Structural and Functional Characterization of the Regulation of Pknb

Mycobacterium tuberculosis responds to diverse environmental changes for survival in the host macrophage. Receptor serine/threonine protein kinases mediate interactions between the cell and its changing environment. The serine/threonine protein kinases are therefore essential disseminators of cellular communication. The M. tuberculosis receptor serine/threonine protein kinase PknB is essential for mycobacterial growth and cell division. To address the role of dimerization in regulating PknB, I used a small molecule to control association of the intracellular PknB kinase and juxtamembrane domain fusion proteins. I found that pairing activates PknB and identified mutations in a known dimerization surface that suppressed autophosphorylation and substrate phosphorylation. We propose a step-wise mechanism for PknB activation in which the unphosphorylated monomer is the inactive species, while the unphosphorylated PknB homodimer is an active species, capable of trans-autophosphorylation of activation loop and juxtamembrane linker residues. We demonstrated that PknB dimerization regulates the kinase activity prior to autophosphorylation and that phosphorylated PknB dimer and monomer are both active species. Unique to Mtb serine/threonine protein kinases, the phosphorylated monomer is able to phosphorylate substrate until the inactive species is regenerated by dephosphorylation with the only Mtb serine/threonine phosphatase, PstP. To explore the mechanism of regulation, we determined the structure of two dimerization-interface mutants, one of which yielded three distinct conformations. These mutations abolished dimerization and distorted the N-lobe and active site organization. In the monomeric mutant kinase domains, the alphaC-helix unwound and moved toward the C-lobe and active site in a direction opposite to the established paradigm for inactive eukaryotic kinases. Additionally, the canonical Lys-Glu salt bridge which is important for Lys coordination of the alpha, beta-phosphates is not formed in any dimerization-interface mutant structure. Metal coordination by nucleotide analogs is also distinct from the wild-type PknB structure. Our structural and functional studies of PknB dimerization-interface mutants indicate that PknB homodimerization is an allosteric regulatory mechanism utilized for activation. Our results establish a general mechanism of prokaryotic kinase regulation and contribute to the understanding of regulation hierarchy achieved at sites distal to the active site.