Inhibition of bacterial ubiquitin ligases by SidJ–calmodulin catalysed glutamylation

Bhogaraju, Sagar; Bonn, Florian; Mukherjee, Rukmini; Adams, Michael; Pfleiderer, Moritz M.; Galej, Wojciech P.; Matkovic, Vigor; López-Mosqueda, Jaime; Kalayil, Sissy; Shin, Dong Hyuk; Đikić, Ivan

doi:10.1038/s41586-019-1440-8

Cited by 106 publications

(113 citation statements)

References 33 publications

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“…However, this unusual deubiquitination activity was not repeatable in another study (Black et al, 2019), as well as in our unpublished studies. The definitive biochemical function of SidJ is now revealed in this study, as well as by recent reports (Bhogaraju et al, 2019; Black et al, 2019; Gan et al, 2019), as a polyglutamylase that adds glutamates to a specific catalytic residue E860 of SdeA and subsequently inhibits the PR-ubiquitination activity of SdeA. An interesting question arises at this point as whether there are other glutamylation substrates, especially from the host, besides the SidE family PR-ubiquitination ligases.…”

Section: Discussionsupporting

confidence: 72%

Protein polyglutamylation catalyzed by the bacterial Calmodulin-dependent pseudokinase SidJ

Sulpizio

Minelli

Wan

et al. 2019

Preprint

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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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Section: Discussionsupporting

confidence: 72%

Protein polyglutamylation catalyzed by the bacterial Calmodulin-dependent pseudokinase SidJ

Sulpizio

Minelli

Wan

et al. 2019

Preprint

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“…In line with the essential roles played by the ubiquitin network in immune signaling (Komander & Rape, ), more than 10 L. pneumophila effectors have been found to modulate the host ubiquitin machinery as E3 ubiquitin ligases that coordinate with E1 and E2 enzymes from the host (Qiu & Luo, ) or as deubiquitinases that remove Gly 76 ‐linked ubiquitin from ubiquitinated substrates (Sheedlo et al , ; Kubori et al , ). In addition, members of the SidE family catalyze ubiquitination by a tightly regulated (Bhogaraju et al , ; Black et al , ; Gan et al , ) mechanism that is unrelated to the classical three‐enzyme cascade of the eukaryotes (Qiu & Luo, ; Song & Luo, ). In these reactions, ubiquitin is first activated by ADP‐ribosylation via a mono‐ADP‐ribosyltransferase activity and is then utilized by a phosphodiesterase activity that attaches phosphoribosyl ubiquitin to serine residues of the substrate proteins (Bhogaraju et al , ; Qiu et al , ; Kotewicz et al , ).…”

Section: Introductionmentioning

confidence: 99%

Legionella pneumophila regulates the activity of UBE 2N by deamidase‐mediated deubiquitination

et al. 2019

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The Legionella pneumophila effector MavC induces ubiquitination of the E2 ubiquitin‐conjugating enzyme UBE2N by transglutamination, thereby abolishing its function in the synthesis of K63‐type polyubiquitin chains. The inhibition of UBE2N activity creates a conundrum because this E2 enzyme is important in multiple signaling pathways, including some that are important for intracellular L. pneumophila replication. Here, we show that prolonged inhibition of UBE2N activity by MavC restricts intracellular bacterial replication and that the activity of UBE2N is restored by MvcA, an ortholog of MavC (50% identity) with ubiquitin deamidase activity. MvcA functions to deubiquitinate UBE2N‐Ub using the same catalytic triad required for its deamidase activity. Structural analysis of the MvcA‐UBE2N‐Ub complex reveals a crucial role of the insertion domain in MvcA in substrate recognition. Our study establishes a deubiquitination mechanism catalyzed by a deamidase, which, together with MavC, imposes temporal regulation of the activity of UBE2N during L. pneumophila infection.

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“…S1B ). Moreover, the activity of SidEs is regulated by SidJ, another effector which inhibits the mono-ADP-ribosyltransferase activity by calmodulin-dependent glutamylation 15–17 ( Fig. S1B ).…”

Section: Introductionmentioning

confidence: 99%

Molecular basis of ubiquitination catalyzed by the bacterial transglutaminase MavC

Guan

et al. 2020

Preprint

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SummaryThe Legionella pneumophila effector MavC is a transglutaminase that carries out atypical ubiquitination of the ubiquitin (Ub) E2 conjugation enzyme UBE2N by catalyzing the formation of an isopeptide bond between Gln40 of Ub and Lys92 (or to a less extent, Lys94) of UBE2N, which results in inhibition of UBE2N signaling in the NF-κB pathway. In the absence of UBE2N, MavC deamidates Ub at Gln40 or catalyzes self-ubiquitination. However, the mechanisms underlying these enzymatic activities of MavC are not fully understood at molecular level. In this study, we obtained the structure of the MavC-UBE2N-Ub ternary complex that represents a snapshot of covalent cross-linking of UBE2N and Ub catalyzed by MavC. The structure reveals the unique way by which the cross-linked catalytic product UBE2N-Ub binds mainly to the Insertion and the Tail domains of MavC prior to its release. Based on our structural, biochemical and mutational analyses, we proposed the catalytic mechanism for both the deamidase and the transglutaminase activities of MavC. Finally, by comparing the structures of MavC and MvcA, the homologous protein that reverses MavC-induced UBE2N ubiquitination, we identified several key regions of the two proteins responsible for their opposite enzymatic activity. Our results provide insights into the mechanisms for substrate recognition and ubiquitination mediated by MavC as well as explanations for the opposite activity of MavC and MvcA.

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Inhibition of bacterial ubiquitin ligases by SidJ–calmodulin catalysed glutamylation

Cited by 106 publications

References 33 publications

Protein polyglutamylation catalyzed by the bacterial Calmodulin-dependent pseudokinase SidJ

Protein polyglutamylation catalyzed by the bacterial Calmodulin-dependent pseudokinase SidJ

Legionella pneumophila regulates the activity of UBE 2N by deamidase‐mediated deubiquitination

Molecular basis of ubiquitination catalyzed by the bacterial transglutaminase MavC

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