Phosphoribosylation of Ubiquitin Promotes Serine Ubiquitination and Impairs Conventional Ubiquitination

Article

Phosphoribosylation of Ubiquitin Promotes Serine Ubiquitination and Impairs Conventional Ubiquitination Graphical Abstract

Authors Sagar Bhogaraju, Sissy Kalayil, Yaobin Liu, Florian Bonn, Thomas Colby, Ivan Matic, Ivan Dikic

Correspondence [email protected]

In Brief Proteins can be ubiquitinated through a serine-linked phosphodiester bond independently of conventional E1 and E2 enzymes.

Highlights d

SdeA catalyzes atypical serine ubiquitination of substrates

d

Arginine phosphoribosylation of ubiquitin is a potent posttranslational signal

d

Phosphoribosylation of ubiquitin prevents activation of E1 and E2 enzymes

d

Phosphoribosylation of ubiquitin impairs several ubiquitindependent processes

Bhogaraju et al., 2016, Cell 167, 1636–1649 December 1, 2016 ª 2016 Elsevier Inc. http://dx.doi.org/10.1016/j.cell.2016.11.019

Article Phosphoribosylation of Ubiquitin Promotes Serine Ubiquitination and Impairs Conventional Ubiquitination Sagar Bhogaraju,1,2 Sissy Kalayil,1,2 Yaobin Liu,1,2 Florian Bonn,1 Thomas Colby,3 Ivan Matic,3 and Ivan Dikic1,2,4,5,* 1Institute

of Biochemistry II, School of Medicine, Goethe University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany Institute for Molecular Life Sciences, Goethe University Frankfurt, Max-von-Laue-Str. 15, 60438 Frankfurt am Main, Germany 3Max Planck Institute for Biology of Ageing, Joseph-Stelzmann-Str. 9b, 50931 Cologne, Germany 4Department of Immunology and Medical Genetics, University of Split, School of Medicine, Soltanska 2, 21000 Split, Croatia 5Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cell.2016.11.019 2Buchmann

SUMMARY

Conventional ubiquitination involves the ATP-dependent formation of amide bonds between the ubiquitin C terminus and primary amines in substrate proteins. Recently, SdeA, an effector protein of pathogenic Legionella pneumophila, was shown to mediate NAD-dependent and ATP-independent ubiquitin transfer to host proteins. Here, we identify a phosphodiesterase domain in SdeA that efficiently catalyzes phosphoribosylation of ubiquitin on a specific arginine via an ADP-ribose-ubiquitin intermediate. SdeA also catalyzes a chemically and structurally distinct type of substrate ubiquitination by conjugating phosphoribosylated ubiquitin to serine residues of protein substrates via a phosphodiester bond. Furthermore, phosphoribosylation of ubiquitin prevents activation of E1 and E2 enzymes of the conventional ubiquitination cascade, thereby impairing numerous cellular processes including mitophagy, TNF signaling, and proteasomal degradation. We propose that phosphoribosylation of ubiquitin potently modulates ubiquitin functions in mammalian cells. INTRODUCTION Ubiquitination is a prevalent post-translational modification that regulates a number of key cellular processes (Grabbe et al., 2011; Yau and Rape, 2016). The known mechanism of conventional ubiquitination proceeds via a universally conserved three-enzyme cascade: ubiquitin (Ub) is first activated by an Ub-activating enzyme (E1) that consumes ATP to adenylate the C terminus of Ub and then forms a thioester bond between the Ub C terminus and its catalytic cysteine residue. This step is followed by the transfer of Ub to the catalytic cysteine of the Ub-conjugating enzyme (E2) and eventually to the substrate lysine with the help of Ub ligases (E3). This final transfer of Ub results in an amide bond between the ε-amino group of a substrate 1636 Cell 167, 1636–1649, December 1, 2016 ª 2016 Elsevier Inc.

lysine and the C-terminal carboxylate of Ub (Hershko et al., 2000). Three major types of E3 ligases are known in eukaryotic cells: (1) really interesting new gene (RING)-type E3s functioning as scaffolds, bringing Ub-charged E2s into close proximity of substrates, thereby facilitating Ub transfer from E2 to a substrate lysine; (2) homologous to the E6–AP C terminus (HECT)-type E3s forming a thioester intermediate with Ub before transferring it directly to the substrate; and (3) RING-in-between-RING (RBR)-type E3s working via a hybrid mechanism (Vittal et al., 2015). Ub itself contains seven lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, Lys63) and an N-terminal methionine, which can be used in iterative ubiquitination reactions to form Ub chains in vivo (Yau and Rape, 2016). Formation of Ub chains is mediated via an amide bond to a primary amine either on the side chain of lysines (Lys-linked ubiquitination) or on the N terminus of Ub (Met1-linked ubiquitination). These differently linked Ub chains have unique structural features that are recognized by Ub-binding domains (UBDs) present in Ub receptors (Husnjak and Dikic, 2012), which mediate distinct cellular functions including proteasomal degradation, protein trafficking, DNA repair, autophagy, innate and adaptive immunity, or defense against pathogens (Di Fiore and von Zastrow, 2014; Finley, 2009; Newton and Dixit, 2012; Rogov et al., 2014; Ulrich and Walden, 2010). Prokaryotes do not possess genes encoding for Ub and lack the Ub-proteasome system (Maculins et al., 2016). Nonetheless, a wide range of bacterial pathogens have evolved potent strategies to counteract inflammatory signaling and microbicidal programs and influence the host Ub system to support their own life cycle (Hicks and Gala´n, 2013; Llosa et al., 2009; Maculins et al., 2016; Pu et al., 2016; Ribet and Cossart, 2015; Sansonetti, 2006). Among gram-negative bacteria, Legionella pneumophila (LP) has the largest arsenal of effector proteins and secretes
300 toxins into host cells via the type IV secretion system (T4SS) (Hubber and Roy, 2010). The SidE family of LP effectors including SidE, SdeA, SdeB, and SdeC was shown to be important for the intracellular replication of Legionella in host cells (Bardill et al., 2005). Intriguingly, the intracellular growth defect observed in a strain lacking all members of the SidE family can be rescued by expressing SdeA alone, indicating that the

members of this protein family perform redundant functions. SdeA was recently shown to ubiquitinate several endoplasmic reticulum-associated human Rab GTPases, resulting in the downregulation of their activity (Qiu et al., 2016). Interestingly, unlike all known bacterial E3 ligases, SdeA does not hijack the host Ub machinery but catalyzes Ub transfer autonomously without the need of host E1 and E2 enzymes. This ATP-independent reaction requires only NAD+ as a co-factor, and ADPribosylation of Ub was suggested to be a possible intermediate step in the transfer reaction. However, the mechanism and the chemistry of the Ub transfer reaction to the substrate remained unknown. We now show that the mono-ADP-ribosyl transferase (mART) domain of SdeA and a newly identified phosphodiesterase (PDE) domain act successively to convert cellular Ub into phosphoribosylated Ub that can be conjugated to a serine residue of a target protein or to SdeA itself. This mechanism of SdeA-mediated ubiquitination is independent of the conventional E1-E2E3 enzymatic cascade and results in the formation of a phosphodiester-bond between phosphoribosylated arginine 42 (Arg42) of Ub and the hydroxyl group of a substrate serine. More importantly, free phosphoribosylated ubiquitin produced by bacterial SdeA inhibits the host ubiquitination machinery, leading to a severe and eventually toxic disturbance of essential Ub-dependent cellular processes such as proteasomal degradation, TNF signaling, and mitophagy. RESULTS AND DISCUSSION SdeA Promotes Phosphoribosylation of Ub SdeA can covalently couple Ub to substrate proteins in an ATPindependent manner and without exploiting the host ubiquitination machinery, using NAD+ as a cofactor instead (Qiu et al., 2016). Thus, SdeA-mediated ubiquitination constitutes a mechanism entirely distinct from the canonical enzymatic cascade of eukaryotic cells. In order to understand the chemistry behind this type of protein ubiquitination, we first analyzed the structural features of SdeA. Previous structural and biochemical studies have shown that SdeA contains an N-terminal deubiquitinase (DUB) domain spanning residues 1 to 200 and an mART domain spanning residues 500 to 1000 (Figure 1A) (Qiu et al., 2016; Sheedlo et al., 2015). Our domain prediction analysis of SdeA revealed that the region between residues 200 and 500 is similar to the predicted PDE domain in the LP effector lpg1496 (Wong et al., 2015) (Figure S1A). It is worth mentioning that even though the structure of lpg1496 shows homology to several canonical PDE domains, attempts to establish its PDE activity toward a number of substrates (30 -AMP, 50 -AMP, ADP, ADP-ribose, 20 ,30 -cAMP, 30 ,50 -cAMP, and 30 ,50 -cGMP) failed. Therefore annotation of the PDE domain in lpg1496 is only based on its structural homology to known PDE domains (Wong et al., 2015). The SdeA PDE region shows 48% sequence similarity to the PDE domain of lpg1496 (167 residues aligned; Figure S1A) and 23% sequence similarity to the PDE domain in the well-characterized cyclic di-30 ,50 -GMP phosphodiesterase PA4781 from Pseudomonas aeruginosa (Rinaldo et al., 2015). Based on these observations, we expected PDE activity for SdeA. We hypothesized that the predicted PDE domain in SdeA may cleave the pyrophosphate bond between

ADP a and b phosphates in ADP-ribosylated Ub, leading to the formation of phosphoribosylated Ub and release of AMP. The PDE domain of SdeA was modeled using the crystal structure of the putative PDE domain of lpg1496 (PDB code: 5BU1), and we mutated the predicted catalytic residues of the PDE domain and the mART domain in SdeA constructs spanning residues 200 to 1005 (DNC SdeA) (Figure 1B). These constructs were then used to modify Ub with biotin-labeled NAD (biotin is attached to the amino group of the adenine base) as per the experimental scheme shown in Figure 1C. Streptavidin blots revealed that the wild-type (WT) protein completely processed ADP-ribosylated Ub to phosphoribosyl-Ub, whereas the PDE domain mutant proteins H284A, H288A, and R344A were defective in cleavage of the pyrophosphate bond in ADP-ribosylated Ub (Figure 1D). As expected, the mART domain mutant (E867A, E869A, or EE/AA) of SdeA failed to ADP-ribosylate Ub. Ub treated with SdeA WT could be visualized with phospho-specific Pro-Q Diamond stain due to the exposed phosphate of the phosphoribose moiety (Daniels et al., 2014), whereas Ub modified by the PDE mutants did not show staining with the phosphoprotein stain (Figures 1C and 1E). We then quantified the AMP released in the reactions catalyzed by DNC SdeA WT and PDE mutant H284A (Figure S1B). Compared to WT, H284A mutant SdeA showed only background level release of AMP in the reaction with NAD and ubiquitin. Together, these biochemical experiments revealed that the PDE domain of SdeA catalyzes conversion of ADP-ribosylated Ub to phosphoribosyl Ub and releases AMP. In addition to the biochemical approaches, we analyzed the Ub modification by DNC SdeA WT and the PDE mutant with high-resolution ETD (electron transfer dissociation) mass spectrometry. As we have recently shown, this mass spectrometric technique allows for precise mapping of ADP-ribosylation and phosphoribosylation on all possible amino acids by preserving the highly labile bonds of these modifications (Leidecker et al., 2016). This unbiased identification of modification sites is important given that ADP-ribosylation has promiscuous aminoacid specificity (Daniels et al., 2015a). We unambiguously mapped the site of ADP-ribosylation (Qiu et al., 2016) as well as phosphoribosylation to arginine 42 of Ub in high-resolution ETD spectra (Figures 1F and 1G). Phosphoribosylation of Ub was detected in DNC SdeA WT reaction mixtures, indicating that SdeA WT is able to fully process ADP-ribosylated Ub (Figure 1F). In contrast, only ADP-ribosylation of Ub was detected in the samples containing the H284A mutant (Figure 1G). Curiously, Qiu et al. have reported that SdeA showed no ADP-ribosylation activity when incubated with mammalian cell lysate in the presence of radiolabeled NAD+ (32P-a-NAD). We think that this is an effect of the PDE domain cleaving off radiolabeled AMP from the ADP-ribosyl group while leaving an unlabeled phosphoribosyl moiety on its substrates, rendering the modification undetectable by autoradiography (Qiu et al., 2016). Atypical Serine Ubiquitination Intriguingly, SdeA can attach Ub lacking the C-terminal diGly motif to substrate proteins devoid of any lysine residue that would serve as acceptor in the canonical ubiquitination reaction (Qiu et al., 2016). Based on the deduced function of PDE domain of SdeA (Figures 1 and S1), we hypothesized that

Cell 167, 1636–1649, December 1, 2016 1637

A

E867A EE/AA E869A

H284A

N DUB

PDE

1

B

{

C125

R344

mART

200

500

H284

C

CC 1000

1506

H288 ADP

Biotin

C

Nicotinamide

Biotin

A

4A

/A

Ubiquitin (Coomassie)

Ubiquitin (Coomassie)

11 Ubiquitin (Pro-Q Diamond phosphoprotein stain)

11 Ubiquitin (α-Biotin) C 14

100

F

ΔNC SdeA

EE

8A

34 R

4A 28

H

T

H

T

-

W

MW kDa 11

ΔNC SdeA

+NAD

28

+NAD

W

A

4A

/A

EE

8A

34 R

4A T

H

T

W

W

-

E

+Biotin-NAD

28

+Biotin-NAD MW kDa 11

Biotin

f -ribose-P Ub f -ADP-ribose Ub phosphodiester bond cleavage in ADP ribose Pro-Q Diamond HRP-streptavidin phosphoprotein stain

28

D

+ NAD

H

SdeA WT

H414

AMP

G c9

100

c9

c8

z4

z3

c 10

z7

z5 c5 y3

z9 c7

c6

z 10

z8

y4

y8

80 60

y8

z3

y 12

y9

C5

0

400

600

800

1000

1200

1400

1600

Z7

C6

y7

Y6

400

600

c 12 y 12 y 13 z 14

z8

C7

0

y6

c 14

c 10

Z5

C8

y7

y5

c 11

c 11 Z4

40

40

C 12

20

z1 14 4 z 13 y 13

relative intensity (%)

60

z6

20

relative intensity (%)

80

z6

800

1000

1200

1400

1600

1800

2000

m/z, Da

m/z, Da

Figure 1. Identification and Biochemical Characterization of a PDE Domain in SdeA (A) Schematic representation of domain organization in SdeA. Domain abbreviations are DUB, deubiquitinase; PDE, phosphodiesterase; mART, Mono-ADPribosyl transferase; and CC, coiled-coil. The putative catalytic residues in each domain are shown. (B) Model of the PDE domain of SdeA. The inset represents the enlarged view of the active site of the PDE domain with putative catalytic residues. ADP was modeled based on the crystal structure of the PDE of lpg1496 bound to ADP (PDB: 5BU2). See also Figure S1A. (C) Experimental scheme showing the usage of biotin-labeled NAD+ to probe cleavage of the phosphodiester bond in ADP-ribosylated ubiquitin. (D) Testing the function of the PDE domain of SdeA. Ubiquitin was incubated with the indicated SdeA constructs in the presence or absence of biotin-labeled NAD+. The reaction components were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and probed with streptavidin-HRP conjugate to detect biotin. (E) The exposed phosphate group remaining on ubiquitin after PDE domain-mediated cleavage of ADP-ribosylated ubiquitin is stained using Pro-Q Diamond phospho-specific gel stain. See also Figure S1B. (F and G) The site of SdeA-dependent ubiquitin modification was determined in the ubiquitination reaction mixture containing SdeA WT (F) or PDE mutant (H284) (G), ubiquitin, and NAD+. Phosphoribosylation and ADP-ribosylation by SdeA WT and SdeA H284A mutant, respectively, could be unambiguously localized to Arg42 by extensive ETD fragmentation.

the phosphoribose would form a bridge between Arg42 of Ub and substrate. We tested whether the PDE activity of SdeA is necessary for substrate ubiquitination by SdeA. The SdeA PDE mutant H284A failed to both auto-ubiquitinate SdeA and ubiquitinate Rab33b (Figure S2A). This indicates that the cleavage of

1638 Cell 167, 1636–1649, December 1, 2016

the pyrophosphate bond in ADP-ribosylated Ub is an essential step in the Ub transfer reaction mediated by SdeA. To gain insight into the chemical nature of SdeA-mediated Ub linkage and identify the modification sites, we performed trypsin digestion and used mass spectrometry to characterize the branched

B

A

D

C

E S9 8 S9 4A 8 S9 6A 8 S9 4,9 36 86 ,9 A 37 ,9 84

,9

86

A

F

G

(legend on next page)

Cell 167, 1636–1649, December 1, 2016 1639

peptides resulting from the covalent attachment of the Ub peptide bearing Arg42 to Rab33b and SdeA peptides. We analyzed the peptides by high-resolution ETD to preserve the phosphoribose bridge and thus allow mapping of modification sites on substrate proteins. We discovered serine as the modified residue on target proteins. In all cases, serine residues of SdeA and Rab33b were bridged to Arg42 of Ub via a phosphoribosyl linker. Specifically, we mapped five serine sites, one in Rab33b (S154) (Figure 2A) and four in the C-terminal region of the SdeA mART domain (S936, S984, S986, S1005) (Figures 2C and S2B–S2D). Accordingly, mutations of several modified serines in SdeA resulted in reduced auto-ubiquitination (Figure 2E, lanes 1 to 11). However, mutating serine 154 of Rab33b did not affect the amount of ubiquitinated Rab33, indicating that there are other sites in Rab33b that are ubiquitinated apart from S154. Next, we set to determine the chemical nature of the linkage between the phosphoribosyl group and the substrate proteins. We reasoned that the phosphate group of phosphoribose is attached to the side chains of target serine residues. To test this hypothesis, we performed a different type of mass spectrometric analysis, called collision-induced dissociation (CID). Whereas ETD allows site mapping by preserving the phosphoribosyl linker, CID induces its cleavage, which we reasoned would provide information on its attachment chemistry. In high-resolution CID spectra of Ub-linked SdeA and Rab33b peptides, fragment ions corresponding to phosphorylated as well as dehydrated forms of the substrate peptide show that SdeA links phosphoribosyl-Ub via a phosphodiester bond to the hydroxyl group of serine residues in its substrates (Figures 2B and 2D). Next, we hypothesized that the pyrophosphate bond cleavage in ADP-ribosylated Ub and the attachment of phosphoribosylated Ub to SdeA substrates are coupled, meaning they take place simultaneously in the active site of the SdeA PDE domain. To test this, we performed in vitro ubiquitination reactions with DNC SdeA WT and H284A mutant in the presence of either ADP-ribosylated or phosphoribosylated Ub, without adding NAD+. ADP-ribosylated and phosphoribosylated Ub were generated as illustrated in Figure S2E and analyzed using phosphoprotein stain (Figure S2F). SdeA carried out auto-ubiquitination and ubiquitination of Rab33b only in the presence of ADP-ribosylated Ub but not in the presence of phosphoribosylated Ub in a PDE domain-dependent manner (Figure 2F). This indicates that

pyrophosphate bond cleavage and ligation of phosphoribosylated Ub to substrate serines are not independent but coupled processes performed by the PDE domain of SdeA. We examined the PDE domain model to find SdeA residues involved in ligation of phosphoribosylated Ub to substrate serines. The conserved D396 and K397 of SdeA are close to the PDE domain’s active site (Figure S2G). Mutating D396 and K397 but not E395 to alanines affected auto-ubiquitination of SdeA (Figure 2E, lanes 12 to 14). Our results provide a deeper understanding of the ubiquitination mechanism by SdeA, where Ub is first ADP-ribosylated on Arg42 by the mART domain. This is followed by the cleavage of the pyrophosphate bond between the a and the b phosphates in ADP-ribosylated Ub coupled to the attachment of the b phosphate to a substrate serine (Figure 2G). Interestingly, a few examples exist where Ub is attached to non-lysine residues such as cysteine, serine, and threonine of substrate proteins via an esterification reaction, and this modification is dependent on the canonical Ub cascade enzymes (Wang et al., 2012). This specific phosphoribosyl-bridged attachment of ubiquitin to serine residues of substrates has not been observed previously and therefore represents a unique biochemical linkage of Ub. Phosphoribosylation of Ub Impairs the Conventional Ubiquitination Cascade Ubiquitination of cellular Rab proteins is the only function of SdeA so far described, but this function does not sufficiently explain its toxic effects in yeast or its strong effect on intracellular bacterial survival (Bardill et al., 2005; Havey and Roy, 2015; Jeong et al., 2015; Qiu et al., 2016). We hypothesized that phosphoribosylation of Ub may have broader consequences in cells. We first tested whether SdeA actively ADP-ribosylates or phosphoribosylates Ub. Top-down mass spectrometry analysis of undigested Ub treated with DNC SdeA revealed that both SdeA WT and H284A mutant proteins can modify, by phosphoribose and ADP-ribose respectively, all Ub molecules in the reaction mixture (Figure 3A). In addition, analysis of our in vitro Ub transfer assays with SdeA revealed that the entire Ub pool is modified regardless of whether Rab33b is present or not, in an NAD+-dependent manner as seen by native-PAGE of the reaction components (Figure 3B). This showed that the modification of Ub is not merely an intermediate step in the ubiquitination of Rab33b but rather represents the primary action of SdeA,

Figure 2. Mechanism of Ubiquitination by SdeA (A) SdeA-catalyzed attachment of ubiquitin to Rab33b was analyzed after FASP digestion by high-resolution ETD fragmentation. (B) By high-resolution CID fragmentation, preferential cleavage of phosphoester bonds was induced. The resulting spectrum contained the Rab33b 154–165 peptide but only in dehydrated (m/z 1252.68) and phosphorylated (m/z 1350.67) states. (C) High-resolution ETD fragmentation data of protease-digested reaction products of NAD+-dependent autoubiquitination of SdeA. See also Figures S2B–S2D. (D) CID fragmentation spectrum of SdeA-ubiquitin cross-linked peptide does not show the intact SdeA peptide (m/z 563.30) but instead its dehydrated (m/z 545.29) and phosphorylated (m/z 643.27) states. (E) In vitro ubiquitination reactions with various SdeA serine mutants and the Rab33b S154A mutant. Altered/decreased ubiquitination pattern compared to the respective WT protein indicates that the mutated residue serves as an ubiquitin attachment site. See also Figure S2G. (F) Testing the ability of SdeA to auto-ubiquitinate and ubiquitinate Rab33b in the presence of ADP-ribosylated ubiquitin (Ubiquitin-ADP ribose) or phosphoribosylated ubiquitin (Ubiquitin-Phosphoribose). Rab33b plus SdeA WT or H284A mutant was incubated with purified ADP-ribosylated ubiquitin or phosphoribosylated ubiquitin. See also Figures S2E and S2F. (G) Stepwise mechanism model of SdeA-mediated ubiquitination. In the first step of the reaction, SdeA ADP-ribosylates Arg42 of ubiquitin by consuming an NAD+ molecule and releasing nicotinamide. Next, cleavage of the pyrophosphate bond in ADP-ribosylated ubiquitin by the PDE domain of SdeA results in the formation of phosphoribosylated ubiquitin and AMP release. This pyrophosphate bond cleavage is coupled to the attachment of phosphoribosylated ubiquitin to the hydroxyl group of substrate serines through a phosphodiester bond.

1640 Cell 167, 1636–1649, December 1, 2016

A

B

C

D

Figure 3. SdeA Phosphoribosylates Ubiquitin and Inactivates the Ubiquitin System (A) After modification reactions with SdeA WT, with SdeA H284A, and from control samples, lacking either SdeA or NAD+, ubiquitin was isolated and analyzed by ESI mass spectrometry. Differentially modified ubiquitin as well as unmodified ubiquitin were found in different charge states, so the spectra were deconvoluted with Xtract for further analysis. After treatment with SdeA-WT,
15% of ubiquitin was ADP-ribosylated (ADPR-Ubiquitin), and the rest was phosphoribosylated (P-Rib-Ubiquitin). In contrast, after incubation with PDE mutant SdeA H284A, only 1% of ADPR-Ub was processed to P-Rib-Ub. In control samples, no modified Ub was detectable. (See also Figure S1B). (B) Quantitative analysis of in vitro Ub modification assays. Indicated SdeA proteins were incubated with excess amounts of ubiquitin in the presence or absence of Rab33b. The reaction mixtures were subjected to SDS-PAGE (upper panel) and native-PAGE (lower panel). Ubiquitin modification can be monitored through the altered electrophoretic mobility of modified ubiquitin in native-PAGE. (C) The activity of Parkin was tested in an in vitro ubiquitination assay using either WT ubiquitin, phosphoribosylated ubiquitin (produced by treatment with WT SdeA), or ADP-ribosylated ubiquitin (produced by treatment with H284A SdeA). Reaction mixtures were separated by SDS-PAGE, and poly-Ub chain formation was visualized by Coomassie (SDS gel, upper panel) and western blotting using an anti-ubiquitin antibody (lower panel). See also Figures S3A and S3B. (D) Effect of SdeA on an ongoing ubiquitination reaction was tested. SdeA was added after 10 min of ubiquitination reaction. E1-Ube1, E2-UbcH5a, E3-Trim56 were used in the in vitro ubiquitination reaction. The reaction mixtures were subjected to SDS-PAGE/western blotting and probed with Ub antibody.

whereas only a subset of the modified host Ub pool is utilized to ubiquitinate substrates such as Rab33b. As several post-translational modifications of Ub are known to regulate various aspects of the Ub system (Herhaus and Dikic, 2015), we sought to identify potential effects of the SdeA-mediated Ub modifica-

tion on the host Ub system. To this end, we performed an in vitro Ub-ligase activity assay with the RBR-type E3 ligase Parkin. The use of both phosphoribosylated and ADP-ribosylated Ub resulted in complete inhibition of Parkin-mediated ubiquitination (Figure 3C). Similar results were obtained with two

Cell 167, 1636–1649, December 1, 2016 1641

unrelated Ub ligases, a RING-type (TRIM56) and a HECT-type (Salmonella effector SopA) E3 ligase (Figures S3A and S3B). SdeA also completely abrogated Ub chain synthesis when added to an ongoing in vitro ubiquitination reaction (Figure 3D). In this experiment, the SdeA full-length protein can reverse Ub chain synthesis using both DUB and mART domains. The SdeA DNC construct lacking the DUB domain leaves already assembled poly-Ub chains intact yet inhibits the fresh incorporation of mono-Ub into chains as seen from the increased levels of unused mono-Ub compared to the control reaction without SdeA. We reasoned that the lack of activity of three different classes of E3 ligases with SdeA-modified Ub points toward defective E1- or E2-related steps of the Ub reaction cascade. To understand the molecular basis of Ub system shutdown by SdeA, we determined the crystal structure of phosphoribosylated Ub at 1.8 A˚ resolution (Figure 4A and Table S1). Electron density corresponding to the ribosyl moiety on Arg42 of Ub was visible in two (chain B and chain C) out of the three molecules in the asymmetric unit (Figure S4A). However, electron density for the phosphate group of the phosphoribosyl moiety was missing. We speculate that the unstable phosphate moiety attached to the ribosyl group is lost due to radiation damage during diffraction data collection. Comparison of the WT Ub structure (PDB: 1UBQ) with the structure of the phosphoribosylated form of Ub revealed that the side chains of Arg42 and Arg72 are shifted from their default positions (Figure 4A). This is a consequence of the side chain of Arg72 making polar contacts with the ribosyl moiety attached to Arg42. A detailed analysis of the structure revealed that chain A (non-modified Ub) adopts a ‘‘loop-out’’ state as seen in most Ub structures (Hospenthal et al., 2013) (Figure S4B), where Leu8 in the flexible loop spanning the b1 and b2 strands is flipped outward and interacts with the Ile44 hydrophobic patch. In contrast, both chain B and chain C are in a ‘‘loop-in’’ conformation as seen in the distal Ub of Lys6-linked diUb. Whereas Leu8 of chain B is not resolved, Leu8 of chain C interacts with the Ile36 hydrophobic patch in the Ub molecule (Figure S4B). Interestingly, both Arg42 and Arg72 have been previously implicated in Ub activation by E1 (Burch and Haas, 1994; Duerksen-Hughes et al., 1987). To understand the role of Arg42 and Arg72 in SdeA-mediated modification of Ub, we performed Ub modification assays with R42A, R72A, and R42_72A Ub mutants. As probed by streptavidin-HRP, phosphoprotein staining, and native-PAGE, R42A Ub showed no modification upon treatment with SdeA (Figures 4B and S4C). Surprisingly, mutating Arg72 also completely abolished Ub modification by SdeA. Accordingly, Arg42 and Arg72 mutants failed to be substrates of SdeA auto-ubiquitination (Figure 4B). We speculate that Arg72 plays a more important role in the catalysis by SdeA than just stabilizing the phosphoribose on Arg42 as seen in the crystal structure. Future structural studies on SdeA alone and in complex with Ub will shed light on the role of Arg72 in Ub modification by SdeA. Arg42 and Arg72 are also directly involved in ionic interactions with E1 as seen in yeast Uba1-Ub complex structure (Lee and Schindelin, 2008). Aligning the phosphoribosylated Ub structure with the structure of Ub bound to Uba1 demonstrated major steric clashes between the modified Ub and the E1 (Figure S4D). We used an enzyme-coupled spectrophotometric assay to measure the kinetics of pyrophosphate

1642 Cell 167, 1636–1649, December 1, 2016

(PPi) release during the Ub activation reaction with E1 and Ub- or SdeA-modified Ub, respectively. In agreement with the structural analysis of SdeA-modified Ub, both phosphoribosylated Ub and ADP-ribosylated Ub exhibited markedly reduced PPi release compared to WT Ub in Ub activation reactions (Figure 4C). Also, E1 was preferentially linked to fluorescein-labeled WT Ub via a thioester bond as opposed to the phosphoribosylated Ub, indicating the incompatibility of the latter in E1-mediated Ub activation (Figure S4E). Furthermore, SdeA-modified Ub cannot be efficiently transferred from E1 to E2 via transthiolesterification (Figure 4D), and the E3-mediated Ub discharge from E2 is compromised as well (Figure 4E). Cellular Effects of Ub Phosphoribosylation In order to analyze the functional consequences of SdeA-mediated Ub modification in cells, we developed a strategy to assay phosphoribosylation of Ub in complex mixtures such as bacterial and human cell lysates. While screening various Ub antibodies, we noticed that the Ub antibody from Cell Signaling Technology (referred to as CS-Ub antibody) recognizes both SdeA-modified and -unmodified Ub with the same efficiency, whereas the antibody from Abcam (referred to as abcam-Ub antibody) selectively recognizes only the unmodified Ub and fails to recognize the modified Ub (Figure 5A). Based on this observation, we first checked whether endogenous SdeA from Legionella possesses Ub modification activity similar to that of recombinantly expressed and purified proteins. Legionella WT strain (lp02) and various mutants were grown in AYE broth until early stationary phase. Ub and NAD+ were added to the clarified bacterial lysates, and Ub modification was probed with the antibody pair strategy described above. Lysates of the lp02 but not the DSidE strains were able to modify Ub, indicating that SidE family members are necessary for modification of Ub (Figure 5B). Importantly, complementing the DSidE strain with WT SdeA but not mART mutant SdeA restores the Ub modification activity, indicating that the endogenous Legionella proteins SdeA and other SidE family members modify Ub in an mART domaindependent manner. We then checked whether ectopically expressed SdeA could modify the cellular Ub pool and affect ubiquitination in cells. Expression of SdeA WT or H284A mutant led to the modification of Ub in cells, but the mART mutant did not (Figure 5C). We also detected the phosphoribosylation and ADPribosylation of endogenous Ub in cells expressing WT and H284A mutant SdeA, respectively, by mass spectrometry (Figures S5A and S5B). The combination of SdeA modification of Ub and the constant recycling of poly-Ub chains in cells should eventually lead to accumulation of modified free-mono Ub that is not able to form chains. Comparing SdeA WT and SdeA H284A, we expected an increase in mono-Ub in H284A samples because the WT SdeA uses Ub to ubiquitinate substrates and itself. To monitor small changes in the levels of mono-Ub we performed fluorescence-activated cell sorting (FACS) of GFP-SdeAtransfected cells. We then compared mono-Ub levels in the cells expressing GFP-SdeA WT, those expressing GFP-SdeA H284A, and non-transfected cells (Figure S5C). As reasoned above, we only observed an increase in the levels of mono-Ub in cells transfected with GFP-SdeA H284A. We next checked whether modified Ub in cells affects ubiquitination similar to the in vitro

A 3.3 Å

se -ribo

R42

+ _

+ _

_

Ubiquitin (1UBQ) Phosphoribosylated Ubiquitin

+ _

+ _

+ _

_

_

_ _

B

R72 r.m.s.d=0.5Å

MW kDa

R42

C

_

+ _ _

+ _

_

+ _

+ _

_

_

_

+

+

+

_

+ _

_

ΔNC SdeA WT ΔNC SdeA H284A

_ _

+ _ _

+ _

_ _

+ _ _

+

+

_

_

Ubiquitin R72A

_

_

_

+

+

Ubiquitin R42_72A

+

+

+

+

+

Biotin-NAD

245 180 135 100 75 63 48 35 25 20 17 11

Ubiquitin

PPi released (nmoles)

11

Ubiquitin (Pro-Q Diamond phosphoprotein stain)

E1+ATP+Ub(ΔNC SdeA WT modified) E1+ATP+Ub(ΔNC SdeA H284A modified)11

4

Ubiquitin R42A

(Coomassie)

E1+ATP+Ub

6

Ubiquitin WT

ΔNC SdeA

8

Ubiquitin (α-Biotin)

E1+ATP+Ub (no DTT) E1+ATP(no Ub)

2

0 0

D

+

+ + + _ MW _ kDa 245 180 135 100 75 63 48 35 25 20 17 11

20 + + + + _

+ + + + +

40 Time (min)

NAD E2 E1 ATP SdeA FL WT

60

80

Discharge of E2 loaded with wildtype ubiquitin

E MW kDa 20 17

0

1

2

5 10 30

Discharge of E2 loaded with phosphoribosylated ubiquitin 0

1

2

5

10 30 Time after E3 addition (min) E2 (HIS)-Ub E2 (HIS) (α-HIS)

20 SdeA FL WT E1 (Ube1)

E2 (HIS)-Ub (α-Ubiquitin)

(Coomassie) Ub-loaded E2 E2 (UbcH7) Ubiquitin

Figure 4. Crystal Structure of SdeA-Modified Ubiquitin and Its Implications (A) The crystal structure of phosphoribosylated ubiquitin (shown in yellow) overlaid onto the WT ubiquitin structure (shown in green, PDB: 1UBQ). Phosphoribosylated Arg42 and the interacting Arg72 of the modified ubiquitin structure are shown. See also Figure S4A. (B) Purified ubiquitin mutants and WT were incubated with SdeA WT and H284A mutant in the presence of biotin-NAD+. The reaction mix was subjected to SDSPAGE followed by Coomassie staining (upper-most panel), phosphoprotein staining with Pro-Q Diamond stain (middle panel), and streptavidin-HRP blotting (lower most panel). See also Figure S4C. (C) A pyrophosphate (PPi) release assay was performed with ubiquitin-activating enzyme E1 and WT ubiquitin or modified ubiquitin. The plot shows nanomoles of PPi released over time. See also Figure S4D and S4E. (D) Effect of SdeA-mediated ubiquitin modification on ubiquitin transfer between E1 and E2. E1, E2, and WT ubiquitin or phosphoribosylated ubiquitin were incubated with ATP and NAD+. Transfer of ubiquitin to the catalytic site of E2 was monitored by SDS-PAGE of reaction mixture components followed by Coomassie staining. (E) E2 (HIS-UbcH5a) discharge assay in presence of either unmodified ubiquitin or phosphoribosylated ubiquitin. E2-Ub discharge was induced by addition of the E3 ligase Trim56. The reaction was stopped at the indicated time points and reaction mixtures were analyzed by SDS-PAGE and western blotting.

Cell 167, 1636–1649, December 1, 2016 1643

_

+

+

+

NAD α-Ubiquitin CS-Ub

11

_

MW kDa 10

A α-Ubiquitin CS-Ub

Flag-HA-SdeA

MW kDa 150 100 10

α-Ubiquitin abcam-Ub 10

α-Ubiquitin abcam-Ub 10

250 150

50

100 75

37

_

ΔN C

MW kDa 11

C

H 28 4A EE /A A

W T W T H 28 4A EE /A A

Legionella lysate

W T

B

ΔNC SdeA

lp 02 ΔS id E ΔS id E+ ΔS Sd eA id E+ W T Sd eA EE /A

A

α-HA α-Ubiquitin CS-Ub α-Ubiquitin abcam-Ub

α-Actin

(Amido black)

50 37 25 20 15 10

245 180 135 100 75 63 48

245 180 135 100 75 63 48 35 25 20 17 11

4 EE A /A A

T

28 H

_

W

4 EE A /A A

T

_

HA-SdeA

28

HA

H

H

28

4 EE A /A A

Ubiquitin

HA-SdeA

W

180

W

MW _ kDa

T

HA-SdeA

D

245 180 135 100 75 63

35

48 35 25 20 17

25 20 17 11

FK2 (Ubiquitin chains) 48

Actin

K63 chains

K48 chains

Figure 5. SdeA Phosphoribosylates Ubiquitin and Blocks Ubiquitination in Cells (A) Unmodified ubiquitin and SdeA-modified ubiquitin are differentially recognized by two commercial ubiquitin antibodies. Antibody from cell signaling (CS-Ub) recognizes both modified and unmodified forms of ubiquitin equally well, whereas the antibody from Abcam (abcam-Ub) selectively recognizes unmodified ubiquitin. Ubiquitin was modified by WT or the indicated mutants of SdeA; reaction mixtures were separated by SDS-PAGE and probed with the abovementioned antibodies. (B) WT (lp02) and mutant Legionella strains were grown until early stationary phase (OD600 = 3), normalized, and lysed in non-denaturing conditions (see STAR Methods). Clarified lysate was used in a reaction mix with ubiquitin and NAD+. Ubiquitin modification was probed using CS-Ub and abcam-Ub antibodies. (C) HEK293T cells were transfected with WT and various mutants of Flag-HA-SdeA. Cell lysates were subjected to SDS-PAGE and probed with CS-Ub and abcam-Ub antibodies to monitor ubiquitin modification. Analysis of ubiquitin signal from both antibodies depicts the dependence of ubiquitin modification on the mART motif of SdeA. See also Figures S5A and S5B. (D) Levels of poly-ubiquitination, K63 ubiquitination, and K48 ubiquitination were assayed in total cell lysates of HEK293T cells transiently expressing SdeA WT, H284A, and EE/AA mutants. See also Figures S5C and S5D.

scenario (Figure 3). Lysates of cells ectopically expressing SdeA WT or H284A mutant or EE/AA mutant were probed for total ubiquitination, K63-Ub chains, and K48-Ub chains using the

1644 Cell 167, 1636–1649, December 1, 2016

respective antibodies (Figure 5D). Ub modification by SdeA severely affected ubiquitination of cellular proteins (Figure 5D). The effect of SdeA seems to be more prominent on K63 chains

CCCP 10µM 1.5 hr

GFP-SdeA

A

10µm

Poly-Ub

10µm

GFP

10µm

GFP-SdeA EE/AA

Mitotracker

Mitotracker ***

B

***

***

100

***

80 10000

% of cells with nuclear p65

***

5000

60 *

40 **

20

N

Sd eA

Sd eA Δ

+ CCCP (10µM, 90 mins)

D

C

W T

H 28 4A

no

Sd eA

TN

F

EE /A A

ΔN

C

Sd eA

Sd eA

Sd eA

H 28 4A

W T

-

Sd eA

-

EE /A A

0

0

Sd eA

Ubiquitination intensity on mitochondria (AU)

*

C

*** 15000

Poly-Ub

GFP

+ TNF

E Flag-HA-SdeA

4A

A

28

/A eA Sd

eA

H

EE

T Sd

Sd

eA

W

ve y Em

MW kDa 100

pt

/A A EE

4A 28 H

T W

Em

pt y

ve ct o

ct or

r

Yeast W303 strain

Glucose HIF1-α Galactose

48 Actin 35

150

HA

(legend on next page)

Cell 167, 1636–1649, December 1, 2016 1645

than on K48 chains, perhaps due to the previously reported specificity of the SdeA DUB domain toward K63 chains (Sheedlo et al., 2015). Proteotoxic stress induced by inhibition of proteasomal degradation leads to a decrease in the levels of ubiquitinated histone H2A as various cellular processes compete for the limited amount of free Ub (Dantuma et al., 2006). We checked whether SdeA-induced proteotoxic stress leads to changes in H2A ubiquitination. Similar to the condition of proteasomal inhibition, SdeA expression also led to decrease in H2A ubiquitination (Figure S5D). Given that Ub is central to many cellular processes including protein homeostasis, autophagy, and signaling, we next checked the effect of SdeA expression on such broad Ub functions in cells. Ubiquitination of the mitochondrial membrane protein fraction by the E3 Ub ligase Parkin is one of the crucial events leading to the autophagy of mitochondria (Geisler et al., 2010; Narendra et al., 2008; Ordureau et al., 2014). In cells with no SdeA transfection, CCCP treatment effectively induced ubiquitination at mitochondria (Figures 6A, 6B and S6A), whereas expression of WT SdeA or H284A mutant severely impaired ubiquitination at mitochondria. Levels of HA-Parkin were similar in all the experiments, indicating that the lack of ubiquitination is a direct consequence of SdeA present in cells (Figure S6B). The general background signal for Ub chains in the cytoplasm also decreased greatly in cells expressing either WT SdeA or the DNC construct of SdeA lacking the N-terminal DUB domain, indicating that SdeA’s inhibitory effect on the host ubiquitination system is mediated mainly through its mART domain (Figures 6A, 6B, and S6A). Accordingly, expression of the mART mutant did not lead to a strong decrease in ubiquitination at the mitochondria. In addition, SdeA expression also affected tumor necrosis factor-a (TNF-a)-dependent activation of the NF-kB pathway (Figure 6C). Ubiquitination of multiple substrates at various stages of TNF signaling is critical to signal transduction and eventual nuclear translocation of the transcription factor p65, leading to induction of its target gene expression (Newton and Dixit, 2012). Expression of SdeA WT, the H284A mutant, or the DNC construct severely affected p65 nuclear translocation, whereas expression of the mART mutant did not significantly affect the translocation of p65 to the nucleus (Figure 6C). We also demonstrated the effect of SdeA expression on proteasomal degradation of intracellular proteins such as hypoxia-inducible factor 1-a (HIF1-a). Under normoxic conditions, HIF1-a constantly undergoes ubiquitination by von Hippel-Lindau protein (pVHL) and subsequent degradation by the Ub pro-

teasome system (Kim and Kaelin, 2003). Expression of SdeA WT or H284A mutant but not the mART mutant led to HIF1-a stabilization in HeLa cells (Figure 6D). These results demonstrate that the SdeA-mediated modification of Ub is a potent and general inhibitor of the cellular Ub system and severely affects multiple Ub-dependent pathways. Our data indicate that SdeA has at least two functions: one is to ubiquitinate substrate proteins such as Rab33b, and the other is to phosphoribosylate Ub, leading to inactivation of the ubiquitin system. To determine which of these two functions are relevant for the observed toxicity of SdeA in yeast cells (Qiu et al., 2016), we performed yeast toxicity assays by transforming galactose-inducible WT SdeA or mART domain or PDE domain mutants into yeast (Figure 6E). WT SdeA was toxic to yeast as shown previously (Qiu et al., 2016). The mART mutant EE/AA is not toxic to yeast, but importantly, the H284A mutant that is defective in substrate ubiquitination but not in ADP-ribosylation of Ub (Figure 1F) is toxic to yeast (Figure 6E). This indicates that SdeA’s toxicity is a result of Ub modification rather than substrate ubiquitination. Chemical Reactions Underlying Serine Ubiquitination Since the initial discovery of ubiquitin as an ATP-dependent tag for proteasomal degradation (Ciehanover et al., 1978), a large body of literature has expanded the scope of Ub and demonstrated its versatility as a signaling molecule in crucial biological processes. So far, only one conjugation chemistry (conjugation of Ub to its substrate proteins through its C-terminal carboxyl) is known to underlie these different modes of actions of Ub. Our data unveil a second chemical reaction resulting in phosphoribosylation of Ub on a specific arginine that is coupled to substrate ubiquitination on a serine residue. This mechanism requires the successive actions of the mART and PDE domains of SdeA. First, the mART domain mediates the attachment of ADP-ribosel to Arg42 of Ub. This is followed by a PDE-mediated cleavage of the pyrophosphate bond between a and b phosphates of ADP-ribose, leading to the formation of phosphoribosylated Ub and the release of AMP. We propose that the cleavage of this energy-rich bond is essential for the Ub transfer reaction linking the phosphoribosyl moiety of Arg42 of Ub to a serine residue in the substrate. Modification of Ub that Blocks the Classical Ubiquitination Cascade Ub is known to undergo several post-translational modifications resulting in the regulation of various aspects of the Ub system

Figure 6. SdeA Impairs Multiple Ubiquitin-Dependent Cellular Processes (A) Detection of mitochondria and poly-Ub after mitophagy induction. HeLa cells stably expressing HA-Parkin and ectopically expressing WT or mutants of GFP-tagged SdeA were treated with 10 mM CCCP for 90 min followed by staining with MitoTracker and FK2 (poly-Ub) antibody. GFP-positive cells are marked with a white arrow. See also Figures S5 and S6. (B) Quantitative analysis of CCCP-induced poly-ubiquitination at mitochondria in presence of SdeA WT or various SdeA mutants. Data are represented as mean ± SEM (see STAR Methods). (C) Monitoring SdeA effect on p65 translocation to nucleus. HeLa cells expressing WT and various mutants of SdeA were treated with TNF-a for 30 min followed by staining with anti-p65 and DAPI. Number of cells with p65 nuclear translocation was quantified in all the samples. Data are represented as mean ± SEM (see STAR Methods). (D) Degradation of HIF1-a under normoxic conditions was probed in HeLa cells expressing WT and various mutants of HA-Flag-SdeA. (E) Galactose-inducible CEN plasmids containing SdeA WT or SdeA EE/AA or SdeA H284A were transformed into yeast W303 strain. Transformed clones were grown and maintained in glucose-containing selective media (URA). Five microliters of 2 OD units cells were spotted on both glucose- and galactosecontaining URA plates. Impaired growth on galactose media indicates toxicity of the induced construct.

1646 Cell 167, 1636–1649, December 1, 2016

(Herhaus and Dikic, 2015). Deamidation of glutamine 40 both in the Ub-like molecule NEDD8 and in Ub by the Burkholderia pseudomallei effector molecule CHBP has been reported to downregulate both the NEDD8 and Ub systems (Cui et al., 2010). Deamidation of Ub does not alter Ub activation or Ub transfer from E1 to E2 but rather blocks the E3-driven discharge of Ub-loaded E2. Here, we show that phosphoribosylation of Ub impairs all three steps of the classical ubiquitination cascade. In line with this, chemical modification or mutation of Arg42 and 72 was previously shown to affect Ub activation (Burch and Haas, 1994; Duerksen-Hughes et al., 1987). Furthermore, non-covalent interactions between Ub and the E2 enzyme CDC34 in its loaded state were shown to create a specific structural architecture that is amenable for deprotonation of substrate lysine and subsequent ubiquitination or chain elongation (Saha et al., 2011). Structural studies indicate that Arg42 is involved in these crucial non-covalent interactions with E2, and phosphoribosylation likely disturbs these E2-Ub interactions, thereby affecting E2 discharge. Taken together, phosphoribosylation of Ub causes defects at multiple steps of the ubiquitination cascade that accumulate to eventually cause complete inhibition of the ubiquitination system in vitro and in cells. Intriguingly, while the host Ub system is shut down, SdeA can utilize the Ub pool to ubiquitinate its preferred substrates. In this way, Legionella can efficiently control the host ubiquitinome. Importance in Bacterial Infection During bacterial infection SdeA appears to have three different effects on the host Ub system: serine ubiquitination of Rab GTPases and potentially other substrates; cleavage of host Ub chains via its DUB domain; and phosphoribosylation of Ub, which impairs the host Ub system. These functions could be spatially and temporally regulated around the Legionella-containing vacuole (LCV) (Sheedlo et al., 2015). Interestingly, several Legionella effectors require an active host Ub system for promotion of intracellular bacterial growth and replication (Xu and Luo, 2013). For example, AnkB assembles K48-linked Ub chains on proteins residing on LCVs whose proteasomal degradation provides bacteria with amino acid resources (Price et al., 2011). Multiple F box and U box domain-containing Ub ligase effectors target host proteins to promote intracellular replication of Legionella (Ensminger and Isberg, 2010; Hsu et al., 2014; Kubori et al., 2008). In light of this, constitutive action of the SidE family of proteins (including SdeA) during infection would potentially negatively affect the function of other bacterial effectors dependent on the conventional ubiquitination system. Interestingly, Legionella strain SuperDP170 (lacking all four members of the SidE protein family plus SidJ), when complemented with overexpression of SdeA, exhibits a dramatic intracellular growth defect that is comparable to the dotA mutant strain (lacking the type IV secretion system). This is consistent with the previously raised idea that prolonged activation of SdeA might be also unfavorable for intracellular growth of bacteria (Jeong et al., 2015). Legionella overcomes this problem by inactivating SidE family effectors in the early stages of infection by using another effector, SidJ (Jeong et al., 2015), but it probably allows its activation in later stages of infection, promoting RabGTPase ubiquitination and preventing fusion of the LCV with the lysosome

(Qiu et al., 2016). Further efforts need to be taken to elucidate how these different activities, exerted simultaneously or consequently, can target the host at specific cellular locations and/or at distinct stages of infection. SdeA-like Enzymes in Mammalian Cells Until now, SdeA was considered to be a unique bacterial virulence factor that is able to promote phosphoribosylation of Ub. Yet, as pathogenic bacteria often acquire and exchange toxins and other effector proteins via horizontal and vertical gene transfer, it is possible that other species harbor proteins with similar capabilities. Moreover, this type of Ub modification could also be utilized during distinct (patho)physiological processes in mammalian cells. Interestingly, protein ADP-ribosylation can be processed into phosphoribosylation in vitro via two families of enzymes: nucleoside diphosphate-linked moiety X (Nudix) hydrolases including the human Nudix-type motif 16 (hNUDT16) and nucleotide pyrophosphatase/phosphodiesterase (NPP) proteins (Daniels et al., 2015b; Palazzo et al., 2016, 2015). Another report identified several arginine phosphoribosylation sites on protein substrates in mouse liver (Matic et al., 2012). Despite all this evidence, the functionality of protein phosphoribosylation remains unknown. The future identification of a mammalian enzymatic system that phosphoribosylates Ub may reveal novel and exciting roles for Ub in biology and medicine. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d

d

d

KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING EXPERIMENTAL MODEL AND SUBJECT DETAILS B Microbe strains B Yeast strain B Cell lines METHOD DETAILS B Protein purification B Purification, crystallization, and structure determination of phosphoribosylated ubiquitin B Ubiquitin modification and Rab33 ubiquitination assays B In vitro ubiquitination reactions with E1, E2, and E3 B Mitophagy B TNF-a experiments B Probing HIF-1a levels B Yeast survival assay B Pyrophosphate release assay B Fluorescent ubiquitin-E1 loading assay B E2 loading and discharge assays B Mass spectrometric analysis of modified ubiquitin B Mass spectrometric analysis of ubiquitin cross-linked to Rab and SdeA B AMP release assay B Modeling SdeA PDE domain B Quantification and statistical analysis DATA AND SOFTWARE AVAILABILITY

Cell 167, 1636–1649, December 1, 2016 1647

SUPPLEMENTAL INFORMATION Supplemental Information includes six figures and one table and can be found with this article online at http://dx.doi.org/10.1016/j.cell.2016.11.019. AUTHOR CONTRIBUTIONS S.B. and I.D. conceived the study and initiated the project. I.D. supervised the project. S.B. designed and performed biochemical experiments, protein purification, and yeast work. S.K. performed the purification, crystallization, and structure determination of phosphoribosylated Ub. Y.L. performed the cell biology and the Legionella experiments with the help of S.B.; F.B. and T.C. performed mass spectrometry experiments with the supervision of I.M. and I.D.; S.B. and I.D. wrote the paper with contributions from all the authors. All authors read and approved the final manuscript. ACKNOWLEDGMENTS

Daniels, C.M., Ong, S.-E., and Leung, A.K.L. (2014). Phosphoproteomic approach to characterize protein mono- and poly(ADP-ribosyl)ation sites from cells. J. Proteome Res. 13, 3510–3522. Daniels, C.M., Ong, S.-E., and Leung, A.K.L. (2015a). The promise of proteomics for the study of ADP-ribosylation. Mol. Cell 58, 911–924. Daniels, C.M., Thirawatananond, P., Ong, S.-E., Gabelli, S.B., and Leung, A.K.L. (2015b). Nudix hydrolases degrade protein-conjugated ADP-ribose. Sci. Rep. 5, 18271. Dantuma, N.P., Groothuis, T.A.M., Salomons, F.A., and Neefjes, J. (2006). A dynamic ubiquitin equilibrium couples proteasomal activity to chromatin remodeling. J. Cell Biol. 173, 19–26. Di Fiore, P.P., and von Zastrow, M. (2014). Endocytosis, signaling, and beyond. Cold Spring Harb. Perspect. Biol. 6, a016865–a016865. Duerksen-Hughes, P.J., Xu, X.X., and Wilkinson, K.D. (1987). Structure and function of ubiquitin: evidence for differential interactions of arginine-74 with the activating enzyme and the proteases of ATP-dependent proteolysis. Biochemistry 26, 6980–6987.

We thank Zhao-Qing Luo for the kind gift of Legionella strains, Marcel Hahn for molecular cloning of SdeA constructs, Evgenij Fiskin for the inducible HA-Parkin Hela cell line, Dr. Harald Hofbauer for gratuitous help in yeast work, Elena Veshkova for her technical assistance, and Sofı´a Rodrı´guez Go´mez for her assistance in protein purification and biochemistry. We are thankful to Daniela Hoeller and Kerstin Koch for critical reading and commenting on the manuscript and Craig Wenger for generously providing a new version of Morpheus implementing Neutral Loss Scoring. We acknowledge the staff of Swiss Light Source (SLS) for their support during diffraction data collection. Work in the I.D. laboratories was supported by the DFG-funded Collaborative Research Centre on Selective Autophagy (SFB 1177); by the DFG-funded Cluster of Excellence ‘‘Macromolecular Complexes’’ (EXC115); by the DFG-funded SPP 1580 program ‘‘Intracellular Compartments as Places of Pathogen-Host-Interactions’’; and by the LOEWE program Ubiquitin Networks (Ub-Net) and the LOEWE Center for Gene and Cell Therapy Frankfurt (CGT), both funded by the State of Hesse/Germany. Work in the I.M. laboratory was supported by the DFG-funded Cluster of Excellence ‘‘Cellular Stress Responses in Aging-Associated Diseases’’ (EXC229).

Emsley, P., and Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132.

Received: September 28, 2016 Revised: October 28, 2016 Accepted: November 10, 2016 Published: December 1, 2016

Havey, J.C., and Roy, C.R. (2015). Toxicity and SidJ-mediated suppression of toxicity require distinct regions in the SidE family of Legionella pneumophila effectors. Infect. Immun. 83, 3506–3514.

REFERENCES

Hershko, A., Ciechanover, A., and Varshavsky, A. (2000). The ubiquitin system. Nat. Med. 6, 1073–1081.

Adams, P.D., Afonine, P.V., Bunko´czi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung, L.-W., Kapral, G.J., Grosse-Kunstleve, R.W., et al. (2010). PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221. Alva, V., Nam, S.-Z., So¨ding, J., and Lupas, A.N. (2016). The MPI bioinformatics Toolkit as an integrative platform for advanced protein sequence and structure analysis. Nucleic Acids Res. 44 (W1), W410–W415. Bardill, J.P., Miller, J.L., and Vogel, J.P. (2005). IcmS-dependent translocation of SdeA into macrophages by the Legionella pneumophila type IV secretion system. Mol. Microbiol. 56, 90–103. Burch, T.J., and Haas, A.L. (1994). Site-directed mutagenesis of ubiquitin. Differential roles for arginine in the interaction with ubiquitin-activating enzyme. Biochemistry 33, 7300–7308. Ciehanover, A., Hod, Y., and Hershko, A. (1978). A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes. Biochem. Biophys. Res. Commun. 81, 1100–1105. Cox, J., and Mann, M. (2008). MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372. Cui, J., Yao, Q., Li, S., Ding, X., Lu, Q., Mao, H., Liu, L., Zheng, N., Chen, S., and Shao, F. (2010). Glutamine deamidation and dysfunction of ubiquitin/NEDD8 induced by a bacterial effector family. Science 329, 1215–1218.

1648 Cell 167, 1636–1649, December 1, 2016

Ensminger, A.W., and Isberg, R.R. (2010). E3 ubiquitin ligase activity and targeting of BAT3 by multiple Legionella pneumophila translocated substrates. Infect. Immun. 78, 3905–3919. Finley, D. (2009). Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu. Rev. Biochem. 78, 477–513. Geisler, S., Holmstro¨m, K.M., Skujat, D., Fiesel, F.C., Rothfuss, O.C., Kahle, P.J., and Springer, W. (2010). PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat. Cell Biol. 12, 119–131. Go¨tze, M., Pettelkau, J., Schaks, S., Bosse, K., Ihling, C.H., Krauth, F., Fritzsche, R., Ku¨hn, U., and Sinz, A. (2012). StavroX–a software for analyzing crosslinked products in protein interaction studies. J. Am. Soc. Mass Spectrom. 23, 76–87. Grabbe, C., Husnjak, K., and Dikic, I. (2011). The spatial and temporal organization of ubiquitin networks. Nat. Rev. Mol. Cell Biol. 12, 295–307.

Herhaus, L., and Dikic, I. (2015). Expanding the ubiquitin code through posttranslational modification. EMBO Rep. 16, 1071–1083.

Hicks, S.W., and Gala´n, J.E. (2013). Exploitation of eukaryotic subcellular targeting mechanisms by bacterial effectors. Nat. Rev. Microbiol. 11, 316–326. Hospenthal, M.K., Freund, S.M.V., and Komander, D. (2013). Assembly, analysis and architecture of atypical ubiquitin chains. Nat. Struct. Mol. Biol. 20, 555–565. Hsu, F., Luo, X., Qiu, J., Teng, Y.-B., Jin, J., Smolka, M.B., Luo, Z.-Q., and Mao, Y. (2014). The Legionella effector SidC defines a unique family of ubiquitin ligases important for bacterial phagosomal remodeling. Proc. Natl. Acad. Sci. USA 111, 10538–10543. Hubber, A., and Roy, C.R. (2010). Modulation of host cell function by Legionella pneumophila type IV effectors. Annu. Rev. Cell Dev. Biol. 26, 261–283. Husnjak, K., and Dikic, I. (2012). Ubiquitin-binding proteins: decoders of ubiquitin-mediated cellular functions. Annu. Rev. Biochem. 81, 291–322. Jeong, K.C., Sexton, J.A., and Vogel, J.P. (2015). Spatiotemporal regulation of a Legionella pneumophila T4SS substrate by the metaeffector SidJ. PLoS Pathog. 11, e1004695. Kabsch, W. (2010). XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132. Kazlauskaite, A., Kondapalli, C., Gourlay, R., Campbell, D.G., Ritorto, M.S., Hofmann, K., Alessi, D.R., Knebel, A., Trost, M., and Muqit, M.M.K. (2014). Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65. Biochem. J. 460, 127–139.

Kim, W., and Kaelin, W.G., Jr. (2003). The von Hippel-Lindau tumor suppressor protein: new insights into oxygen sensing and cancer. Curr. Opin. Genet. Dev. 13, 55–60. Kubori, T., Hyakutake, A., and Nagai, H. (2008). Legionella translocates an E3 ubiquitin ligase that has multiple U-boxes with distinct functions. Mol. Microbiol. 67, 1307–1319. Lee, I., and Schindelin, H. (2008). Structural insights into E1-catalyzed ubiquitin activation and transfer to conjugating enzymes. Cell 134, 268–278. Leidecker, O., Bonfiglio, J.J., Colby, T., Zhang, Q., Atanassov, I., Zaja, R., Palazzo, L., Stockum, A., Ahel, I., and Matic, I. (2016). Serine is a new target residue for endogenous ADP-ribosylation on histones. Nat. Chem. Biol. Published online October 10, 2016. http://dx.doi.org/10.1038/nchembio.2180. Llosa, M., Roy, C., and Dehio, C. (2009). Bacterial type IV secretion systems in human disease. Mol. Microbiol. 73, 141–151. Maculins, T., Fiskin, E., Bhogaraju, S., and Dikic, I. (2016). Bacteria-host relationship: ubiquitin ligases as weapons of invasion. Cell Res. 26, 499–510. Matic, I., Ahel, I., and Hay, R.T. (2012). Reanalysis of phosphoproteomics data uncovers ADP-ribosylation sites. Nat. Methods 9, 771–772. Narendra, D., Tanaka, A., Suen, D.-F., and Youle, R.J. (2008). Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J. Cell Biol. 183, 795–803. Newton, K., and Dixit, V.M. (2012). Signaling in innate immunity and inflammation. Cold Spring Harb. Perspect. Biol. 4, a006049–a006049. Ordureau, A., Sarraf, S.A., Duda, D.M., Heo, J.-M., Jedrychowski, M.P., Sviderskiy, V.O., Olszewski, J.L., Koerber, J.T., Xie, T., Beausoleil, S.A., et al. (2014). Quantitative proteomics reveal a feedforward mechanism for mitochondrial PARKIN translocation and ubiquitin chain synthesis. Mol. Cell 56, 360–375. Palazzo, L., Thomas, B., Jemth, A.-S., Colby, T., Leidecker, O., Feijs, K.L.H., Zaja, R., Loseva, O., Puigvert, J.C., Matic, I., et al. (2015). Processing of protein ADP-ribosylation by Nudix hydrolases. Biochem. J. 468, 293–301. Palazzo, L., Daniels, C.M., Nettleship, J.E., Rahman, N., McPherson, R.L., Ong, S.-E., Kato, K., Nureki, O., Leung, A.K.L., and Ahel, I. (2016). ENPP1 processes protein ADP-ribosylation in vitro. FEBS J. 283, 3371–3388. Price, C.T.D., Al-Quadan, T., Santic, M., Rosenshine, I., and Abu Kwaik, Y. (2011). Host proteasomal degradation generates amino acids essential for intracellular bacterial growth. Science 334, 1553–1557. Pu, Y., Zhao, Z., Li, Y., Zou, J., Ma, Q., Zhao, Y., Ke, Y., Zhu, Y., Chen, H., Baker, M.A.B., et al. (2016). Enhanced efflux activity facilitates drug tolerance in dormant bacterial cells. Mol. Cell 62, 284–294. Qiu, J., Sheedlo, M.J., Yu, K., Tan, Y., Nakayasu, E.S., Das, C., Liu, X., and Luo, Z.-Q. (2016). Ubiquitination independent of E1 and E2 enzymes by bacterial effectors. Nature 533, 120–124. Ribet, D., and Cossart, P. (2015). How bacterial pathogens colonize their hosts and invade deeper tissues. Microbes Infect. 17, 173–183. Richter, B., Sliter, D.A., Herhaus, L., Stolz, A., Wang, C., Beli, P., Zaffagnini, G., Wild, P., Martens, S., Wagner, S.A., et al. (2016). Phosphorylation of OPTN by TBK1 enhances its binding to Ub chains and promotes selective autophagy of damaged mitochondria. Proc. Natl. Acad. Sci. USA 113, 4039–4044.

Rinaldo, S., Paiardini, A., Stelitano, V., Brunotti, P., Cervoni, L., Fernicola, S., Protano, C., Vitali, M., Cutruzzola`, F., and Giardina, G. (2015). Structural basis of functional diversification of the HD-GYP domain revealed by the Pseudomonas aeruginosa PA4781 protein, which displays an unselective bimetallic binding site. J. Bacteriol. 197, 1525–1535. Rogov, V., Do¨tsch, V., Johansen, T., and Kirkin, V. (2014). Interactions between autophagy receptors and ubiquitin-like proteins form the molecular basis for selective autophagy. Mol. Cell 53, 167–178. Saha, A., Lewis, S., Kleiger, G., Kuhlman, B., and Deshaies, R.J. (2011). Essential role for ubiquitin-ubiquitin-conjugating enzyme interaction in ubiquitin discharge from Cdc34 to substrate. Mol. Cell 42, 75–83. Sansonetti, P.J. (2006). The bacterial weaponry: lessons from Shigella. Ann. N Y Acad. Sci. 1072, 307–312. Sheedlo, M.J., Qiu, J., Tan, Y., Paul, L.N., Luo, Z.-Q., and Das, C. (2015). Structural basis of substrate recognition by a bacterial deubiquitinase important for dynamics of phagosome ubiquitination. Proc. Natl. Acad. Sci. USA 112, 15090–15095. So¨ding, J., Biegert, A., and Lupas, A.N. (2005). The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 33, W244–W248. Storoni, L.C., McCoy, A.J., and Read, R.J. (2004). Likelihood-enhanced fast rotation functions. Acta Crystallogr. D Biol. Crystallogr. 60, 432–438. Ulrich, H.D., and Walden, H. (2010). Ubiquitin signalling in DNA replication and repair. Nat. Rev. Mol. Cell Biol. 11, 479–489. Vittal, V., Stewart, M.D., Brzovic, P.S., and Klevit, R.E. (2015). Regulating the regulators: recent revelations in the control of E3 ubiquitin ligases. J. Biol. Chem. 290, 21244–21251. Vizcaı´no, J.A., Csordas, A., Del-Toro, N., Dianes, J.A., Griss, J., Lavidas, I., Mayer, G., Perez-Riverol, Y., Reisinger, F., Ternent, T., et al. (2016). 2016 update of the PRIDE database and its related tools. Nucleic Acids Res. Published online September 28, 2016. gkw880. Wang, X., Herr, R.A., and Hansen, T.H. (2012). Ubiquitination of substrates by esterification. Traffic 13, 19–24. Wenger, C.D., and Coon, J.J. (2013). A proteomics search algorithm specifically designed for high-resolution tandem mass spectra. J. Proteome Res. 12, 1377–1386. Winn, M.D., Ballard, C.C., Cowtan, K.D., Dodson, E.J., Emsley, P., Evans, P.R., Keegan, R.M., Krissinel, E.B., Leslie, A.G.W., McCoy, A., et al. (2011). Overview of the CCP4 suite and current developments. Acta Crystallogr. Sect. D Biol. Crystallogr. 67, 235–242. Wisniewski, J.R., Zougman, A., Nagaraj, N., and Mann, M. (2009). Universal sample preparation method for proteome analysis. Nat. Methods 6, 359–362. Wong, K., Kozlov, G., Zhang, Y., and Gehring, K. (2015). Structure of the Legionella effector, lpg1496, suggests a role in nucleotide metabolism. J. Biol. Chem. 290, 24727–24737. Xu, L., and Luo, Z.-Q. (2013). Cell biology of infection by Legionella pneumophila. Microbes Infect. 15, 157–167. Yau, R., and Rape, M. (2016). The increasing complexity of the ubiquitin code. Nat. Cell Biol. 18, 579–586.

Cell 167, 1636–1649, December 1, 2016 1649

STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies Ubiquitin Ubi-1

abcam

ab7254; RRID: AB_305802

Ubiquitin

Cell Signaling Technology

3936S; RRID: AB_10691572

Actin

Sigma-Aldrich

A4700; RRID: AB_476730

Vinculin

Sigma-Aldrich

V4505; RRID: AB_477617

HA

Cell Signaling

2367S; RRID: AB_10691311

GFP (B-2)

Santa Cruz Biotechnology

sc-9996; RRID: AB_627695

K63-Chain

Genentech

APU3.A8

K48-Chain

Genentech

APU2.07

Streptavidin-HRP

ThermoFischer Scientific

21130

Mono- and poly-ubiquitinylated proteins (FK2)

Biomol

BML-PW8810; RRID: AB_10541840

Ubiquityl-Histone H2A

Millipore

05-678; RRID: AB_309899

Histone H2A

Abcam

ab18255; RRID: AB_470265

HIF-1a

R&D systems

MAB1536; RRID: AB_2116983

b-Nicotinamide adenine dinucleotide sodium salt (NAD)

Sigma-Aldrich

N0632

Biotin-NAD

TREVIGEN

4670-500-01

Adenosine 50 -triphosphate (ATP)

Roth

K054.4

Mitotracker Red Deep Red FM

ThermoFischer Scientific

M22426

Chemicals, Peptides, and Recombinant Proteins

Pro-Q Diamond phosphoprotein gel stain

ThermoFischer Scientific

P33300

Penicillin-streptomycin (10,000 U/mL)

ThermoFischer Scientific

15140122

Hygromycin B

Invivogen

ant-hm-1

Doxycyclin

Sigma-Aldrich

D9891

Inositol hexakisphosphate

Sigma-Aldrich

P8810

Isopropyl-b-D-thiogalactopyranoside

Roth

CN08.4

MBP-PINK1

The University of Dundee

DU34701

PARKIN

The University of Dundee

DU40847

Ubiquitin N-terminal Fluorescein

Boston Biochem

U-580

UbcH5a (human)

Enzo Lifesciences

BML-UW9050-0100

Critical Commercial Assays Enzcheck Phosphate assay kit

ThermoFischer Scientific

E6646

AMP glow assay

Promega

V5011

Structure of SdeA-modified ubiquitin

This study

PDB: 5M93

Mass spectrometry data (ubiquitin modification, ubiquitin linkage to substrate serines)

This study

ProteomeXchange: PXD005240

Deposited Data

Experimental Models: Cell Lines HeLa

ATCC

CCL-2

HEK293T

ATCC

CRL-3216

Flp-In T-Rex HeLa-HA-Parkin

Dikic lab: Richter et al., 2016

N/A

Escherichia coli T7 express

New England Biolabs

C2566H

Escherichia coli NEB Turbo

New England Biolabs

C2984H

Experimental Models: Organisms/Strains

(Continued on next page)

e1 Cell 167, 1636–1649.e1–e7, December 1, 2016

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

Legionella pneumophila lp02 WT strain

Gift of Zhao-Qing Luo: Qiu et al., 2016

N/A

Legionella pneumophila DSidE strain (lacking all 4 members of SidE family)

Gift of Zhao-Qing Luo: Qiu et al., 2016

N/A

Legionella pneumophila DSidE strain + SdeA WT

Gift of Zhao-Qing Luo: Qiu et al., 2016

N/A

Legionella pneumophila DSidE strain + SdeA (EE/AA)

Gift of Zhao-Qing Luo: Qiu et al., 2016

N/A

Yeast W303-1A

Gift of Robert Ernst, Goethe University, Frankfurt

N/A

Recombinant DNA pGEX-6P1

GE Healthcare Lifesciences

28-9546-48

pGEX-6P1 human ubiquitin

This study

N/A

pGEX-6P1 human ubiquitin R42A

This study

N/A

pGEX-6P1 human ubiquitin R72A

This study

N/A

pGEX-6P1 human ubiquitin R42_72A

This study

N/A

pET28b mouse Ube1

Dikic lab

N/A

pET23a UbcH7

Dikic lab

N/A

pET21a-CPD tag

This study

N/A

pET21a-CPD-SdeA (NCBI reference sequence: WP_010947868.1)*

This study

N/A

pET21a-CPD-SdeA (EE/AA)

This study

N/A

pET21a-CPD-SdeA (200-1005)

This study

N/A N/A

pET21a-CPD-SdeA (200-1005) H284A

This study

pET21a-CPD-SdeA (200-1005) EE/AA

This study

N/A

pET21a-CPD-SdeA (200-1005) H288A

This study

N/A

pET21a-CPD-SdeA (200-1005) R344A

This study

N/A

pET21a-CPD-SdeA (200-1005) S936A

This study

N/A

pET21a-CPD-SdeA (200-1005) S937A

This study

N/A

pET21a-CPD-SdeA (200-1005) S936_937A

This study

N/A

pET21a-CPD-SdeA (200-1005) S984A

This study

N/A

pET21a-CPD-SdeA (200-1005) S986A

This study

N/A

pET21a-CPD-SdeA (200-1005) S984_986A

This study

N/A

pET21a-CPD-SdeA (200-1005) S936_937_984_986A

This study

N/A

pET21a-CPD-SdeA (200-1005) D396A

This study

N/A

pET21a-CPD-SdeA (200-1005) D396A_K397A

This study

N/A

pET21a-CPD-SdeA (200-1005) E395A

This study

N/A

pET21a-CPD-SopA (163-782)

Dikic lab

N/A

pET21a-CPD-Trim56 (1-207)

Dikic lab

N/A

pGREG598 Yeast CEN plasmid

EUROSCARF

P30378

pGREG598- SdeA

This study

N/A

pGREG598- SdeA H284A

This study

N/A

pGREG598- SdeA EE/AA

This study

N/A

pcDNA5 FRT/TO HA-Parkin

This study

N/A

pEGFPC1-SdeA

This study

N/A

pEGFPC1-SdeA H284A

This study

N/A

pEGFPC1-SdeA EE/AA

This study

N/A

pEGFPC1-SdeA DDUB

This study

N/A

pHAC1-SdeA

This study

N/A (Continued on next page)

Cell 167, 1636–1649.e1–e7, December 1, 2016 e2

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

pHAC1-SdeA H284A

This study

N/A

pHAC1-SdeA EE/AA

This study

N/A

pHAC1-SdeADDUB

This study

N/A

pET21a-Rab33b (C-terminal HIS)

This study

N/A

pET21a-Rab33b S154A (C-terminal HIS)

This study

N/A

HHpred

Alva et al., 2016

https://toolkit.tuebingen.mpg.de/hhpred

Modeller

Alva et al., 2016

https://toolkit.tuebingen.mpg.de/modeller

XDS

Kabsch, 2010

http://xds.mpimf-heidelberg.mpg.de/

CCP4

Winn et al., 2011

https://www.ccp4.ac.uk/

Phenix

Adams et al., 2010

https://www.phenix-online.org/

Graphpad Prism 5.0

Graphpad Software

http://www.graphpad.com/

Fiji

ImageJ

https://fiji.sc/

Pymol

The PyMOL Molecular Graphics System, Version 1.7.6.0, Schro¨ dinger, LLC

https://www.pymol.org/

Coot

Emsley and Cowtan, 2004

https://www2.mrc-lmb.cam.ac.uk/personal/ pemsley/coot/

MaxQuant 1.5.3

Cox and Mann, 2008

http://www.maxquant.org

Xcalibur (including Freestyle and Xtract)

ThermoFisher

https://www.thermofisher.com/order/catalog/ product/OPTON-20502

StavroX 3.6

Go¨tze et al., 2012

http://www.stavrox.com

Morpheus 1.68

Wenger and Coon, 2013

http://cwenger.github.io/Morpheus/

Software and Algorithms

CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for reagents may be directed to and will be fulfilled by the Lead Contact Ivan Dikic (ivan.dikic@ biochem2.de). EXPERIMENTAL MODEL AND SUBJECT DETAILS Microbe strains T7 express E.coli chemically competent cells were obtained from NEB (catalog number: C2566H). These cells were stored at 80 C and grown in LB medium at 37 C. T7 express cells were used for protein expression. NEB Turbo E.coli chemically completent cells (catalog number: C2984H) were obtained from NEB. These cells were stored at 80 C and grown in LB medium at 37 C. NEB Turbo cells were used for molecular cloning and plasmid amplification. Following four different strains of Legionella pneumophila were used in this study: These strains were a kind gift of Zhao-Qing Luo (Purdue University). 1. 2. 3. 4.

lp02 (WT strain of Legionella) DSidE strain lacking all 4 members of SidE family DSidE strain complemented with WT SdeA DSidE strain complemented with SdeA EE/AA mutant

Legionella were grown in AYE broth and are maintained on CYE plates at 37 C. Yeast strain Yeast strain W303-1A was used for all the experiments. Yeast was grown at 30 C in YPD for transformation or appropriate selective media lacking uracil and containing either 2% glucose or galactose as carbon source. Cell lines Doxycycline-inducible stable HeLa Flp-In TRex HA-parkin cell line was generated by transfection of HeLa FRT/TO cells with pcDNA5 FRT/TO HA-Parkin and Flp-recombinase expression vector pOG44. 36 hr after the transfection, cells were then subcultured in a

e3 Cell 167, 1636–1649.e1–e7, December 1, 2016

selection medium containing 15 mg/ml blasticidin and 250 mg/mL hygromycin. Resistant cell colonies were expanded and tested for doxycycline inducibility of the transgene. Parkin stable cell lines were maintained in DMEM supplemented with 10% FBS, 100 I.U./mL penicillin and 100 mg/mL streptomycin (Pen/Strep), 10 mg/mL blasticidin and 200 mg/mL hygromycin, at 37 C, 5% CO2 (Richter et al., 2016). HeLa and HEK293T cells were cultured in DMEM supplemented with 10% FBS, 100 I.U./mL penicillin and 100 mg/ml streptomycin (Pen/Strep) at 37 C, 5% CO2. METHOD DETAILS Protein purification SdeA FL and DNC constructs were cloned into a modified pET21a vector with a C-terminal CPD tag. The currently annotated SdeA sequence entry in Uniprot (Uniprot: Q5ZTK4) is curated as erroneously initiated and is seven amino acids shorter (at the N terminus) than the NCBI reference sequence for SdeA (NCBI: WP_010947868.1) that we used in this study. Rab33b was cloned into pET21a vector with a C-terminal HIS tag. GST-tagged ubiquitin-expressing plasmid pGEX6P1-Ub was a kind gift of Dr. Anja Bremm. Histagged E1 and untagged ubiquitin expression vectors were a kind gift of Dr. Masato Akutsu. For protein purification, the expression vectors were transformed into T7 express E.coli chemically competent cells. Cultures were allowed to grow at 37 C until the OD 0.6. Protein expression was induced by the addition of 0.3 mM IPTG overnight at 18 C. Cells were lysed in the buffer 50 mM Tris pH7.5, 10% Glycerol, 300 mM NaCl containing 1mM PMSF. Subsequently, the cleared cell lysate was loaded onto Talon metal affinity beads and the bound proteins were either eluted with 200 mM imidazole (in the case of HIS-tagged proteins) or cleaved off the beads using 100 mM inositol-6-phosphate (in the case of CPD-tagged proteins). The proteins were then loaded onto the size exclusion chromatography column in buffer 10 mM HEPES pH 7.5, 150 mM NaCl. Proteins were concentrated and flash frozen in liquid nitrogen and stored at 80 C until usage. His-tagged E1 and E2 proteins were also purified using the procedure mentioned above. Untagged ubiquitin was purified using the standard procedure. SopA (163-782) and Trim56 (1-207) constructs used in the in vitro ubiquitination reactions were also purified as described above. Purification, crystallization, and structure determination of phosphoribosylated ubiquitin Phosphoribosylated ubiquitin was synthesized by an in vitro ADP-ribosylation reaction consisting of 5 mM DNC SdeA WT (residues 200 to 1005), 165mM GST-tagged ubiquitin and 2 mM NAD+. The reaction was supplemented with 3C protease to cleave the GST tag off the ubiquitin. The reaction was set up at 37 C for 4 hr in a final volume of 2 mL. The reaction mixture was subjected to size exclusion chromatography using a Superdex 75 (16/60) column pre-equilibriated with 10 mM HEPES, 150 mM NaCl and 1 mM DTT. The fractions corresponding to ubiquitin were pooled and concentrated using a 3 kDa cut-off concentrator. The protein was then diluted with water to give a final buffer composed of 1 mM HEPES, 15 mM NaCl, and 0.1 mM DTT and re-concentrated to 19 mg/mL for crystallization. Crystals of phosphoribosylated ubiquitin were grown at 18 C in 0.1M sodium acetate pH 4 - 5.5, 0.2M lithium sulfate and 30% PEG 8000. Crystals were flash cooled in liquid nitrogen using mother liquor supplemented with 15% glycerol as cryoprotectant. Diffraction data were collected at the Swiss light source (SLS, Villigen, Switzerland). The best crystal diffracted to a resolution of 1.8A˚ and belonged to the space group P1211, with 3 molecules in the asymmetric unit. Data were processed using the XDS package and the structure was solved by molecular replacement with ubiquitin (PDB: 1UBQ) in PHASER (Kabsch, 2010; Storoni et al., 2004). The structure was refined with iterative rounds of manual rebuilding in COOT (Emsley and Cowtan, 2004) and maximum likelihood energy minimization and isotropic B-factor refinement in PHENIX (Adams et al., 2010) and Refmac (Winn et al., 2011). All structure figures were generated with PyMOL (The PyMOL Molecular Graphics System, Version 1.7.6.0 Schro¨dinger, LLC.). Purification of modified ubiquitin used for the assay shown in Figure 2F was also generated using a similar strategy but the DNC SdeA (WT to generate phosphoribosyl Ub and H284A mutant to generate ADP-ribosyl Ub) proteins were incubated with purified untagged ubiquitin for this purpose (Figure S2E). Ubiquitin modification and Rab33 ubiquitination assays For ubiquitin modification and auto-ubiquitination of SdeA experiments, 25 mM of purified untagged ubiquitin was incubated with 1–2 mM of SdeA FL or DNC construct of SdeA at 37 C for 1hr in the presence or absence of 1 mM NAD+ in a buffer containing 50 mM Tris pH7.5, 50 mM NaCl. The reaction mixture is subjected to SDS-PAGE or native PAGE followed by Coomassie staining. Alternatively, reaction mixture was subjected to SDS-PAGE followed by western blotting using ubiquitin antibodies. CS-Ub is mouse monoclonal antibody against ubiquitin that was purchased from Cell Signaling (catalog number: 3936s). Abcam-Ub is also a mouse monoclonal antibody against ubiquitin that was purchased from abcam (catalog number: ab 7254). Biotin-labeled NAD+ was purchased from Trevigen (catalog number: 4670-500-01). Experiments using Biotin-NAD+ were analyzed by transferring the reaction components onto a nitrocellulose membrane following the SDS-PAGE and probing the membrane with Streptavidin-HRP conjugate. 4 mM of Rab33b was included in the reaction mixture where indicated. For producing the ADP-ribosylated ubiquitin, SdeA (H284A) was used instead of WT protein. For phospho-specific staining of phosphoribosylated ubiquitin, Pro-Q Diamond stain was used as per manufacturer’s instructions (Daniels et al., 2014). For ubiquitination modification reactions using Legionella lysates, four different strains of Legionella were grown in 3 mL AYE broth until they reached early stationary phase (OD600 = 3). Cultures of different strains were normalized and lysed by incubating the cell pellet in 100 mL lysis buffer (50 mM HEPES pH7.5, 200 mM NaCl, 0.1% Triton X-100, 1 mM EDTA, 1 mM PMSF and 0.1 mg/ml Cell 167, 1636–1649.e1–e7, December 1, 2016 e4

Lysozyme) for 30 min at room temperature. After lysis, cell debris was removed by centrifuging the samples at 15,000 rpm for 15 min. 0.5 mL of 25 mg/ml ubiquitin and 2 mM NAD+ was added to each lysate and incubated at 37 C. The reaction was stopped using SDS loading dye and the samples were analyzed using CS-Ub and abcam-Ub antibodies. In vitro ubiquitination reactions with E1, E2, and E3 0.2 mM E1, 2 mM E2, and 5 mM E3 were incubated in the buffer 50 mM Tris pH7.5, 50 mM NaCl, 0.5 mM DTT, 1 mM MgCl2. WT ubiquitin or SdeA-modified ubiquitin were used in the reaction mixtures. Reaction was initiated by the addition of 1mM ATP. For reactions using SopA as E3 ligase, its substrate Trim56 was added to the reactions. For reactions using Parkin, PINK1 was used to activate Parkin as previously described (Kazlauskaite et al., 2014). The reactions were terminated by the addition of SDS-PAGE loading buffer. Ubiquitination was probed with SDS-PAGE analysis followed by Coomassie staining and/or western blotting followed by detection of poly-ubiquitination using CS-Ub antibody described above. For testing the effect of the DUB domain, in vitro ubiquitination reactions were setup as described above but with unmodified ubiquitin. 0.2 mM of FL SdeA or SdeA DNC proteins were added to the reaction mixture along with 1mM NAD+ at 10 min after the start of ubiquitination reaction. Samples were collected at indicated time points and the reaction was stopped using SDS loading dye. Ubiquitination in various samples was probed as described above. Mitophagy Full-length WT or mutant SdeA constructs and truncations were cloned into pEGFP-C1 vector for expression in mammalian cells. 4 3 105 HeLa Flp-In TRex HA-Parkin cells were seeded on a sterile coverslip placed in a 6-well plate. After allowing the cells to settle overnight, GFP-SdeA plasmids were transfected using polyethyleneimine. 24 hr after transfection, cells were checked for the expression of SdeA with green fluorescence. 1 mg/mL of doxcycline (Clontech) was added to the cells to induce the expression of HA-Parkin. After 6 hr of treatment with doxycycline, mitochondria were stained with 100nM MitoTracker (Invitrogen). Cells were then treated with 10 mM CCCP for 90 min to induce mitophagy. Slides were then fixed with formaldehyde and stained with FK2 antibody (EMD-Millipore) using standard procedures. Cells surrounding the glass coverslip were lysed and probed for HA-Parkin and GFP-SdeA using HA and GFP antibodies respectively. Images for all conditions were acquired under same laser power and settings using Zeiss LSM 780 microscope. Ubiquitination signal on mitochondria in acquired images was quantified using ImageJ. Briefly, to resolve mitochondria, mitotracker channel image threshold was adjusted first using default threshold option. Using wand tracking tool in imageJ, mitochondria of cells were then marked as regions of interest (ROIs) and added to ROI manager. We then moved to FK2 channel and using ROI manager quantified the poly-ubiquitination signals in the ROIs (mitochondria previously selected). Mean intensity was used for quantification according to the methods described in quantification and statistical analysis described below. TNF-a experiments HeLa cells were seeded on a sterile coverslip placed in a 6-well plate. After allowing the cells to settle overnight they were transfected with various SdeA constructs using polyethyleneimine. 24 hr after the transfection, the culture medium was changed to DMEM without FBS to starve cells overnight. 25 ng/mL of TNF-a was added to induce the signaling. After 20 min, cells were fixed and stained for p65 and DAPI using standard procedures. Experiments in each condition represented were performed in biological triplicates. Images were acquired for all conditions under same settings in Zeiss LSM 780 microscope. The quantification was done as described in quantification and statistical analysis section below. Probing HIF-1a levels HeLa cells were cultured and transfected with various SdeA constructs as described above using polyethyleneimine. 24 hr after the transfection, cells were lysed and the levels of HIF-1a were probed with antibody against the protein (R&D systems). Yeast survival assay Yeast strain W303-1A was used for all the experiments. Yeast was grown at 30 C in YPD for transformation or appropriate selective media lacking uracil and containing either 2% glucose or galactose as carbon source. pGREG598 vector (CEN, URA3) was purchased from EUROSCARF, Frankfurt, Germany. SdeA WT, SdeA E867A, E869A, and SdeA H284A constructs were cloned into pGREG598. 0.5 mg of plasmid DNA was used to transform yeast cells using the standard lithium acetate method. For yeast viability experiments, W303-1A strain carrying the defined plasmids were grown overnight in synthetic media lacking uracil and containing 2% glucose (repression conditions). OD600 2 units of cells were harvested, washed once with sterile water and resuspended in 1ml of sterile water (OD600 = 2/mL). 5mL aliquots of this suspension were spotted onto solid synthetic defined media lacking uracil and containing either 2% glucose or 2% galactose for protein expression. Spotted cells were grown at 30 C and images were acquired after 3 days of growth on galactose and after 2 days of growth on glucose media. Pyrophosphate release assay This assay was performed using EnzChek Pyrophosphate Assay Kit from Molecular probes (catalog number, E-6645) as per manufacturer’s instructions with slight modifications. Briefly, in a final reaction volume of 300 mL, 30 pmoles of E1, 10 mM ubiquitin or SdeA-modified ubiquitin, 0.2 mM 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG), 6 mL of 3U/ml inorganic pyrophosphatase, 3 mL of 100U/ml purine nucleoside phosphorylase were added in the buffer 50 mM Tris pH7.5, 1 mM MgCl2 and 50 mM e5 Cell 167, 1636–1649.e1–e7, December 1, 2016

DTT where indicated. 1 mM ATP was added to start the reaction and the enzymatic conversion of MESG to ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine was probed using the continuous measurement of the reaction mixture’s absorbance at 360 nm wavelength using a TECAN infinite 200 Pro plate reader. The absorbance was converted into moles of PPi released based on the standard curve obtained using various amounts of sodium pyrophosphate. Fluorescent ubiquitin-E1 loading assay N-terminally fluorescein labeled WT ubiquitin was purchased from Boston Biochem. The reaction mixture was composed of 0.5 mM E1, 1.2 mM fluorescent ubiquitin and the indicated amounts of unlabeled WT ubiquitin or SdeA-modified ubiquitin. The reaction was initiated by adding 0.5 mM ATP. After 10 min, the reaction was stopped by adding non-reducing SDS-PAGE loading buffer. The mixture was then subjected to SDS-PAGE and imaged using Biorad Chemidoc MP. E2 loading and discharge assays 2 mM of purified UbcH7 and 25 mM ubiquitin was added to 0.2 mM Ube1 in the reaction buffer containing 50 mM Tris pH7.5 50 mM NaCl and 1 mM MgCl2. E2-loading assays were started by the addition of 1 mM ATP and the reaction was stopped after 10 min by the addition of SDS-PAGE loading buffer and heating the samples at 96 C for 2 min. The reaction mixture was then subjected SDS-PAGE followed by Coomassie staining to assay the ubiquitin loading of E2. For E2 discharge assays, WT ubiquitin or phosphoribosylated ubiquitin loaded E2 (UbcH5a) was prepared as described above except that the reaction mixtures were incubated for 1 hr to allow complete loading of E2 with the modified ubiquitin. The reaction mixture was incubated with 1 unit of Apyrase for 30 min to completely deplete the ATP. 3mM E3 was then added to initiate the discharge of ubiquitin from E2. Samples were collected at indicated time points and SDS-PAGE loading buffer was added to stop the reaction. Reaction mixtures were subjected to SDS-PAGE, blotted and probed with antibodies against HIS tag and ubiquitin to monitor the discharge of ubiquitin loaded HIS-UbcH5a. Mass spectrometric analysis of modified ubiquitin Proteins from reaction mixtures were separated by 1D PAGE, the ubiquitin band excised and subjected to in-gel trypsin digestion. Tryptic peptides were desalted by StageTip cleanup and analyzed by Orbitrap Fusion mass spectrometry as described recently (Leidecker et al., 2016). In brief, the peptides were separated by C18 reversed phase chromatography with an Easy nLC 1000 (Thermo) coupled to an Orbitrap Fusion mass spectrometer (Thermo). For ADPR analysis, Adenine-triggered and targeted high-resolution ETD fragmentation was applied; phospho-ribosylation was verified by data-dependent high-resolution ETD fragmentation. Spectra were searched with MaxQuant and modification sites verified by manual validation. For analysis of intact modified Ubiquitin, SdeA was removed by Amicon Ultracel 30 kD centrifugal filters (Merck) and ubiquitin was enriched and desalted by C18 StageTip cleanup. Intact Ubiquitin was analyzed by mass spectrometry as described above with minor modifications. MS-spectra were collected and deconvoluted using Xtract and Freestyle to determine the exact protein mass, highresolution ETD MS/MS spectra were manually inspected to verify the sequence. Analysis of endogenous ubiquitin from HEK293T cells was performed as described for purified samples with minor modifications. In brief the low molecular weight proteins in the cell lysate were isolated by SDS-PAGE, disulfide bridges reduced with DTT and cysteines alkylated with iodoacetamide. After ingel digestion, the tryptic peptides were separated with a 90 min non-linear gradient and analyzed with high-resolution HCD and ETD fragmentation on an Orbitrap Fusion. Mass spectrometric analysis of ubiquitin cross-linked to Rab and SdeA In vitro mixtures containing Ubiquitin, SdeA and Rab33 were run through 30 kDa MW cutoff filters to remove the majority of ubiquitin following the reaction. The proteins retained above the filters were digested by FASP (Wi sniewski et al., 2009) and analyzed by LC MS/MS to obtain initial high-resolution HCD spectra for all peptide species. Bridged peptide candidates were subject to targeted CID and ETD fragmentation using inclusion lists to yield high-quality spectra for determining bridge structure and localization, respectively. Initial HCD spectra were searched using open search methodology using the search engine Morpheus 1.68 (Wenger and Coon, 2013) against the proteins in the reaction mix – first against ubiquitin, then against the other proteins. A fictional fixed labile modification of 1000 Da and 1000 Da precursor mass tolerance yielded an effective open search error of + 0 to 2000 Da. Spectra with strong matches in both searches were used to determine the mass of the bridging structure. Once the binding chemistry was established, the ubiquitin-peptide with the ribose-phosphate bridge modification were defined in Morpheus 1.68 and in Stavrox 3.6 (Go¨tze et al., 2012) and used to re-search the datasets for additional bridged peptide candidates. Targeted ETD spectra for bridged peptides were searched and annotated using Stavrox 3.6. CID spectra for selected confirmed bridged peptides were manually examined to gain insights into the nature of the covalent bridge structure. AMP release assay 1 mM of SdeA DNC WT or the mutants were incubated with 25mM of ubiquitin in the presence of 1mM NAD at 37 C for 1 hr. This reaction was performed in 25 mL reaction volume in buffer containing 50 mM Tris pH7.5 50 mM NaCl. AMP released in the reaction was measured using a luminescent-based commercially available kit AMP-glow (Promega). The assay was performed according to manufacturer’s instructions. Briefly, all the reaction mixtures were transferred to different wells of a white flat-bottomed plate and 25 mL of AMP-Glow reagent I was added to each. Plate was incubated at room temperature for 1 hr. After this, 50 mL of AMP detection solution Cell 167, 1636–1649.e1–e7, December 1, 2016 e6

was added to each well and the plate was incubated at room temperature for an additional 1 hr. Luminescent signal from samples was measured using TECAN infinite M200 plate reader. Three independent experiments were performed for each condition and luminescent signal was converted to concentration of AMP using an AMP standard curve obtained. The resulting values were entered fed into Graphpad PRISM software and analyzed as described in the quantification and statistical analysis section below. Modeling SdeA PDE domain HHpred server (https://toolkit.tuebingen.mpg.de/hhpred) for protein homology detection and structure prediction was used to first detect functional domains in SdeA (So¨ding et al., 2005). This analyis picked up several pdb structures with a phosphodiesterase domain, which showed homology to SdeA region between residues 200 and 500. The top hit was the PDE domain of Legionella protein lpg1496. This particular structure was selected to generate an alignment of homologous SdeA sequence with lpg1496 sequence. The resulting alignment was modified manually and fed into the connecting Modeller server as the template for building a 3D model. The final output model of SdeA PDE domain was analyzed by the accompanying quality control programs VERIFY3D and ANOLEA and deemed of good quality (Alva et al., 2016). Quantification and statistical analysis Data shown in Figures 6B and 6C and Figure S1B are analyzed using Graphpad Prism 5 software and the error bars shown in the graphs represent SEM. Unpaired t test was used to analyze the datasets quantified in Figures 6B and 6C. *** represent a t test p value (two-tailed) of < 0.0001. ** represent a p value of < 0.01 and * represent a p value of < 0.05. For Figure 6B, poly-ubiquitination signal was quantified on mitochondria of approximately 50 transfected cells per condition. These values were normalized based on the average intensities in untransfected cells in each condition. Normalized values were then fed into Graphpad Prism software for analysis as described above. For p65 translocation quantification, three biological replicate experiments were performed for each condition. Approximately 50 cells were examined for each replicate of a condition and cells with nuclear translocation of p65 were counted. Triplicate values were fed into Graphpad Prism and analyzed as described above. DATA AND SOFTWARE AVAILABILITY PDB co-ordinates of SdeA-modified ubiquitin were deposited in Protein Data Bank under PDB: 5M93. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Vizcaı´no et al., 2016) partner repository with the dataset identifier ProteomeXchange: PXD005240.

e7 Cell 167, 1636–1649.e1–e7, December 1, 2016

Supplemental Figures

Figure S1. Identification of PDE Domain in SdeA, Related to Figure 1 (A) Sequence alignment of homologous regions of PDE domains of SdeA and lpg1496. Predicted catalytic residues are marked with a red asterisk. (B) Ubiquitin was treated with SdeA WT and mutants H284A or EE/AA in the presence of NAD+. The released AMP was measured using AMP-Glo assay (Promega). Each experiment was done in triplicate and the data are represented as mean ± SEM (see STAR Methods).

Figure S2. Ubiquitin Linkage Is Mediated through PDE Domain of SdeA, Related to Figure 2 (A) SdeA PDE mutant (H284A) was tested for its ability to ubiquitinate the substrate Rab33b. Reaction mixtures containing WT SdeA or the PDE mutant were subjected to SDS-PAGE and western blotting. Ubiquitinated Rab33b was detected by Coomassie staining (left panel) of the SDS-gel and using an anti-ubiquitin antibody (right panel). (B–D) High-resolution ETD spectra of SdeA cross-linked to ubiquitin were analyzed with StavroX showing arginine 42 of ubiquitin attached via a phospho-ribosyl linker to serine 984, serine 986, and serine 1005 residues of SdeA. (E) Schematic describing how ubiquitin-ADP-ribose and ubiquitin-phosphoribose were generated. (F) Purified ADP-ribosylated ubiquitin (ADP-R Ub) and phosphoribosylated ubiquitin (P-R Ub) proteins were stained with Pro-Q diamond phosphoprotein stain as is and after treatment with SdeA. (G) Model of PDE domain of SdeA, inset represents the enlarged view of the active site of the PDE domain with putative catalytic residues. Additional residues E395, D396, and K397 are close to the PDE domain and therefore may be important for ubiquitin transfer to the substrate via phosphodiester bond.

A Inhibition of HECT-E3 ligase SopA’s activity

MW kDa 135 100 75 63 48 35 25 20 17 11

+ _

+ _

+ + _

+ + +

_ + + + _

_ + + + +

_ _ + + +

B

ΔNC SdeA WT ΔNC SdeA H284A Ubiquitin E1+E2+E3+ATP NAD

Ube1 ΔNC SdeA SopA (Coomassie) UbcH7

Inhibition of RING-E3 ligase Trim56’s activity

MW kDa

+ _

+ _

+ + _

+ + +

_ + + + _

_ + + + +

135 100 75 63 48 35 25 20 17 11

_ ΔNC SdeA WT _ ΔNC SdeA H284A + Ubiquitin + E1+E2+E3+ATP + NAD

Ube1 ΔNC SdeA

(Coomassie) Trim56 (1-207) UbcH5a Ubiquitin

Ubiquitin

245 180 135 100 75 63 48 35 25 20 17

(α-Ubiquitin)

135 100 75 63 48 35 25 20 17 11

11

Figure S3. SdeA Phosphoribosylates Ubiquitin and Shuts down Canonical Ubiquitination, Related to Figure 3 (A) Effect of phosphoribosylation or ADP-ribosylation of ubiquitin on in vitro ubiquitination by the HECT-type E3 ligase SopA. (B) Effect of phosphoribosylation or ADP-ribosylation of ubiquitin on in vitro ubiquitination by the RING type E3 ligase Trim56.

(α-Ubiquitin)

A

chain A - unmodified ubiquitin

B

chain C phosphoribosylated ubiquitin R42

R42

I44

Arg42 ribose

I44

I36

I36 L8

C

WT + _

+ _

_

+

R42A _

_

+

+ _

+ _

+

+ _

+

_

+

+

_

R72A + _ _ + +

_

+

Ubiquitin (1UBQ) - “loop-out”

R42_72A Ubiquitin + _ ΔNC SdeA WT _ + ΔNC SdeA H284A

+ _

+

+

L8

Ubiquitin (3ZLZ) - “loop-in”

NAD

D

Ubiquitin SdeA Ubiquitin

E1 (Uba1) Ubiquitin Phosphoribosylated ubiquitin ribose

Ubiquitin (native-PAGE Coomassie)

R72 R42

E

100kDa

no ATP

(native-PAGE α-Ubiquitin)

Ub (WT) 0

0.5 1

2

Ub (SdeA treated) 5

10

0

0.5 1

2

5

10

µM E1 charged with fluorescent Ub

Figure S4. Crystal Structure of SdeA-Modified Ub and Its Implications, Related to Figure 4 (A) Difference map (Fo-Fc) contoured at 3s showing electron density for a ribosyl moiety attached to arginine 42 of ubiquitin. (B) Superposition of chain A and chain C of modified ubiquitin structure with ubiquitin structures in ‘‘loop-out’’ conformation (mono-ubiquitin, PDB: 1UBQ) and ‘‘loop-in’’ conformation (K6-linked tri-ubiquitin, PDB: 3ZLZ). Ile36 and Ile44 are indicated in orange and blue, respectively. (C) Ubiquitin WT and indicated mutants were treated with DNC SdeA WT and H284A mutant. Samples were subjected to native-PAGE followed by Coomassie staining and western blot analysis with ubiquitin antibody. (D) Superimposition of phosphoribosylated ubiquitin with WT ubiquitin in complex with yeast Uba1 (PDB: 3CMM) showing the potential clashes between the phosphoribosyl moiety attached to arginine 42 of ubiquitin and E1. (E) 0.6 mM E1 was incubated with a mixture of 1.2 mM N-terminal fluorescein labeled ubiquitin and indicated amounts of WT or phosphoribosylated ubiquitin. Reaction was initiated by the addition of ATP. Reaction mixture was subjected to SDS-PAGE followed by imaging with Biorad Chemidoc MP.

A

B [XM+H]+∙

relative intensity (%)

relative intensity (%)

[XM+H]+∙

[XM+2H]2+∙

[XM+2H]2+∙

m/z, Da

D

GFP

150

10

α-Ubiquitin CS-Ub

10

α-Ubiquitin abcam-Ub

150 100

Vinculin

A /A

4A

_

EE

MW kDa

28

WT H284A

T

-

Flag-HA-SdeA

H

MW kDa 250 150

GFP-SdeA

W

C

m/z, Da

HA

17 11 25 20

Histone H2A Ubiquitinated Histone H2A

150 100

Vinculin

Figure S5. SdeA Phosphoribosylates Ubiquitin in Cells, Related to Figure 5 (A and B) Phosphoribosylation or ADP-ribosylation of ubiquitin arginine 42 was detected in cells expressing SdeA WT or SdeA H284A mutant by LC-MS analysis on an Orbitrap Fusion mass spectrometer. By high resolution ETD fragmentation, the peptide backbone could be mapped efficiently without losing the attached modification. The localization of P-Rib (A) as well as ADP-Rib (B) could be mapped to arginine 42 of endogenous ubiquitin. XM marks coeluting precursor ions with an m/z at charge state +2 very close to that of the modified ubiquitin peptide in the +3 state, which are thus co-isolated. Due their lower charge state, they are inefficiently fragmented by ETD and instead undergo charge reduction, so they appear mainly as singly-charged ions. (C) GFP-SdeA WT and H284A mutant expressing HE293T cells were FACS sorted and the mono-ubiquitin in the cells was monitored using CS-Ub and Abcam-Ub antibodies. (D) HEK293T cells transfected with HA-tagged WT SdeA or indicated mutants. Ubiquitinated H2A in SdeA-transfected cells was probed with the corresponding antibody.

A

No transfection

CCCP 10µM 1.5 hr

Poly-Ub

GFP-ΔNC SdeA

Mitotracker

10µm

GFP

10µm

Poly-Ub

10µm

GFP-SdeA H284A

Mitotracker

Poly-Ub

Sd MW kDa 250 150

Sd

eA

B

GFP

W eA T Sd H eA 284 Sd E A eA E/A ΔN A C

Mitotracker

_

+

+

+

+

+

Doxycycline (1µg/mL, 8 h)

_

+

+

+

+

+

CCCP (10µM, 90 min)

*

Anti-GFP (SdeA)

100 70 50

Anti-HA (Parkin)

Figure S6. Effect of SdeA on Ubiquitination on Mitochondria during Mitophagy, Related to Figure 6 (A) HeLa cells stably expressing HA-Parkin and ectopically expressing WT and various mutants of SdeA were treated with 10 mM CCCP for 90 min followed by staining with mitotracker and FK2 (poly-Ub) antibody. (B) HA-Parkin expression was probed in samples used for the experiment shown in Figure 4D. *GFP-tagged mART mutant (E867, E869A) shows greater electrophoretic mobility perhaps due to degradation of the FL protein.