Structural basis for the specific inhibition of protein kinase G, a virulence factor of Mycobacterium tuberculosis

Abstract

The pathogenicity of mycobacteria such as Mycobacterium tuberculosis is closely associated with their capacity to survive within host macrophages. A crucial virulence factor for intracellular mycobacterial survival is protein kinase G (PknG), a eukaryotic-like serine/threonine protein kinase expressed by pathogenic mycobacteria that blocks the intracellular degradation of mycobacteria in lysosomes. Inhibition of PknG with the highly selective low-molecular-weight inhibitor AX20017 results in mycobacterial transfer to lysosomes and killing of the mycobacteria. Here, we report the 2.4 Å x-ray crystal structure of PknG in complex with AX20017. The unique multidomain topology of PknG reveals a central kinase domain that is flanked by N- and C-terminal rubredoxin and tetratrico-peptide repeat domains, respectively. Directed mutagenesis suggests that the rubredoxin domain functions as a regulator of PknG kinase activity. The structure of PknG-AX20017 further reveals that the inhibitor is buried deep within the adenosine-binding site, targeting an active conformation of the kinase domain. Remarkably, although the topology of the kinase domain is reminiscent of eukaryotic kinases, the AX20017-binding pocket is shaped by a unique set of amino acid side chains that are not found in any human kinase. Directed mutagenesis of the unique set of residues resulted in a drastic loss of the compound's inhibitory potency. Our results explain the specific mode of action of AX20017 and demonstrate that virulence factors highly homologous to host molecules can be successfully targeted to block the proliferation of M. tuberculosis .

Eukaryote-Like Serine/Threonine Kinases and Phosphatases in Bacteria

“…Second, the conserved Arg immediately preceding the invariant catalytic Asp residue is absent in the PknG catalytic loop (see above and Ser/Thr Protein Kinases). Activation of PknG is controlled by its rubredoxin domain, which is often found in electron transfer proteins, suggesting that the redox status of the environment may regulate PknG (153).…”

“…The M. tuberculosis soluble kinase PknG has a unique modular organization, with the kinase domain sandwiched between an N-terminal rubredoxin domain and a C-terminal tetratricopeptide repeat domain (TPRD) (153) (Fig. 2C).…”

Biochemical and Spatial Coincidence in the Provisional Ser/Thr Protein Kinase Interaction Network of Mycobacterium tuberculosis*

“…These two activation mechanisms result in an autophosphorylated active kinase. In contrast, PknG contains phosphorylation sites in the C-terminal domain that may be necessary for activity (30,31). These biochemical and structural insights suggest a general model for activation in which ligand binding to the extracellular domain promotes KD dimerization and intermolecular autophosphorylation (26,32).…”

Efforts Towards the Development of New Antitubercular Agents: Potential for Thiolactomycin Based Compounds

“…Recently some new targets such as signaling kinase inhibitors have been investigated. The survival of M. tuberculosis against the macrophage phagocytosis relies not only on a thick cell wall but also on many mycobacterial kinases and phosphatases which disrupt the host-cell defence mechanism against parasitism (92)(93)(94). Histidine kinase is the focus for the specific inhibition of two component signal transduction system in mycobacteria (95)(96)(97)(98).…”