The bacterial pathogen Legionella pneumophila creates an intracellular niche permissive for its replication by extensively modulating host-cell functions using hundreds of effector proteins delivered ...by its Dot/Icm secretion system
. Among these, members of the SidE family (SidEs) regulate several cellular processes through a unique phosphoribosyl ubiquitination mechanism that bypasses the canonical ubiquitination machinery
. The activity of SidEs is regulated by another Dot/Icm effector known as SidJ
; however, the mechanism of this regulation is not completely understood
. Here we demonstrate that SidJ inhibits the activity of SidEs by inducing the covalent attachment of glutamate moieties to SdeA-a member of the SidE family-at E860, one of the catalytic residues that is required for the mono-ADP-ribosyltransferase activity involved in ubiquitin activation
. This inhibition by SidJ is spatially restricted in host cells because its activity requires the eukaryote-specific protein calmodulin (CaM). We solved a structure of SidJ-CaM in complex with AMP and found that the ATP used in this reaction is cleaved at the α-phosphate position by SidJ, which-in the absence of glutamate or modifiable SdeA-undergoes self-AMPylation. Our results reveal a mechanism of regulation in bacterial pathogenicity in which a glutamylation reaction that inhibits the activity of virulence factors is activated by host-factor-dependent acyl-adenylation.
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.
Synopsis
Transglutaminase MavC of the bacterial pathogen Legionella pneumophila catalyzes atypical ubiquitination of the host cell E2 ubiquitin conjugation enzyme UBE2N. Here, the L. pneumophila ubiquitin deamidase MvcA is found to reverse MavC‐induced UBE2N ubiquitination and to restore its activity to promote host cell survival at later stages of L. pneumophila infection.
Expression of MvcA reduces MavC‐induced UBE2N ubiquitination.
MvcA promotes synthesis of K63‐type polyubiquitin chains by UBE2N during L. pneumophila infection.
MvcA is a UBE2N‐specific deubiquitinase that cleaves the isopeptide bond between Gln40 of ubiquitin and Lys92 of UBE2N.
Structure of the MvcA‐UBE2N‐Ub complex reveals involvement of the MvcA “insertion domain” in substrate recognition.
MvcA counteracts MavC activity at the late stage of infection to promote intracellular replication of L. pneumophila.
Dynamic regulation of activity and atypical ubiquitination of the host E2 enzyme UBE2N via bacterial effectors MavC and MvcA over the course of infection is important for pathogen replication.
The bacterial effector MavC modulates the host immune response by blocking Ube2N activity employing an E1-independent ubiquitin ligation, catalyzing formation of a γ-glutamyl-ε-Lys (Gln40
-Lys92
) ...isopeptide crosslink using a transglutaminase mechanism. Here we provide biochemical evidence in support of MavC targeting the activated, thioester-linked Ube2N~ubiquitin conjugate, catalyzing an intramolecular transglutamination reaction, covalently crosslinking the Ube2N and Ub subunits effectively inactivating the E2~Ub conjugate. Ubiquitin exhibits weak binding to MavC alone, but shows an increase in affinity when tethered to Ube2N in a disulfide-linked substrate that mimics the charged E2~Ub conjugate. Crystal structures of MavC in complex with the substrate mimic and crosslinked product provide insights into the reaction mechanism and underlying protein dynamics that favor transamidation over deamidation, while revealing a crucial role for the structurally unique insertion domain in substrate recognition. This work provides a structural basis of ubiquitination by transglutamination and identifies this enzyme's true physiological substrate.
Members of the SidE effector family from Legionella pneumophila represent a new paradigm in the ubiquitin world. These enzymes catalyze ubiquitination of target proteins via a mechanism different ...from that of conventional E1-E2-E3 biochemistry and play important roles in L. pneumophila virulence. They combine mono-ADP-ribosylation and phosphodiesterase activities to attach ubiquitin onto substrates, in great contrast to the orthodox pathway. A series of recent structural and mechanistic studies have clarified the action of these enzymes. Herein, we summarize the key insights into the structure and function of these proteins, emphasizing their modular nature, and discuss the biochemical implications of these proteins as well as areas of further exploration.
The SidE family of Legionella pneumophila effectors can ubiquitinate host protein substrates on Ser residues via a novel mechanism considerably different from that of the canonical E1-E2-E3 pathway.
X-ray crystallographic data of these proteins reveal a unique three-domain arrangement and identify Ub and cofactor binding sites.
Biochemical studies have provided insights into the SidE mechanism and also discovered that flexible regions of proteins containing Ser residues can serve as ubiquitination targets.
Ubiquitination regulates many aspects of host immunity and thus is a common target for infectious agents. Recent studies have revealed that members of the SidE effector family of the bacterial ...pathogen Legionella pneumophila attack several small GTPases associated with the endoplasmic reticulum by a novel ubiquitination mechanism that does not require the E1 and E2 enzymes of the host ubiquitination machinery. In this case, ubiquitin is first activat- ed by ADP-ribosylation at Arg42 by a mono-ADP-ribosyltransferase activity; the intermediate is then cleaved by a phosphodiesterase activity also residing within SdeA, concomitant with the attachment of ubiquitin to serine residues of substrate proteins via a phosphoribosyl linker. Here we demonstrate that the effect of SidEs is antagonized by SidJ, an effector encoded by a gene situated in the locus coding for three members of the SidE family （SdeC, SdeB and SdeA）. SidJ reverses ubiquitination of SidEs-modified substrates by cleaving the phosphodiester bond that links phosphoribosylated ubiquitin to protein substrates. SidJ also displays classical deubiquitinase activity but does not require catalytic cysteine residues. Further, these deubiquitinase activities of SidJ are essential for its role in L. pneu- mophila infection. Finally, the activity of SidJ is required for efficiently reducing the abundance of ubiquitinated Rab33b in infected cells within a few hours after bacterial uptake. Our results establish SidJ as a ubiquitin-deconju- gating enzyme that functions to impose temporal regulation on the activity of SidE effectors. SidJ may be important in future studies of signaling cascades mediated by this unique ubiquitination, one that also potentially regulates cel- lular processes in eukaryotic cells.
he modification of proteins by the covalent attachment of ubiquitin is a natural process that crucially regulates a wide range of eukaryotic signaling outcomes. This process has been understood as ...the linking of the C-terminus of ubiquitin to the lysine residue of a target protein via an isopeptide linkage, catalyzed by the coordinated effort by E1, E2, and E3 enzymes. Importantly, ubiquitination has only been observed to be a eukaryotic phenomenon. In recent years though, intracellular bacteria, including human pathogens, have been observed to possess ubiquitininteracting proteins in their genomes. These proteins serve to subdue and manipulate their hosts’ ubiquitin signaling for their own benefit. While some of these proteins act within the eukaryotic context, more recent findings reveal the existence of prokaryotic enzymes that catalyze ubiquitination using mechanisms never before seen in nature. These remarkable processes utilize different cofactors and target different amino acid residues of both ubiquitin as well as substrate protein. The findings reported in this Thesis involve structural and biochemical studies on two new ubiquitinating proteins, the only two proteins known to catalyze ubiquitination outside of the canonical pathway. Both proteins are present in the genome of the intracellular human pathogen Legionella pneumophila: the SidE family, which catalyzes ubiquitination via a mechanism combining ADP-ribosylation and phosphodiesterase activities, and MavC, which utilizes a mechanism reminiscent of transglutaminases. Key insights provided in this document include the discovery that SidE enzymes can modify multiple ubiquitin moieties within a ubiquitin chain, and that modified ubiquitin chains are resistant to hydrolytic cleavage from many deubiquitinating enyzmes. Also, the development of a robust, continuous assay for SidE-catalyzed ubiquitination using a synthetic substrate is described. The catalytic action of MavC, which differs from both canonical E1/E2/E3 ubiquitination and SidE ubiquitination is also here elucidated. The crystal structure of MavC in complex with its ubiquitinated product is presented and provides an atomic view into the basis of substrate recognition. These findings bring to light a new dimension of hostpathogen interactions, where pathogenic ubiquitinating enzymes have appeared to arise from convergent evolution. The regulation of these pathogenic enzymes by other effectors is also discussed, as well as biochemical studies of these regulators. Further, these findings describe possible new drug discovery strategies, as well as possible techniques for discovering similar enzymes in organisms besides Legionella.
The SidE family of Legionella pneumophila effectors is a unique group of ubiquitin-modifying enzymes. Along with catalyzing NAD+-dependent ubiquitination of certain host proteins independent of the ...canonical E1/E2/E3 pathway, they have also been shown to produce phosphoribosylated free ubiquitin. This modified ubiquitin product is incompatible with conventional E1/E2/E3 ubiquitination processes, with the potential to lock down various cellular functions that are dependent on ubiquitin signaling. Here, we show that in addition to free ubiquitin, Lys63-, Lys48-, Lys11-, and Met1-linked diubiquitin chains are also modified by SdeA in a similar fashion. Both the proximal and distal ubiquitin moieties are targeted in the phosphoribosylation reaction. Furthermore, this renders the ubiquitin chains unable to be processed by a variety of deubiquitinating enzymes. These observations broaden the scope of SdeA’s modulatory functions during Legionella infection.
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