Letter
https://doi.org/10.1038/s41586-019-1440-8Inhibition of bacterial ubiquitin ligases by
SidJ–calmodulin catalysed glutamylation
Sagar Bhogaraju1,2,3, Florian Bonn^1 , rukmini Mukherjee1,2, Michael Adams^3 , Moritz M. Pfleiderer^3 , Wojciech P. Galej^3 ,
Vigor Matkovic1,2, Jaime Lopez-Mosqueda1,4, Sissy Kalayil1,2, Donghyuk Shin1,2,5 & Ivan Dikic1,2,5
The family of bacterial SidE enzymes catalyses phosphoribosyl-
linked serine ubiquitination and promotes infectivity of Legionella
pneumophila, a pathogenic bacteria that causes Legionnaires’
disease^1 –^3. SidE enzymes share the genetic locus with the Legionella
effector SidJ that spatiotemporally opposes the toxicity of these
enzymes in yeast and mammalian cells, through a mechanism that
is currently unknown^4 –^6. Deletion of SidJ leads to a substantial
defect in the growth of Legionella in both its natural hosts (amoebae)
and in mouse macrophages^4 ,^5. Here we demonstrate that SidJ is a
glutamylase that modifies the catalytic glutamate in the mono-ADP
ribosyl transferase domain of the SdeA, thus blocking the ubiquitin
ligase activity of SdeA. The glutamylation activity of SidJ requires
interaction with the eukaryotic-specific co-factor calmodulin, and
can be regulated by intracellular changes in Ca^2 + concentrations.
The cryo-electron microscopy structure of SidJ in complex
with human apo-calmodulin revealed the architecture of this
heterodimeric glutamylase. We show that, in cells infected with
L. pneumophila, SidJ mediates the glutamylation of SidE enzymes on
the surface of vacuoles that contain Legionella. We used quantitative
proteomics to uncover multiple host proteins as putative targets of
SidJ-mediated glutamylation. Our study reveals the mechanism by
which SidE ligases are inhibited by a SidJ–calmodulin glutamylase,
and opens avenues for exploring an understudied protein
modification (glutamylation) in eukaryotes.
L. pneumophila contains about 300 effector proteins that modulate
the cellular processes of the host through diverse activities, to aid in the
growth and survival of this infectious pathogen^1. Fourteen of these effec-
tors, including SidJ, have previously been shown to directly suppress the
activities of other Legionella effectors^4 ,^6 ,^7. SidJ opposes the toxicity of the
SidE class of ubiquitin ligases (comprising SdeA, SdeB, SdeC and SidE)
in yeast and mammalian cells. Deletion of SidJ in Legionella elicits sub-
stantial growth defects, probably owing to the unhinged toxicity of SidE
enzymes^4. In a recent report, SidJ has been shown to act as a deubiq-
uitinase for canonical ubiquitination and for phosphoribosyl-linked
ubiquitination mediated by SidE, which might account for the anti-SidE
activity of SidJ^8. However, we could not detect any intrinsic deubiq-
uitinase activity of SidJ expressed in Escherichia coli or in mammalian
cells (Extended Data Fig. 1a–c). Instead, we identified SidJ-associated
proteins with deubiquitinase activities that co-precipitated with SidJ
isolated from Legionella lysate, which potentially explains the previ-
ous reported observations regarding its anti-SidE activity (Extended
Data Fig. 1c). SidE enzymes contain a mono-ADP-ribosyl transferase
(mART) domain and a phosphodiesterase (PDE) domain, which
sequentially perform ADP ribosylation of ubiquitin and substrate
phosphoribosyl-linked ubiquitination, respectively^2 ,^3. In HEK293T
cells, expression of SidJ with SdeA abolished the SdeA-mediated
phosphoribosylation of ubiquitin, and nullified the ADP ribosylation of
ubiquitin that is catalysed by the PDE-defective mutant SdeA(H277A)
(which retains mART activity)^3 (Fig. 1a). Consistently, the yeast toxicity
exerted by wild-type SdeA and the SdeA(H277A) mutant was
rescued by co-expressing SidJ (Fig. 1b). Thus, we hypothesized that
SidJ inhibits the first step of SdeA-mediated ubiquitination of sub-
strates (that is, mono-ADP ribosylation of ubiquitin). As expected,
although SdeA alone showed robust ε-NAD+ hydrolysis, SdeA
co-expressed with SidJ (hereafter, SidJ-treated SdeA) did not show
mART activity (Fig. 1c). Lysate of HEK293T cells that express
SidJ—but not lysate of untransfected cells or SidJ-expressing
cell lysate that is depleted of SidJ—were able to block in vitro
ε-NAD+ hydrolysis mediated by SdeA (Fig. 1d). The addition
of 10 mM ethylenediaminetetraacetic acid (EDTA) to lysates
of SidJ-expressing HEK293T cells decreased the effect of these
lysates on SdeA activity. Moreover, SidJ purified from E. coli was(^1) Institute of Biochemistry II, Faculty of Medicine, Goethe University, Frankfurt am Main, Germany. (^2) Buchmann Institute for Molecular Life Sciences, Goethe University, Frankfurt am Main, Germany.
(^3) European Molecular Biology Laboratory, Grenoble, France. (^4) Department of Biology and Microbiology, South Dakota State University, Brookings, SD, USA. (^5) Max Planck Institute of Biophysics,
Frankfurt am Main, Germany. *e-mail: [email protected]; [email protected]
HA–SdeA
HA–SdeA(H277A)
GFP–SidJ
––
–++
++
–+ ++
––––––111145135Unmodi ed Ub
IB: Abcam UbIB: GFPIB: HAIB: actinTotal UbIB: CS Uba bcd240
135kDa
SdeA
SdeA(H277A)
SdeA(E860A/E862A)Empty vectorSidJ + empty vector
SidJ + SdeA
SidJ + SdeA(H277A)
Empty vectorDextrose (off) Galactose (on)0 500 1,000 1,500 2,0006
5
4
3
26
5
4
3SdeA
SdeA + SidJ
BufferTime (s)Fluorescence(AU) (×^104 )0 500 1,000 1,500 2,000GST–SdeA + SidJ lysate
GST–SdeA + SidJ lysate + EDTA
GST–SdeA + SidJ depleted lysate
GST–SdeA
GST–beadsFluorescence(AU) (×^104 )Time (s)
Fig. 1 | SidJ inhibits the ubiquitin–ADP ribosylation activity of SdeA.
a, SidJ and SdeA constructs were expressed as indicated in HEK293T
cells and ubiquitin modification was probed using the ubiquitin
(Ub) antibodies Abcam Ub and Cell Signaling (CS) Ub, as previously
described^3. IB, immunoblot. b, Yeast strain W303 was transformed
using the indicated combination of constructs (H277A, PDE defective;
E860A/E862A, mART defective). Serial dilutions of transformed yeast
were spotted on plates containing dextrose (repressing) or galactose
(inducing). c, Purified SdeA, from HEK293T cells that express SdeA
alone or in combination with SidJ, was used in ε-NAD+ hydrolysis assays.
The increase in the fluorescence indicates ubiquitin–ADP ribosylation^17.
d, Glutathione-S-transferase-tagged SdeA (GST–SdeA) purified from
E. coli was incubated with HEK293T cell lysate that contained SidJ or was
depleted of SidJ. SdeA was subsequently purified using glutathione agarose
beads and used in ε-NAD+ hydrolysis assays. Experiments in a–d were
repeated three times independently with similar results. For gel source
data, see Supplementary Fig. 1.382 | NAtUre | VOL 572 | 15 AUGUSt 2019