Abstract
The transfer of a phosphate from ATP to a protein substrate, a modification known as protein phosphorylation, is catalyzed by protein kinases. Protein kinases play a crucial role in virtually every cellular activity. Recent studies of atypical protein kinases have highlighted the structural similarity of the kinase superfamily despite notable differences in primary amino acid sequence. Here, using a bioinformatics screen, we searched for putative protein kinases in the intracellular bacterial pathogen Legionella pneumophila and identified the type 4 secretion system effector Lpg2603 as a remote member of the protein kinase superfamily. Employing an array of biochemical and structural biology approaches, including in vitro kinase assays and isothermal titration calorimetry, we show that Lpg2603 is an active protein kinase with several atypical structural features. Importantly, we found that the eukaryote-specific host signaling molecule inositol hexakisphosphate (IP6) is required for Lpg2603 kinase activity. Crystal structures of Lpg2603 in the apo-form and when bound to IP6 revealed an active-site rearrangement that allows for ATP binding and catalysis. Our results on the structure and activity of Lpg2603 reveal a unique mode of regulation of a protein kinase, provide the first example of a bacterial kinase that requires IP6 for its activation, and may aid future work on the function of this effector during Legionella pathogenesis.
Highlights
The transfer of a phosphate from ATP to a protein substrate, a modification known as protein phosphorylation, is catalyzed by protein kinases
Using a bioinformatics screen, we searched for putative protein kinases in the intracellular bacterial pathogen Legionella pneumophila and identified the type 4 secretion system effector Lpg2603 as a remote member of the protein kinase superfamily
Employing an array of biochemical and structural biology approaches, including in vitro kinase assays and isothermal titration calorimetry, we show that Lpg2603 is an active protein kinase with several atypical structural features
Summary
Using a bioinformatics screen, we searched for putative protein kinases in the intracellular bacterial pathogen Legionella pneumophila and identified the type 4 secretion system effector Lpg2603 as a remote member of the protein kinase superfamily. In search of atypical protein kinases in L. pneumophila, we identified a kinase domain in the type 4 secretion system effector protein, Lpg2603 (lem, sdmB), which has been predicted to have a kinase fold by Burstein et al [24]. A conserved phosphatidylinositol-4-phophate (PI4P)– binding domain at the C terminus of Lpg2603 was identified and shown to localize the protein to the Legionella-containing vacuole during infection [25] This localization is shared by L. pneumophila type 4 secretion system effectors Lpg1101/Lem and DrrA/sidM, which harbor the conserved PI4P-binding domain at their C termini but differ in their N-terminal domains [25]. Our results provide insights into the mechanism by which Lpg2603 catalyzes phosphorylation and highlight a unique mechanism of allosteric regulation of kinases by IP6
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