Auto-activation mechanism of the Mycobacterium tuberculosis PknB receptor Ser/Thr kinase

Many Ser/Thr protein kinases are activated by autopho-sphorylation, but the mechanism of this process has not been defined. We determined the crystal structure of a mutant of the Ser/Thr kinase domain (KD) of the myco-bacterial sensor kinase PknB in complex with an ATP competitive inhibitor and discovered features consistent with an activation complex. The complex formed an asymmetric dimer, with the G helix and the ordered activation loop of one KD in contact with the G helix of the other. The activation loop of this putative ‘substrate’ KD was disordered, with the ends positioned at the en-trance to the partner KD active site. Single amino-acid substitutions in the G-helix interface reduced activation-loop phosphorylation, and multiple replacements abol-ished KD phosphorylation and kinase activation. Phos-phorylation of an inactive mutant KD was reduced by G-helix substitutions in both active and inactive KDs, as predicted by the idea that the asymmetric dimer mimics a trans-autophosphorylation complex. These results support a model in which a structurally and functionally asym-metric, ‘front-to-front ’ association mediates autophosphor-ylation of PknB and homologous kinases