Structural Insights into Non-canonical Ubiquitination Catalyzed by SidE

Wang, Yong; Shi, Miao; Han, Feng; Zhu, Yalan; Liu, Songqin; Gao, Ang; Gao, Pu

doi:10.1016/j.cell.2018.04.023

Cited by 63 publications

(85 citation statements)

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“…However, unlike SusF, 298 overexpression of SidJ is toxic to eukaryotic cells (39). The SidE family of effectors also contributes 299 directly to intracellular replication through a novel mechanism of phosphoribosyl-ubiquitin 300 conjugation to host substrates (40)(41)(42). Unlike the SidE effectors, loss of sidI or the sidI-lpg2505 301 operon has no effect on L. pneumophila intracellular growth (6), suggesting that SidI functions 302 redundantly within macrophages.…”

Section: Discussion 248mentioning

confidence: 99%

TheLegionella pneumophilametaeffector Lpg2505 (SusF) regulates SidI-mediated translation inhibition and GDP-dependent glycosyltransferase activity

Joseph

Pohl

Ball

et al. 2019

Preprint

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19Legionella pneumophila, the etiological agent of Legionnaires Disease, employs an arsenal of 20 hundreds of Dot/Icm-translocated effector proteins to facilitate replication within eukaryotic 21 phagocytes. Several effectors, called metaeffectors, function regulate the activity of other 22 Dot/Icm-translocated effectors during infection. The metaeffector Lpg2505 is essential for L. 23 pneumophila intracellular replication only when its cognate effector, SidI, is present. SidI is a 24 cytotoxic effector that interacts with the host translation factor eEF1A and potently inhibits 25 eukaryotic protein translation by an unknown mechanism. Here, we evaluated the impact of 26Lpg2505 on SidI-mediated phenotypes and investigated the mechanism of SidI function. We 27 determined that Lpg2505 binds with nanomolar affinity to SidI and suppresses SidI-mediated 28 inhibition of protein translation. SidI binding to eEF1A and SusF is not mutually exclusive and 29 these proteins bind distinct regions of SidI. We also discovered that SidI possesses GDP-30 dependent glycosyltransferase activity and that this activity is regulated by Lpg2505. We have 31 therefore renamed Lpg2505, SusF (suppressor of SidI function). This work reveals novel 32 enzymatic activity for SidI and provides insight into how intracellular replication of L. 33 pneumophila is regulated by a metaeffector. 34 35 Introduction 36Legionella pneumophila is the etiological agent of Legionnaires' Disease, a severe 37 inflammatory pneumonia that results from uncontrolled bacterial replication within alveolar 38 macrophages. Upon phagocytosis, L. pneumophila avoids lysosomal degradation through 39 establishment of an endoplasmic reticulum-derived compartment called the Legionella-containing 40 vacuole (LCV) (1). For biogenesis of the LCV and acquisition of nutrients from the host cell, L. 41 pneumophila is dependent on a massive arsenal of over 300 individual effector proteins that are 42 translocated directly into the host cell through a Dot/Icm Type IVB secretion system (T4BSS) (2). 43The cellular functions of the majority of effectors have yet to be elucidated, due in part to their 44 functional redundancy within macrophages (3). 45Metaeffectors have emerged as a common theme in L. pneumophila pathogenesis and are 46 used by L. pneumophila to regulate effector function (4). The first metaeffector described was LubX, 47 which temporally regulates function of its cognate effector SidH by hijacking host ubiquitination 48 machinery to facilitate proteasomal degradation of SidH (5). At least 20 of the over 300 identified 49 L. pneumophila effectors are metaeffectors (4) and two of these -SidJ and Lpg2505 -are members 50 of a small group of effectors that are individually important for L. pneumophila intracellular 51 replication within macrophages (6-8). SidJ is a glutamylase that covalently modifies and 52 abrogates the function of the SidE family of effector ubiquitin ligases (9-11). Lpg2505 is a 53 metaeffector of unknown function that suppresses toxicity of its cognate...

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Section: Discussion 248mentioning

confidence: 99%

TheLegionella pneumophilametaeffector Lpg2505 (SusF) regulates SidI-mediated translation inhibition and GDP-dependent glycosyltransferase activity

Joseph

Pohl

Ball

et al. 2019

Preprint

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“…Among the large cohort of Legionella effectors, the SidE family of effectors have recently been identified as a group of novel Ub ligases that act independently of ATP, Mg 2+ or E1 and E2 enzymes (Bhogaraju et al, 2016; Kotewicz et al, 2017; Qiu et al, 2016). This unusual SidE family ubiquitin ligases contain multiple domains including a mono-ADP-ribosyl transferase (mART) domain, which catalyzes ubiquitin ADP-ribosylation to generate mono-ADP-ribosyl ubiquitin (ADPR-Ub), and a phosphodiesterase (PDE) domain, which conjugates ADPR-Ub to serine residues on substrate proteins (phosphoribosyl-ubiquitination) (Akturk et al, 2018; Dong et al, 2018; Kalayil et al, 2018; Kim et al, 2018; Wang et al, 2018). Interestingly, the function of SidEs appears to be antagonized by SidJ (Lpg2155), an effector encoded by a gene resides at the same locus with genes encoding three members of the SidE family (Lpg2153, Lpg2156, and Lpg2157) (Liu and Luo, 2007).…”

Section: Introductionmentioning

confidence: 99%

Protein polyglutamylation catalyzed by the bacterial Calmodulin-dependent pseudokinase SidJ

Sulpizio

Minelli

Wan

et al. 2019

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23Pseudokinases are considered to be the inactive counterparts of conventional protein 24 kinases and comprise approximately 10% of the human and mouse kinomes. Here we report the 25 crystal structure of the Legionella pneumophila effector protein, SidJ, in complex with the 26 eukaryotic Ca 2+ -binding regulator, Calmodulin (CaM). The structure reveals that SidJ contains a 27 protein kinase-like fold domain, which retains a majority of the characteristic kinase catalytic 28 motifs. However, SidJ fails to demonstrate kinase activity. Instead, mass spectrometry and in vitro 29 biochemical analysis demonstrate that SidJ modifies another Legionella effector SdeA, an 30 unconventional phosphoribosyl ubiquitin ligase, by adding glutamate molecules to a specific 31 residue of SdeA in a CaM-dependent manner. Furthermore, we show that SidJ-mediated 32 polyglutamylation suppresses the ADP-ribosylation activity. Our work further implies that some 33 pseudokinases may possess ATP-dependent activities other than conventional phosphorylation. 34 35 36 KEYWORDS 37 SidJ; polyglutamylation; Legionella pneumophila; SdeA; phosphoribosyl ubiquitination; 38 ubiquitin 39 40 5effectors (Havey and Roy, 2015; Jeong et al., 2015; Urbanus et al., 2016). Furthermore, SidJ has 87 been shown to act on SidE proteins and releases these effectors from the LCV (Jeong et al., 2015). 88A recent study reported that SidJ functions as a unique deubiquitinase that counteracts the SidE-89 mediated phosphoribosyl-ubiquitination by deconjugating phosphoribosyl-ubiquitin from 90 modified proteins (Qiu et al., 2017). However, our recent results do not support this SidJ-mediated 91 deubiquitinase activity (Wan et al., 2019) and the exact function of SidJ remains elusive. 92The goal of the present study is to elucidate the molecular function of SidJ and to 93 investigate the mechanism that underlies how SidJ antagonizes the PR-ubiquitination activity of 94 SidEs. Here we report the crystal structure of SidJ in complex with human Calmodulin 2 (CaM) 95 and reveal that SidJ adopts a protein kinase-like fold. A structural comparison allowed us to 96 identify all the catalytic motifs conserved in protein kinases. However, SidJ failed to demonstrate 97 protein kinase activity. Using SILAC (Stable Isotope Labeling by Amino acids in Cell culture) 98 based mass spectrometry approach, we discovered that SidJ modifies SdeA by attaching the amino 99 acid glutamate to a key catalytic residue on SdeA. Moreover, we found that this glutamylation 100 activity by SidJ is CaM dependent and the glutamylation of SdeA suppresses its PR-ubiquitination 101 activity. Thus our work provides molecular insights of a key PR-ubiquitination regulator in 102 Legionella infection. We anticipate that our work will also have impact on the studies of 103 pseudokinases and CaM-regulated cellular processes. 104 6 Results 105 SidJ Binds CaM through its C-terminal IQ Motif 106To elucidate the biological function of SidJ, we performed sequence analyses and found 107 that the C-terminus of SidJ c...

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“…Magnetic forms of graphene would be useful for spintronics, a technology that forms the basis of today's magnetic data storage 3,4 . But the main interest in generating magnetic edge states in graphene is for quantum technologies.…”

Section: F E R N a N D O Lu I S And E U G E N I O C O R O N A D Omentioning

confidence: 99%

“…Once the nicotinamide group from NADH is released from the enzyme, a conformational change can occur, allowing Arg42 to replace Arg72 in the active site. This model explains why ADPR attaches selectively to Arg42 and not to , Dong et al 2 and Kalayil et al 3 report the structure of the enzyme SdeA, and Wang et al 4 present the structure of the enzyme SidE. a, In the first step, the enzyme's mART domain processes NAD + and adds an adenosine diphosphate ribose (ADPR) group to the amino-acid residue arginine 42 (Arg42) of ubiquitin.…”

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confidence: 99%