Pseudokinases are considered to be the inactive counterparts of conventional protein kinases and comprise approximately 10% of the human and mouse kinomes. Here, we report the crystal structure of the Legionella pneumophila effector protein, SidJ, in complex with the eukaryotic Ca2+-binding regulator, calmodulin (CaM). The structure reveals that SidJ contains a protein kinase-like fold domain, which retains a majority of the characteristic kinase catalytic motifs. However, SidJ fails to demonstrate kinase activity. Instead, mass spectrometry and in vitro biochemical analyses demonstrate that SidJ modifies another Legionella effector SdeA, an unconventional phosphoribosyl ubiquitin ligase, by adding glutamate molecules to a specific residue of SdeA in a CaM-dependent manner. Furthermore, we show that SidJ-mediated polyglutamylation suppresses the ADP-ribosylation activity. Our work further implies that some pseudokinases may possess ATP-dependent activities other than conventional phosphorylation.

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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Glutamylation is a posttranslational modification where the amino group of a free glutamate amino acid is conjugated to the carboxyl group of a glutamate side chain within a target protein. SidJ is a Legionella kinase-like protein that has recently been identified to perform protein polyglutamylation of the Legionella SdeA Phosphoribosyl-Ubiquitin (PR-Ub) ligase to inhibit SdeA's activity. The attachment of multiple glutamate amino acids to the catalytic glutamate residue of SdeA by SidJ inhibits SdeA's modification of ubiquitin (Ub) and ligation activity. In this protocol, we will discuss a SidJ non-radioactive, in vitro glutamylation assay using its substrate SdeA. This will also include a second reaction to assay the inhibition of SdeA by using both modification of free Ub and ligation of ADP-ribosylated Ubiquitin (ADPR-Ub) to SdeA's substrate Rab33b. Prior to the identification and publication of SidJ's activity, no SdeA inhibition assays existed. Our group and others have demonstrated various methods to display inhibition of SdeA's activity. The alternatives include measurement of ADP-ribosylation of Ub using radioactive NAD, NAD hydrolysis, and Western blot analysis of HA-Ub ligation by SdeA. This protocol will describe the inhibition of both ubiquitin modification and the PR-Ub ligation by SdeA using inexpensive standard gels and Coomassie staining.