Abstract
The Mycobacterium tuberculosis kinase PknB is essential for growth and survival of the pathogen in vitro and in vivo. Here we report the results of our efforts to elucidate the mechanism of regulation of PknB activity. The specific residues in the PknB extracytoplasmic domain that are essential for ligand interaction and survival of the bacterium are identified. The extracytoplasmic domain interacts with mDAP-containing LipidII, and this is abolished upon mutation of the ligand-interacting residues. Abrogation of ligand-binding or sequestration of the ligand leads to aberrant localization of PknB. Contrary to the prevailing hypothesis, abrogation of ligand-binding is linked to activation loop hyperphosphorylation, and indiscriminate hyperphosphorylation of PknB substrates as well as other proteins, ultimately causing loss of homeostasis and cell death. We propose that the ligand-kinase interaction directs the appropriate localization of the kinase, coupled to stringently controlled activation of PknB, and consequently the downstream processes thereof.
Highlights
The Mycobacterium tuberculosis kinase PknB is essential for growth and survival of the pathogen in vitro and in vivo
The prevailing hypothesis suggests that the interaction of the extracytoplasmic domain with the ligand results in the dimerization of intracellular kinase domain, which is required for the activation of the kinase through activation loop phosphorylations (Fig. 1b)[13]
We employed previously described Mycobacterium tuberculosis (Mtb) conditional mutant of pknB (RvΔB), wherein the native locus has been modified to bring its expression under pristinamycin inducible promoter, which allows the bacterium to grow efficiently in the presence of pristinamycin but not in its absence[5,17] (Supplementary Fig. 1a)
Summary
The Mycobacterium tuberculosis kinase PknB is essential for growth and survival of the pathogen in vitro and in vivo. A particular class of receptor-type serine-threonine kinase called PASTA (Penicillin binding proteins And Serine Threonine Associated) kinase is widespread across gram-positive firmicutes and actinomycetes and is known for its functions associated with bacterial cell growth[1] These protein kinases have an intracellular kinase domain, which shows sequence and structural homology to the eukaryotic serine/threonine kinases, and an extracytoplasmic (Ec) domain made up of varying number of PASTA domains. The hypothesis is based on the front-to-front and back-to-back dimeric crystal structures of cytosolic kinase domain[14,15] and surface plasmon resonance-based in vitro binding experiments of PASTA domain with the muropeptides[11] In consonance with this we have previously reported that the extracytoplasmic PASTA domains are indispensable for the function of PknB and survival of Mtb[5].
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