Auto-Activation Mechanism of a Mycobacterial Ser/Thr Protein Kinase.

Despite the widespread presence of eukaryotic-like serine/threonine protein kinases (STPKs) throughout the bacterial kingdom, their predominant functions and mechanisms of regulation have not been elucidated to the degree of their eukaryotic counterparts. A chemical genetics approach previously used to study eukaryotic protein kinase function was applied to the Ser/Thr kinase domain (KD) of the PknB receptor kinase from M. tuberculosis (Mtb), an important regulator of growth and morphology. The PknB active site was engineered to confer high specificity towards a designed ATP competitive inhibitor. The mutant kinase-inhibitor crystal structure highlighted the high degree of structural and functional conservation between prokaryotic and eukaryotic protein kinases. This work establishes a general strategy for in vivo inhibition studies of PknB and other bacterial homologs. Many STPKs are activated by autophosphorylation, but the mechanism of this process has not been elucidated. The crystal structure of an active-site mutant of the PknB KD in complex with an ATP competitive inhibitor was determined, and features consistent with an activation complex were discovered. The complex formed an asymmetric dimer mediated by the conserved G helix. Analogous to the effects of mutations in the N-lobe dimerization interface of PknB, substitutions in the G-helix interface reduced activation-loop phosphorylation and KD activity. Multiple G-helix replacements abolished KD phosphorylation. As predicted by the idea that the asymmetric dimer mimics an autophosphorylation complex, PknB autophosphorylation was shown to occur in trans. Unexpectedly, intermolecular phosphorylation of the activation-loop occurred in a preferred order. These results support a model in which two separate interfaces in PknB are involved in activation: N-lobe dimerization activates the unphosphorylated KD and an asymmetric, "front-to-front" association through the C-lobe G-helix mediates sequential, intermolecular autophosphorylation of PknB and homologous prokaryotic and eukaryotic kinases. To explore the role of activation--loop phosphorylation, three different crystal structures of the PknB-KD were solved with unprecedented, ordered activation-loops. These structures, ranging from inactive to active-like states, revealed that large changes in activation-loop conformation occur in response to phosphorylation, support testable new ideas about protein substrate recognition, and imply that prokaryotic and eukaryotic STPKs are regulated by conserved allosteric mechanisms.