane and extracellular domains of PknA, although dispensable for phosphorylation activities, are crucial in responding to signals. Thus, our results for the first time establish the significance of different domains in a bacterial eukaryotic type Ser/Thr kinase for reconstitution of its functionality.