Biology subjects

Pawlowski, K.

Publications and source records attributed to Pawlowski, K..

At least 19 recordsLinked to original sources

bioRxiv2026

MoALE1, an ART-like effector, contributes to rice blast virulence through host immune suppression

Rice blast, caused by the hemibiotrophic fungus Magnaporthe oryzae, is one of the most destructive diseases affecting rice production worldwide. During infection, M. oryzae secretes effector proteins that suppress host immunity and facilitate colonization. Here, we characterized MoALE1, a predicted effector that is structurally similar to members of the heat-labile enterotoxin/cholera toxin family. Using a yeast secretion trap assay, we demonstrated that MoALE1 contains a functional signal peptide. A MoALE1-GFP fusion localized to the biotrophic interfacial complex, indicating that MoALE1 is a cytoplasmic effector. Expression analyses revealed that MoALE1 is expressed during biotrophy, from 16 to 48 h post-inoculation. Transient expression in Nicotiana benthamiana showed that MoALE1 suppresses BAX-mediated cell death, suggesting a role in host immune suppression. To investigate its contribution to pathogenicity, a{Delta} moale1 mutant was generated and evaluated in rice infection assays. Deletion of MoALE1 significantly reduced disease severity but did not affect fungal growth, stress tolerance, sporulation, appressorium development, host penetration, or early invasive growth. In contrast, infection by the {Delta}moale1 mutant resulted in enhanced host H2O2 accumulation and increased expression of ROS-associated defense genes. Together, these findings identify MoALE1 as a biotrophy-associated cytoplasmic effector that promotes rice blast pathogenicity by suppressing host immune responses and provide new insight into the function of ADP-ribosyltransferase (ART)-like effector candidates in M. oryzae .

microbiology↗

bioRxiv2026

YfiM is a lipid-linked oligosaccharide pyrophosphatase

Lipid-linked oligosaccharides (LLO), such as Lipid II and O-antigen precursors in bacteria, and dolichol-linked oligosaccharides (DLO) in eukaryotes, are universal metabolic intermediates required for cell wall assembly and protein glycosylation across all domains of life. The oligosaccharide moiety of LLO is synthesized through the sequential addition of various monosaccharides, a process catalyzed by a series of glycosyltransferases, to form the final mature structure. However, the existence of a homeostasis and quality control mechanism to degrade surplus and aberrant LLO has long remained a mystery. Although we recently identified the yeast LLP1 as an LLO pyrophosphatase involved in the homeostasis and quality control of DLO in fungi, the corresponding mechanism in bacteria and plants has remained elusive. Here, we identify YfiM and AT1G15900 as LLO pyrophosphatases in Escherichia coli and Arabidopsis thaliana, respectively. Phylogenetic profiling revealed that while YfiM is broadly conserved among Gram-negative bacilli, its plant ortholog displays a unique lineage-specific distribution shaped by selective gene loss. In vitro, purified YfiM and AT1G15900 exhibit LLO pyrophosphatase activity. In vivo, deletion mutant of yfiM caused vancomycin sensitivity in E. coli. Conversely, overexpression of YfiM triggered osmotic lysis under hypotonic conditions. Moreover, overexpression of YfiM in an outer membrane-compromised strain partially rescued detergent-chelator sensitivity, revealing a compensatory envelope stress response. Together, our findings uncover an ancient, conserved cross-kingdom system for LLO quality control bridging prokaryotes and plants.

microbiology↗

bioRxiv2026

TIR-like NADases act in bacterial immunity and the RNA vault

Across all domains of life, organisms exploit NAD+ metabolism as a central line of defense against invading pathogens. Here, we show that domain of unknown function 4062 (DUF4062) is a widespread family of TIR-like NADases that hydrolyze NAD+ to ADP-ribose and nicotinamide. In bacteria, DUF4062 homologs form a previously unrecognized antiphage defense system, which we name Swaro[z]yc, that assembles with the phage portal into a supramolecular NADase complex to induce abortive infection. In eukaryotes, DUF4062 is found in TEP1, which we demonstrate functions as an active NADase within the RNA vault, an enigmatic organelle-like structure. Single-particle cryo-electron microscopy reveals ADP-ribose bound within the shoulder of both reconstituted and human brain vaults, while cryo-electron tomography positions TEP1 along the central axis at the shoulder. Thus, TEP1, like bacterial Swaro[z]yc, functions by depleting NAD+, providing new insight into the long-standing mystery of vault function.

biochemistry↗

bioRxiv2026

Proteomics-Enhanced AI-Digital Pathology in Metastatic Mucinous Colorectal Carcinoma: A Case Report

Mucinous colorectal carcinoma (CRC) is a distinct histomorphological subtype characterized by abundant extracellular mucin that may promote immune evasion and chemoresistance. We describe a metastatic mucinous CRC case integrating digital pathology and proteomics to investigate disease progression and therapy resistance. Formalin-fixed paraffin-embedded samples from the primary tumor, peritoneal metastasis, and hepatoduodenal ligament metastasis of a 56-year-old patient were analyzed. Whole-slide imaging with QuPath-based AI enabled detailed histological annotation, while data-independent acquisition mass spectrometry identified over 6,000 proteins. Digital pathology revealed extensive mucin pools, architectural evolution from heterogeneous glandular patterns in the primary tumor to cribriform morphology in advanced metastases, and immune cell exclusion from mucin-rich regions. Proteomics revealed metabolic reprogramming, suppressed antigen presentation, and stage-specific activation of inflammatory, angiogenic, EMT, and PI3K/AKT/mTOR-MYC signaling pathways, consistent with proliferative and therapy-resistant phenotypes.Integration of AI-assisted histopathology with spatial proteomics highlighted the mucin barrier as a key mediator of immune evasion and chemoresistance. These findings support a personalized therapeutic framework targeting mucin-associated mechanisms alongside pathway-directed inhibitors, suggesting that spatial multi-omics may guide precision management strategies for aggressive mucinous colorectal cancer.

pathology↗

bioRxiv2025

ACAD10 encodes two orphan enzymes in the ether lipid biosynthetic and salvage pathways

Ether lipids play critical roles in membrane dynamics, antioxidant defense, and signaling. They comprise ~20% of mammalian phospholipids, and disruptions in their metabolism cause severe genetic disorders and are associated with neurodegenerative and metabolic diseases. Ether lipids are synthesized de novo from glycolytic intermediates or salvaged from the diet. While the products of these pathways are known, several key enzymes remain unidentified, including the 1-O-alkylglycerol kinase and the 1-O-alkyl-2-acetyl-sn-glycero-3-phosphate phosphatase. Here, we show that acyl-CoA dehydrogenase member 10 (ACAD10) catalyzes the phosphorylation of 1-O-alkylglycerols and the dephosphorylation of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphate. Worms and mice lacking ACAD10 have reduced ether lipid levels and cannot salvage dietary alkylglycerols. Furthermore, individuals from the Akimel O'odham (Pima) tribe carrying ACAD10 polymorphisms also show decreased plasma ether lipid levels. Collectively, our findings resolve two long-standing gaps in ether lipid biochemistry and reveal a mechanistic link between ether lipid metabolism and a population-associated risk factor for type 2 diabetes.

biochemistry↗

bioRxiv2024

Covalent inhibition of the SARS-CoV-2 NiRAN domain via an active-site cysteine

The kinase-like NiRAN domain of nsp12 in SARS-CoV-2 catalyzes the formation of the 5 RNA cap structure. This activity is required for viral replication, offering a new target for the development of antivirals. Here, we develop a high-throughput assay to screen for small molecule inhibitors targeting the SARS-CoV-2 NiRAN domain. We identified NCI-2, a compound with a reactive chloromethyl group that covalently binds to an active site cysteine (Cys53) in the NiRAN domain, inhibiting its activity. NCI-2 can enter cells, bind to, and inactivate ectopically expressed nsp12. A cryo-EM reconstruction of the SARS-CoV-2 replication-transcription complex (RTC) bound to NCI-2 offers a detailed structural blueprint for rational drug design. Although NCI-2 showed limited potency against SARS-CoV-2 replication in cells, our work lays the groundwork for developing more potent and selective inhibitors targeting the NiRAN domain. This approach presents a promising therapeutic strategy for effectively combating COVID-19 and potentially mitigating future coronavirus outbreaks.

biochemistry↗

bioRxiv2024

A survey of ADP-ribosyltransferase families in the pathogenic Legionella

BackgroundADP-ribosyltransferases (ARTs) are a superfamily of enzymes implicated in various cellular processes, including pathogenic mechanisms. The Legionella genus, known for causing Legionnaires disease, possesses diverse ART-like effectors. This study explores the proteomes of 41 Legionella species to bioinformatically identify and characterise novel ART-like families, providing insights into their potential roles in pathogenesis and host interactions. MethodsWe conducted a comprehensive bioinformatic survey of 41 Legionella species to identify proteins with significant sequence or structural similarity to known ARTs. Sensitive sequence searches were performed to detect candidate ART-like families. Subsequent validation, including structure prediction of such families, was achieved using artificial intelligence-driven tools, such as AlphaFold. Comparative analyses were performed to assess sequence and structural similarities between the novel ART-like families and known ARTs. ResultsOur analysis identified 63 proteins with convincing similarity to ARTs, organised into 39 ART-like families, including 26 novel families. Key findings include: O_LIDUF2971 family: exhibits sequence similarity to DarT toxins and other DNA-acting ARTs. C_LIO_LIDUF4291 family: the largest newly identified family shows structural and sequence similarity to the diphtheria toxin, suggesting the ability to modify proteins. C_LI Most members of the novel ART families are predicted effectors. Although experimental validation of the predicted ART effector functions is necessary, the novel ART-like families identified present promising targets for understanding Legionella pathogenicity and developing therapeutic strategies. We publish a complete catalogue of our results in the astARTe database: http://bioinfo.sggw.edu.pl/astarte/.

bioinformatics↗

bioRxiv2024

Predicting immune checkpoint therapy response in three independent metastatic melanoma cohorts

While Immune checkpoint inhibition (ICI) therapy shows significant efficacy in metastatic melanoma, only about 50% respond, lacking reliable predictive methods. We introduce a panel of six proteins aimed at predicting response to ICI therapy. Evaluating previously reported proteins in two untreated melanoma cohorts, we used a published predictive model (EaSIeR score) to identify potential proteins distinguishing responders and non-responders. Six proteins initially identified in the ICI cohort correlated with predicted response in the untreated cohort. Additionally, three proteins correlated with patient survival, both at the protein, and at the transcript levels, in an independent immunotherapy treated cohort. Our study identifies predictive biomarkers across three melanoma cohorts, suggesting their use in therapeutic decision-making. O_FIG O_LINKSMALLFIG WIDTH=164 HEIGHT=200 SRC="FIGDIR/small/592032v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@754668org.highwire.dtl.DTLVardef@1616263org.highwire.dtl.DTLVardef@827009org.highwire.dtl.DTLVardef@1899ba7_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Biochemical and structural insights into a 5' to 3' RNA ligase reveal a potential role in tRNA ligation

ATP-grasp superfamily enzymes contain a hand-like ATP-binding fold and catalyze a variety of reactions using a similar catalytic mechanism. More than 30 protein families are categorized in this superfamily, and they are involved in a plethora of cellular processes and human diseases. Here we identify C12orf29 as an atypical ATP-grasp enzyme that ligates RNA. Human C12orf29 and its homologs auto-adenylate on an active site Lys residue as part of a reaction intermediate that specifically ligates RNA halves containing a 5-phosphate and a 3-hydroxyl. C12orf29 binds tRNA in cells and can ligate tRNA within the anticodon loop in vitro. Genetic depletion of c12orf29 in female mice alters global tRNA levels in brain. Furthermore, crystal structures of a C12orf29 homolog from Yasminevirus bound to nucleotides reveal a minimal and atypical RNA ligase fold with a unique active site architecture that participates in catalysis. Collectively, our results identify C12orf29 as an RNA ligase and suggest its involvement in tRNA biology. Significance StatementATP-grasp enzymes share an atypical ATP-binding fold and catalyze a diverse set of reactions involved in many essential cellular processes. We identified C12orf29 as an atypical ATP-grasp enzyme. Our biochemical and structural characterizations reveal this enzyme to be a 5 to 3 RNA ligase, structurally and functionally similar to the phage T4 RNA ligase. C12orf29 can ligate tRNAs in vitro and C12orf29 knockout female mice have altered tRNA levels in brain. We also report structures of C12orf29, which have revealed critical insights into the mode of ATP binding and catalysis. Our work suggests that C12orf29 may be a new player in the regulation of tRNAs.

biochemistry↗

bioRxiv2024

Identifying Ferroptosis Inducers, HDAC, and RTK Inhibitor Sensitivity in Melanoma Subtypes through Unbiased Drug Target Prediction

The utilization of PD1 and CTLA4 inhibitors has revolutionized the treatment of malignant melanoma (MM). However, resistance to targeted and immune-checkpoint-based therapies still poses a significant problem. Here we mine large scale MM proteogenomic data integrating it with MM cell line dependency screen, and drug sensitivity data to identify druggable targets and forecast treatment efficacy and resistance. Leveraging protein profiles from established MM subtypes and molecular structures of 82 cancer treatment drugs, we identified nine candidate hub proteins, mTOR, FYN, PIK3CB, EGFR, MAPK3, MAP4K1, MAP2K1, SRC and AKT1, across five distinct MM subtypes. These proteins serve as potential drug targets applicable to one or multiple MM subtypes. By analyzing transcriptomic data from 48 publicly accessible melanoma cell lines sourced from Achilles and CRISPR dependency screens, we forecasted 162 potentially targetable genes. We also identified genetic resistance in 260 genes across at least one melanoma subtype. In addition, we employed publicly available compound sensitivity data (Cancer Therapeutics Response Portal, CTRPv2) on the cell lines to assess the correlation of compound effectiveness within each subtype. We have identified 20 compounds exhibiting potential drug impact in at least one melanoma subtype. Remarkably, employing this unbiased approach, we have uncovered compounds targeting ferroptosis, that demonstrate a striking 30x fold difference in sensitivity among different subtypes. This implies that the proteogenomic classification of melanoma has the potential to predict sensitivity to ferroptosis compounds. Our results suggest innovative and novel therapeutic strategies by stratifying melanoma samples through proteomic profiling, offering a spectrum of novel therapeutic interventions and prospects for combination therapy. Highlights(1) Proteogenomic subtype classification can define the landscape of genetic dependencies in melanoma (2) Nine proteins from molecular subtypes were identified as potential drug targets for specified MM patients (3) 20 compounds identified that show potential effectiveness in at least one melanoma subtype (4) Proteogenomics can predict specific ferroptosis inducers, HDAC, and RTK Inhibitor sensitivity in melanoma subtypes O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/579424v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@73dd77org.highwire.dtl.DTLVardef@d0cb8eorg.highwire.dtl.DTLVardef@1e4117forg.highwire.dtl.DTLVardef@38b727_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

cancer biology↗

bioRxiv2023

Histopathology-assisted proteogenomics provides foundations for stratification of melanoma metastases

Here we describe the histopathology-driven proteogenomic landscape of 142 treatment-naive metastatic melanoma samples. We identified five proteomic subtypes that integrate the immune and stroma microenvironment components, and associate with clinical and histopathological parameters, providing foundations for an in-depth molecular classification of melanoma. Our study shows that BRAF V600 mutated melanomas display heterogeneous biology, where the presence of an oncogene-induced senescence-like phenotype improves patient survival. Therefore, we propose a mortality-risk-based stratification, which may contribute to a more personalized approach to patient treatment. We also found a strong association between tumor microenvironment composition, disease progression, and patient outcome supported by single-cell omic signatures that point to straightforward histopathological connective tissue-to-tumor ratio assessment for better informed medical decisions. A melanoma-associated signature of single amino acid variants (SAAV) responsible for remodeling the extracellular matrix was uncovered together with SAAV-derived neoantigen candidates as targets of anti-tumor immune responses. Overall, this study offers comprehensive stratifications of melanoma metastases that may help develop tailored strategies for diagnosing and treating the disease.

cancer biology↗

bioRxiv2023

Comparative Phylogenomics and Phylotranscriptomics Provide Insights into the Genetic Complexity of Nitrogen Fixing Root Nodule Symbiosis

Plant root nodule symbiosis (RNS) with mutualistic nitrogen-fixing bacteria is restricted to a single clade of angiosperms, the Nitrogen-Fixing Nodulation Clade (NFNC), and is best understood in the legume family. It is widely accepted that nodulation originated through the assembly of modules recruited from existing functions, such as mycorrhizal symbiosis, polar growth, and lateral root development. Because nodulating species are scattered within the NFNC, the number of times nodulation has evolved or has been lost has been a matter of considerable speculation. This interesting evolutionary question has practical implications concerning the ease with which nodulation might be engineered in non-nodulating crop plants. Nodulating species share many commonalities, due either to divergence from a common ancestor over 100 million years ago or to convergence or deep homology following independent origins over that same time period. In either case, comparative analyses of diverse nodulation syndromes can provide insights into constraints on nodulation--what must be acquired or cannot be lost for a functional symbiosis--and what the latitude is for variation in the symbiosis. However, much remains to be learned about nodulation, especially outside of legumes. Here we present new information across the spectrum of nodulating groups. We find no evidence for convergence at the level of amino acid residues or gene family expansion across the NFNC. Our phylogenomic analyses further emphasize the uniqueness of the transcription factor, NIN, as a master regulator of nodulation, and identify key mutations affecting its function across the NFNC. We find that nodulation genes are over-represented among orthologous gene groups (OGs) present in the NFNC common ancestor, but that lineage-specific OGs play major roles in nodulation. We identified over 900,000 conserved noncoding elements (CNEs), of which over 300,000 were unique to NFNC species. A significant proportion of these are associated with nodulation-related genes and thus are candidates for transcriptional regulators.

evolutionary biology↗

bioRxiv2022

Structural insights into regulation of the PEAK3 scaffold by 14-3-3

The three members of the PEAK family of pseudokinases (PEAK1, PEAK2, and PEAK3) are molecular scaffolds that have recently emerged as important regulatory nodes in signaling pathways that control cell migration, morphology, and proliferation, and they are increasingly found to be mis-regulated in human cancers. While no structures of PEAK3 have been solved to date, crystal structures of the PEAK1 and PEAK2 pseudokinase domains revealed their dimeric organization. It remains unclear how dimerization plays a role in PEAK scaffolding functions, as no structures of PEAK family members in complex with their binding partners have been solved. Here, we report the cryo-EM structure of the PEAK3 pseudokinase, also adopting a dimeric state, and in complex with an endogenous 14-3-3 heterodimer purified from mammalian cells. Our structure reveals an asymmetric binding mode between PEAK3 and 14-3-3 stabilized by one pseudokinase domain and the Split HElical Dimerization (SHED) domain of the PEAK3 dimer. The binding interface is comprised of a canonical primary interaction involving two phosphorylated 14-3-3 consensus binding sites located in the N-terminal domains of the PEAK3 monomers docked in the conserved amphipathic grooves of the 14-3-3 dimer, and a unique secondary interaction between 14-3-3 and PEAK3 that has not been observed in any previous structures of 14-3-3/client complexes. Disruption of these interactions results in the relocation of PEAK3 to the nucleus and changes its cellular interactome. Lastly, we identify Protein Kinase D as the regulator of the PEAK3/14-3-3 interaction, providing a mechanism by which the diverse functions of the PEAK3 scaffold might be fine-tuned in cells.

biophysics↗

bioRxiv2022

Gene losses resulting in specialized metabolism in the earliest divergent symbiotic Frankia clade can be linked to its low saprotrophic capabilities

Frankia cluster-2 are diazotrophs that engage in root nodule symbiosis with host plants of the Cucurbitales and the Rosales. They are rarely found in the soil in the absence of their hosts. Previous studies have shown that an assimilated nitrogen source, presumable arginine, is exported to the host in nodules of Datisca glomerata (Cucurbitales), but not in the nodules of Ceanothus thyrsiflorus (Rosales). To investigate if an assimilated nitrogen form is commonly exported by cluster-2 strains, and which nitrogen source would then be exported to C. thyrsiflorus, gene expression levels, metabolite profiles and enzyme activities were analysed. We found that the export of assimilated nitrogen in symbiosis is a common feature for Frankia cluster-2 strains, but which source is host-plant dependent. We also identified several gene losses. The ammonium assimilation via the GS/GOGAT cycle for export to the host, entails a high demand of 2-oxoglutarate from the TCA cycle. This specialised metabolism seems to have led to genome reduction: we show that Frankia cluster-2 strains have lost the glyoxylate shunt and succinate semialdehyde dehydrogenase, leading to a linearization of the TCA cycle. This could explain the low saprotrophic potential of Frankia cluster-2.

plant biology↗

bioRxiv2022

The mechanism of RNA capping by SARS-CoV-2

The SARS-CoV-2 RNA genome contains a 5-cap that facilitates translation of viral proteins, protection from exonucleases and evasion of the host immune response1-4. How this cap is made is not completely understood. Here, we reconstitute the SARS-CoV-2 7MeGpppA2-O-Me-RNA cap using virally encoded non-structural proteins (nsps). We show that the kinase-like NiRAN domain5 of nsp12 transfers RNA to the amino terminus of nsp9, forming a covalent RNA-protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers RNA to GDP, forming the cap core structure GpppA-RNA. The nsp146 and nsp167 methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replication-transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system8 that the N-terminus of nsp9 and the kinase-like active site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19.

biochemistry↗

bioRxiv2021

Structural and mechanistic basis for protein glutamylation by the kinase fold

The kinase domain transfers phosphate from ATP to substrates. However, the Legionella effector SidJ adopts a kinase fold yet catalyzes calmodulin (CaM)-dependent glutamylation to inactivate the SidE ubiquitin ligases. The structural and mechanistic basis in which the kinase domain catalyzes protein glutamylation is unknown. Here we present cryo-EM reconstructions of SidJ:CaM:SidE reaction intermediate complexes. We show that the kinase-like active site of SidJ adenylates an active site Glu in SidE resulting in the formation of a stable reaction intermediate complex. An insertion in the catalytic loop of the kinase domain positions the donor Glu near the acyl-adenylate for peptide bond formation. Our structural analysis led us to discover that the SidJ paralog SdjA is a glutamylase that differentially regulates the SidE-ligases during Legionella infection. Our results uncover the structural and mechanistic basis in which the kinase fold catalyzes non-ribosomal amino acid ligations and reveal an unappreciated level of SidE-family regulation.

biochemistry↗

bioRxiv2020

A novel predicted ADP-ribosyltransferase family conserved in eukaryotic evolution

The presence of many completely uncharacterized proteins, even in well-studied organisms such as humans, seriously hampers full understanding of the functioning of the living cells. ADP-ribosylation is a common post-translational modification of proteins; also nucleic acids and small molecules can be modified by the covalent attachment of ADP-ribose. This modification, important in cellular signalling and infection processes, is usually executed by enzymes from the large superfamily of ADP-ribosyltransferases (ARTs) Here, using bioinformatics approaches, we identify a novel putative ADP-ribosyltransferase family, conserved in eukaryotic evolution, with a divergent active site. The hallmark of these proteins is the ART domain nestled between flanking leucine-rich repeat (LRR) domains. LRRs are involved in innate immune surveillance. The novel family appears as likely novel ADP-ribosylation "writers", previously unnoticed new players in cell signaling by this emerging post-translational modification. We propose that this family, including its human member LRRC9, may be involved in an ancient defense mechanism, with analogies to the innate immune system, and coupling pathogen detection to ADP-ribosyltransfer signalling.

bioinformatics↗

bioRxiv2020

A Legionella effector kinase is activated by host inositol hexakisphosphate

The transfer of a phosphate from ATP to a protein substrate, a modification known as 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. We searched for putative protein kinases in the intracellular bacterial pathogen, Legionella pneumophila and identified the Type-4 secretion system (T4SS) effector, Lpg2603 as a remote member of the protein kinase superfamily. We show that Lpg2603 is an active protein kinase with several atypical structural features. Importantly, we find that the eukaryotic-specific host signaling molecule, inositol hexakisphosphate (IP6) is required for Lpg2603 kinase activity. Crystal structures of Lpg2603 in the apo-form and bound to IP6 reveal 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 protein kinases and will aid future work into the function of this effector during Legionella pathogenesis.

biochemistry↗