Legionella pneumophila effector Lem4 is a membrane-associated protein tyrosine phosphatase

Beyrakhova, Ksenia; Li, Lei; Xu, Caishuang; Gagarinova, Alla; Cygler, Mirosław

doi:10.1074/jbc.ra118.003845

Cited by 15 publications

(14 citation statements)

References 64 publications

(68 reference statements)

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“…The 3xFLAG‐RavA was overexpressed in HEK293T cells as described above. Protein complexes were cross‐linked with dithiobis(succinimidylpropionate) (DSP) and affinity‐purified (AP) for analysis by mass spectrometry (MS) essentially as described previously 42 . Control (3xFLAG) and experimental (3xFLAG‐RavA) AP‐MS experiments were independently performed three times.…”

Section: Methodsmentioning

confidence: 99%

“…Protein complexes were cross-linked with dithiobis(succinimidylpropionate) (DSP) and affinity-purified (AP) for analysis by mass spectrometry (MS) essentially as described previously. 42 Control (3xFLAG) and experimental (3xFLAG-RavA) AP-MS experiments were independently performed three times. Purified complexes were divided into two parts and proteolyzed by trypsin or LysN (Thermo Fisher Scientific).…”

Section: Mass Spectrometrymentioning

confidence: 99%

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Structural and functional study of Legionella pneumophila effector RavA

Chung

Tyurin

et al. 2021

Protein Science

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Legionella pneumophila is an intracellular pathogen that causes Legionnaire's disease in humans. This bacterium can be found in freshwater environments as a free‐living organism, but it is also an intracellular parasite of protozoa. Human infection occurs when inhaled aerosolized pathogen comes into contact with the alveolar mucosa and replicates in alveolar macrophages. Legionella enters the host cell by phagocytosis and redirects the Legionella‐containing phagosomes from the phagocytic maturation pathway. These nascent phagosomes fuse with ER‐derived secretory vesicles and membranes forming the Legionella‐containing vacuole. Legionella subverts many host cellular processes by secreting over 300 effector proteins into the host cell via the Dot/Icm type IV secretion system. The cellular function for many Dot/Icm effectors is still unknown. Here, we present a structural and functional study of L. pneumophila effector RavA (Lpg0008). Structural analysis revealed that the RavA consists of four ~85 residue long α‐helical domains with similar folds, which show only a low level of structural similarity to other protein domains. The ~90 residues long C‐terminal segment is predicted to be natively unfolded. We show that during L. pneumophila infection of human cells, RavA localizes to the Golgi apparatus and to the plasma membrane. The same localization is observed when RavA is expressed in human cells. The localization signal resides within the C‐terminal sequence C409WTSFCGLF417. Yeast‐two‐hybrid screen using RavA as bait identified RAB11A as a potential binding partner. RavA is present in L. pneumophila strains but only distant homologs are found in other Legionella species, where the number of repeats varies.

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Section: Methodsmentioning

confidence: 99%

Section: Mass Spectrometrymentioning

confidence: 99%

Structural and functional study of Legionella pneumophila effector RavA

Chung

Tyurin

et al. 2021

Protein Science

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“…While Lem4 and Lem28 localize to the LCV through their PI(4)P-binding domains, they do not act on Rab1. Lem4 was recently demonstrated to be a phosphotyrosine phosphatase [78], although how this enzymatic function contributes to infection has yet to be determined.…”

Section: Legionella Pneumophila Effectors Targeting Pi(3)p and Pi(4)pmentioning

confidence: 99%

Exploitation of Phosphoinositides by the Intracellular Pathogen,Legionella pneumophila

Pike¹,

Noll²,

Neunuebel³

2020

Pathogenic Bacteria

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Manipulation of host phosphoinositide lipids has emerged as a key survival strategy utilized by pathogenic bacteria to establish and maintain a replication-permissive compartment within eukaryotic host cells. The human pathogen, Legionella pneumophila, infects and proliferates within the lung's innate immune cells causing severe pneumonia termed Legionnaires' disease. This pathogen has evolved strategies to manipulate specific host components to construct its intracellular niche termed the Legionella-containing vacuole (LCV). Paramount to LCV biogenesis and maintenance is the spatiotemporal regulation of phosphoinositides, important eukaryotic lipids involved in cell signaling and membrane trafficking. Through a specialized secretion system, L. pneumophila translocates multiple proteins that target phosphoinositides in order to escape endolysosomal degradation. By specifically binding phosphoinositides, these proteins can anchor to the cytosolic surface of the LCV or onto specific host membrane compartments, to ultimately stimulate or inhibit encounters with host organelles. Here, we describe the bacterial proteins involved in binding and/or altering host phosphoinositide dynamics to support intracellular survival of L. pneumophila.

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“…DrrA is a multidomain effector with a nucleotidyltransferase domain and a guanine nucleotide exchange factor (GEF) domain in addition to the PI4P binding domain (26,27). In contrast, Lpg1101 contains an N-terminal haloacid dehalogenase (HAD) domain with tyrosine phosphatase activity (28). However, the structure and biochemical activity of Lpg2603 remained uncharacterized.…”

Section: Introductionmentioning

confidence: 99%

A Legionella effector kinase is activated by host inositol hexakisphosphate

Sreelatha

Nolan

Park

et al. 2020

Journal of Biological Chemistry

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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.

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Legionella pneumophila effector Lem4 is a membrane-associated protein tyrosine phosphatase

Cited by 15 publications

References 64 publications

Structural and functional study of Legionella pneumophila effector RavA

Structural and functional study of Legionella pneumophila effector RavA

Exploitation of Phosphoinositides by the Intracellular Pathogen,Legionella pneumophila

A Legionella effector kinase is activated by host inositol hexakisphosphate

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