A Legionella effector kinase is activated by host inositol hexakisphosphate

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.

The Shigella type III effector protein OspB is a cysteine protease

“…Using a genetic approach, we exclude inositol pyrophosphate species as being necessary for OspB activity and define the inositol phosphate species requirement as IP 6 ; however, it is possible that an inositol pyrophosphate species may be sufficient for stimulating activity in vitro , as found for IP 7 and C. difficile TcdB (Savidge et al, 2011). The requirement for a host-specific cofactor, such as IP 6 , ubiquitin, calmodulin, or cyclophilins, for the activation bacterial effectors is increasingly appreciated (Anderson et al, 2011; Coaker et al, 2005; Drum et al, 2002; Mittal et al, 2010; Sreelatha et al, 2020; Tyson & Hauser, 2013) and necessarily restricts enzymatic activity to the context of host infection. Together, these findings provide strong evidence that OspB is a cysteine protease in the family of proteases represented by the cysteine protease domains of the MARTX toxins and large clostridial cytotoxins.…”

Structural biology of the invasion arsenal of Gram‐negative bacterial pathogens

“…S1B). IP6 binding in the allosteric site showed that binding of a highly negatively charged IP6 coordinates several lysine side chains (Lys 76, Lys107, and Lys111) from the previously disordered region of the N-lobe and restores the ATP binding site [52].…”

ALegionellaeffector ADP-ribosyltransferase inactivates glutamate dehydrogenase

“…Our lab has previously taken a bioinformatic approach to identify atypical and uncharacterized members of the protein kinase superfamily. Several of the outlying kinase families we have characterized are components of bacterial conflict systems, including the HopBF1 substrate of the Pseudomonas syringae type III secretion system (1) and the Lpg2603 and SidJ substrates of the Legionella pneumophila Type IV secretion system (T4SS) (2,3). Legionella is a gram-negative environmental pathogen and the causative agent of a potentially fatal pneumonia called Legionnaire's disease.…”