Legionella maintains host cell ubiquitin homeostasis by effectors with unique catalytic mechanisms

Fu, Jiaqi; Li, Siying; Guan, Hongxin; Li, Chuang; Zhao, Yan-Bo; Chen, Tao-Tao; Xian, Wei; Zhang, Zhengrui; Liu, Yao; Guan, Qingtian; Wang, Jingting; Lu, Qiuhua; Kang, Lina; Zheng, Si-Ru; Li, Jinyu; Cao, Shoujing; Das, Chittaranjan; Liu, Xiaoyun; Song, Lei; Ouyang, Songying; Luo, Zhao-Qing

doi:10.1038/s41467-024-50311-2

Legionella maintains host cell ubiquitin homeostasis by effectors with unique catalytic mechanisms

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Published: 15 July 2024

Volume 15, article number 5953, (2024)
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Legionella maintains host cell ubiquitin homeostasis by effectors with unique catalytic mechanisms

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Abstract

The intracellular bacterial pathogen Legionella pneumophila modulates host cell functions by secreting multiple effectors with diverse biochemical activities. In particular, effectors of the SidE family interfere with host protein ubiquitination in a process that involves production of phosphoribosyl ubiquitin (PR-Ub). Here, we show that effector LnaB converts PR-Ub into ADP-ribosylated ubiquitin, which is further processed to ADP-ribose and functional ubiquitin by the (ADP-ribosyl)hydrolase MavL, thus maintaining ubiquitin homeostasis in infected cells. Upon being activated by actin, LnaB also undergoes self-AMPylation on tyrosine residues. The activity of LnaB requires a motif consisting of Ser, His and Glu (SHxxxE) present in a large family of toxins from diverse bacterial pathogens. Thus, our study sheds light on the mechanisms by which a pathogen maintains ubiquitin homeostasis and identifies a family of enzymes capable of protein AMPylation.

Ubiquitination independent of E1 and E2 enzymes by bacterial effectors

Article 06 April 2016

Exploiting the ubiquitin and phosphoinositide pathways by the Legionella pneumophila effector, SidC

Article 03 October 2015

Methods for NAD-Dependent Ubiquitination Catalyzed by Legionella pneumophila Effector Proteins

Introduction

Successful pathogens use virulence factors to actively modulate host processes to evade cell-autonomous defense and immune detection. It is now recognized that the activity of virulence factors is a double-edged sword for the pathogen: on the one hand, these factors are essential for colonizing their hosts; on the other hand, cellular damage inflicted by pathogenic factors can be detected by specific receptors, leading to robust immune responses¹. To minimize the chance of virulence factors being used as immune agonists, pathogens have evolved various regulatory mechanisms, including tight regulation of gene expression, precise targeting to specific cellular compartments, and/or the use of additional virulence factors to dampen the immune response^2,3,4.

In some scenarios, virulence factors may disrupt host cell homeostasis, thus making the cell less suitable for pathogen replication. One such example is the intracellular bacterial pathogen Legionella pneumophila. This bacterium creates an intracellular niche permissive for its replication utilizing a large cohort of effectors to modulate host processes by diverse biochemical activities⁵. Among the scores of effectors that target the ubiquitin network, members of the SidE effector family pose unique challenges to host cells. These effectors catalyze protein ubiquitination by a mechanism involving the production of ADP-ribosylated ubiquitin (ADPR-Ub) as the reaction intermediate using their monoADP-ribosyltransferase (mART) activity⁶. ADPR-Ub is then used to modify substrate proteins by their phosphodiesterase (PDE) domains^7,8. These two reactions are uncoupled, and ADPR-Ub may accidentally release into the cytosol of infected cells. Furthermore, the reversal of SidEs-induced ubiquitination by DupA and DupB produces phosphoribosyl ubiquitin (PR-Ub)^9,10. Neither ADPR-Ub nor PR-Ub can be used in canonical ubiquitination reactions⁷, and their accumulation may interfere with key signaling cascades in infected cells and ultimately impede bacterial replication. These challenges necessitate mechanisms to maintain ubiquitin homeostasis in infected cells.

Here we show that the bacterium utilizes two sequential reactions catalyzed by additional effectors to convert PR-Ub into functional ubiquitin. After being activated by the host protein Actin, LnaB reduces PR-Ub to ADPR-Ub, which is hydrolyzed into ADP-ribose and native ubiquitin. We also show that LnaB represents a large family of bacterial toxins potentially involved in cell signaling by protein AMPylation by a mechanism that requires catalysis induced by a motif consisting of Serine, Histidine, and Glutamate.

Results

The macro domain protein MavL is an (ADP-ribosyl)hydrolase against ADPR-Ub

We attempted to identify additional L. pneumophila effectors involved in ubiquitin signaling using biotin-mediated proximal labeling (Turbo ID)¹¹, the BirA*-Ub fusion (BirA*, the promiscuous BirA mutant)¹¹ was expressed in L. pneumophila and biotinylated proteins were identified by mass spectrometry (Supplementary Fig.1a). Not surprisingly, a number of Dot/Icm effectors known to be involved in ubiquitin signaling were identified. In addition, a few effectors of unknown function were detected with high confidence (Supplementary Fig. 1b). Among these, MavL(Lpg2526) has been studied in a recent study which reveals that this protein binds ADP-ribose and structural overlay with poly-(ADP-ribose) glycohydrolases (PARGs) identified two aspartate residues potentially involved in catalysis¹². This study also found that MavL interacts with the mammalian ubiquitin-conjugating enzyme UBE2Q1, suggesting a role in ubiquitin signaling¹². In light of these observations and the fact that MavL was identified in our screening strategy based on ubiquitin interaction, we further pursued the hypothesis that MavL acts on proteins that have been modified by ubiquitination, ADP-ribosylation, or both. That APDR-Ub is inactive in canonical ubiquitination reactions (Supplementary Fig. 1c) prompted us to examine whether it is a substrate of MavL. Indeed, recombinant MavL effectively hydrolyzed ADPR-Ub into ADPR and ubiquitin, which is in contrast to DupA and DupB (Fig. 1a). Like most Dot/Icm effectors, MavL is not required for bacterial intracellular growth (Supplementary Fig. 1d).

**Fig. 1: MavL is a macro domain protein that converts ADPR-Ub into ADP-ribose and ubiquitin.**

To understand the catalytic mechanism of the ADP-ribosylhydrolase (ARH) activity of MavL, we screened its active truncation mutants and succeeded in solving the structure of the MavL_40-404-ADPR complex at a resolution of 2.38 Å, which revealed that several pairs of hydrogen bonds and a π-π stacking interaction provided by surrounding amino acids or water molecules are involved in interactions between MavL and ADPR (Supplementary Fig. 1e). The negatively charged residues D315, D323, and D333 likely form a catalytic loop (β8-α8) that directly interacts with the hydroxyl groups of ribose by hydrogen bonding (Supplementary Fig. 2a). Mutational analysis indicated that all three residues are critical for the ARH activity of MavL against ADPR-Ub (Fig. 1b). The role of D323 and D333 in catalysis has been predicted in an earlier structural study of MavL¹². Furthermore, the mutants MavL_D315A and MavL_D323A displayed higher affinity toward ADPR-Ub and ADPR (Fig. 1cand Supplementary Figs. 2b, 3, 4). Further efforts using these mutants allowed us to obtain the structure of the MavL_{40-404(D315A)}-ADPR-Ub complex at a 1.93 Å resolution (Fig. 1d, left). The combined surface area of the interface between MavL and ubiquitin is approximately 867.6 Å². In the complex, ADPR-Ub engages MavL in such a way that the ADPR moiety linked to the side chain of R42 of ubiquitin is inserted into the active-site pocket and the binding is coordinated by several pairs of hydrogen bonds provided by G223, G225, C226, F227, K236, P264, N322, D323, T331, D332, and D333 of MavL with D39 and R42 of ubiquitin (Supplementary Fig. 2c). E107 and D323 of MavL hold R42 of ubiquitin in a position suitable for catalysis via hydrogen bond interactions (Fig. 1d, middle). In addition, the side chain of D323 points to the 1”-OH of the ADPR moiety and the -NH2 of R42 in ubiquitin at a distance of 2.65 Å and 2.69 Å, respectively, thus is most likely the residue key for catalysis (Fig. 1d, middle). Unexpectedly, the N-glycosidic bond between ADPR and R42 of ubiquitin was cleaved in our structure, which may be caused by the residual activity of the D315A mutant. (Fig. 1d, right).

Comparison of the structures of apo-MavL, MavL-ADPR, and MavL_{40-404(D315A)}-ADPR-Ub revealed that the side chains of several residues, including F105, C226, Y232, and Q330 are involved in the formation of the activity pocket of apo-MavL that faces outwards, keeping the binding pocket in an open state (Fig. 1e). Among these, the side chains of C226 and Q330 form steric hindrance with the side chains of D39 and Q40 on ubiquitin loop1 and E51 and D52 on ubiquitin loop2, respectively, preventing ubiquitin from binding to apo-MavL(Supplementary Fig. 5a). In addition, we observed four water molecules around the ADPR moiety, two of which (H₂O-1 and H₂O-2) are also found in other macro domain ARHs (Supplementary Fig. 5b). These two water molecules form a hydrogen bond network with the α-phosphate of ADPR and the O1” site of the distal ribose group, wherein the α-phosphate activates H₂O-1 to initiate a nucleophilic attack on the O1” glycosidic bond, leading to its cleavage (Fig. 1d, middle).

We examined substrate specificity of MavL using several ADP-ribosylated proteins, including ADPR-Ub_T66 produced by CteC of Chromobacterium violaceum¹³, ADPR-Actin induced by SpvB of Salmonella enterica¹⁴, ADPR-ANT1 catalyzed by Ceg3 of L. pneumophila¹⁵, and ADPR-PARP1 catalyzed by Sirt6¹⁶. In addition to ADPR-Ub produced by SidEs, ADP-ribosyl hydrolysis against ADPR-ANT1 and ADPR-Actin also detectably occurred (Supplementary Fig. 2d). We further examined the physiological role of MavL by probing ADPR-Ub in cells infected with relevant L. pneumophila strains. ADPR-Ub was detected in cells infected by wild-type L. pneumophila and its level was elevated in samples infected with the ∆mavL mutant, and complementation with MavL but not the inactive mutant MavL_D323A restored the phenotype (Fig. 1f), indicating that MavL functions to reduce cellular ADPR-Ub. Our attempt to determine the distribution of MavL in cells infected with L. pneumophila by immunostaining was not successful, probably due to low protein abundance or the quality of our antibodies. Importantly, infection of cells transfected to express HA-MavL revealed a clear accumulation of protein on the Legionella-containing vacuole (LCV) in a manner that required a functional Dot/Icm system (Supplementary Fig. 6), suggesting that the effector mainly acts on the cytoplasmic surface of the bacterial phagosome.

The effector LnaB is an adenylyltransferase that converts PR-Ub into ADPR-Ub

Efficient conversion of ADPR-Ub into ADPR and ubiquitin by MavL suggests that this enzyme functions to return modified ubiquitin to its native form. Yet, MavL cannot remove the phosphoribosyl group from PR-Ub (Supplementary Fig. 2e), we thus considered the possibility that PR-Ub is first converted into ADPR-Ub prior to hydrolysis by MavL.

Dot/Icm effectors of relevant functions often are encoded by genes of close proximity on the chromosome^5,17. The gene upstream of mavL is mavK(lpg2525), which appears to harbor an F-box motif known to be involved in ubiquitination¹⁸. The genes coding for LnaB (Lpg2527) and the deubiquitinase Lem27 (Lpg2529) (also known as LotC)¹⁹ are separated by a gene predicted to code for an α-amylase (Supplementary Fig. 7a). We then examined the hypothetical proteins, MavK and LnaB, for the ability to convert PR-Ub into ADPR-Ub. Incubation of recombinant MavK¹⁸ or LnaB with PR-Ub and ATP did not detectably produce ADPR-Ub (Supplementary Fig. 7b). Some L. pneumophila effectors are known to require host co-factors for their activity^20,21,22,23, we thus added lysates of mammalian cells to these reactions and found that native, but not heat-treated lysates enabled LnaB to produce ADPR-Ub from PR-Ub (Fig. 2a). Thus, LnaB has the capacity to convert PR-Ub into ADPR-Ub in the presence of a eukaryotic cell-specific molecule, which likely is a protein due to its sensitivity to heat treatment.

**Fig. 2: Conversion of PR-Ub into ADPR-Ub by LnaB requires Actin as a co-factor.**

To identify the host factor required for LnaB activity, we identified interacting proteins by immunoprecipitation from lysates of cells transfected to express Flag-LnaB by mass spectrometric analysis. Among the proteins specifically enriched by Flag-LnaB, Actin was identified as the most differed and abundant hit (Fig. 2b, Supplementary Fig. 7c and Supplementary Dataset 1). In line with these results, LnaB and Actin formed a complex in cells that was readily detectable by immunoprecipitation and by analytic ultracentrifugation (Supplementary Fig. 7d, e). ITC analysis revealed that these two proteins bind each other with a K_d of ∼ 1.24 µM (Fig. 2c). More importantly, inclusion of Actin in reactions containing PR-Ub, ATP and LnaB led to the production of ADPR-Ub (Fig. 2d). PR-Ub differs from ADPR-Ub only by an adenosine monophosphate (AMP) moiety (Fig. 2e), suggesting that LnaB catalyzes a reaction at the α phosphate center of ATP to transfer the AMP moiety onto PR-Ub. Indeed, the inclusion of ³²P-α-ATP in the reaction led to the production of radio-labeled ADPR-Ub, again in an Actin-dependent manner (Fig. 2f).

ATP analogs containing a cleavable α phosphate, including adenylyl-imidodiphosphate (AMPPNP) and adenosine 5’-(γ-thio)triphosphate (ATPγS), supported the full activity of LnaB. In line with its partially susceptible α phosphate²⁴, ATPαS also supported the activity (Fig. 2g). In contrast, adenosine 5’-(α, β-methylene)triphosphate (ApCpp), which has an uncleavable α-site, was unable to serve as the nucleotide donor (Fig. 2g).

Similar results were obtained when the product of the reaction was analyzed by mass spectrometry, which revealed that the tryptic peptide of ubiquitin (-E₃₄GIPPDQQRLIFAGK₄₈-) derived from PR-Ub in which R42 was modified by phosphoribosylation had been converted into ADP-ribosylation after incubation with ATP, LnaB, and Actin (Fig. 3a–c). Importantly, ADPR-Ub produced by LnaB from PR-Ub can be used in ubiquitination induced by the PDE activity of SidEs. Incubation of the product with SdeA_E/A (a SdeA mutant without mART activity but retaining its PDE function) led to Rab33b ubiquitination at levels comparable to reactions receiving native ADPR-Ub (Fig. 3d). Furthermore, ubiquitin produced from PR-Ub by LnaB and MavL was active in canonical ubiquitination reactions (Fig. 3e).

**Fig. 3: LnaB and MavL sequentially convert PR-Ub into ADPR and active ubiquitin.**

LnaB utilizes an S-HxxxE motif to catalyze the conversion of PR-Ub into ADPR-Ub

We explored the mechanism of action of LnaB using PSI-BLAST²⁵ searches to identify proteins that may have common functional motifs, which revealed that LnaB belongs to a family of toxins of at least 103 members that harbor a conserved S-HxxxE (x, any amino acid) motif of unknown biochemical activity (Supplementary Fig. 8). Notably, the lengths of the space between the conserved Serine and Histidine residues vary greatly among members of the protein family (Supplementary Fig. 8a). These proteins are encoded by diverse bacterial pathogens of a wide range of hosts, particularly a large set of proteins categorized as Making caterpillar floppy (MCF) toxins found in insect pathogens (Supplementary Fig. 8b)²⁶. Sequence alignment revealed that in LnaB, the S-HxxxE motif is composed of S261, H305, and E309 (Supplementary Fig. 8a). We examined the role of this predicted motif in the activity of LnaB by creating substitution mutants for each of these residues and testing their activity in converting PR-Ub into ADPR-Ub. Mutations in S261, H305, or E309 completely abolished the enzymatic activity (Fig. 4a, b).

**Fig. 4: The reaction catalyzed by LnaB required an S-HxxxE motif.**

To determine the role of LnaB during L. pneumophila infection, we employed mass spectrometric analysis to probe PR-Ub in cells infected with relevant bacterial strains. Very weak signals of PR-Ub were detected in cells infected with wild-type bacteria, but it became abundant in cells infected with the ∆lnaB mutant. Complementation with LnaB but not the LnaB_S261A mutant rendered PR-Ub undetectable (Fig. 4c), indicating that LnaB functions to eliminate PR-Ub in infected cells.

We also examined the distribution of LnaB in cells infected with L. pneumophila but were not able to detect signals using our antibodies specific to this protein. Yet, results from infection of cells transfected to express 4Flag-LnaB indicated that the protein was enriched on the LCV and such enrichment was dependent upon an active Dot/Icm system as vacuoles containing the dotA mutant did not detectably recruit Flag-LnaB (Supplementary Fig. 9a). These results suggest that similar to MavL, LnaB likely acts on the surface of the bacterial phagosome.

Similar to earlier experiments, in cells infected with wild-type L. pneumophila, ADPR-Ub is detected, and adding LnaB led to a slight increase in its abundance (Fig. 4d). Importantly, ADPR-Ub was not detectable in cells infected with the ∆lnaB mutant but became abundant after adding recombinant LnaB (Fig. 4d), which is consistent with the notion that PR-Ub was accumulated in these cells. PR-Ub accumulation caused by infections with strain ∆lnaB can be reversed by complementation with LnaB but not the LnaB_S261A mutant (Fig. 4d). LnaB-dependent elimination of PR-Ub in infected cells by converting it into ADPR-Ub with recombinant LnaB was also determined. We also probed the ratio of PR-Ub in cells infected with the ∆lnaB mutant by mass spectrometric analysis, which revealed that more than 30% of ubiquitin was modified in the PR-Ub form under our experimental conditions (Fig. 4e). Finally, PR-Ub was not detected in cells infected with the ∆dupA∆dupB mutant (Supplementary Fig. 9b), expression of either gene but not their enzymatically inactive mutants in this strain restored its accumulation in infected cells, indicating that PR-Ub is produced by reversal of SidEs-induced ubiquitination. Together, these results establish that LnaB functions to covert PR-Ub into ADPR-Ub in infected cells.

The conversion of PR-Ub and ADPR-Ub into native ubiquitin suggests that the accumulation of these ubiquitin derivatives interferes with the intracellular replication of L. pneumophila. We thus examined the growth of relevant L. pneumophila strains in Dictyostelium discoideum. Consistent with results from an earlier study²⁷, deletion of lnaB did not detectably impact intracellular bacterial replication (Fig. 4f). Overexpression of pSdeA in the wild-type strain led to a reduction in bacterial growth, and such growth defects became more pronounced when SdeA was expressed in the ∆lnaB strain. In contrast, expression of the mART-defective mutant SdeA_E/A or SdeA_H/A, the mutant defective in the phosphodiesterase (PDE) activity in the ∆lnaB mutant did not cause such defect (Fig. 4f). Importantly, the growth defect can be complemented by expressing LnaB but not its enzymatically inactive mutant (Fig. 4f). Taken together, these results suggest that accumulation of PR-Ub in host cells is detrimental to intracellular bacterial growth and the effects became more severe when the expression level of SidEs such as SdeA was increased in the bacterium, and that LnaB ameliorates such impact by eliminating it in infected cells.

LnaB and tested members of the S-HxxxE family catalyze protein AMPylation

Incubation of ³²P-α-ATP with LnaB in the absence of PR-Ub generated radio-labeled LnaB (Fig. 2f, 2^nd lane), suggesting that this enzyme is capable of catalyzing protein AMPylation by transferring the AMP moiety from ATP onto one or more of its own residues. Indeed, mass spectrometric analysis revealed that both Y196 and Y247 were AMPylated (Fig. 5a, b and Supplementary Fig. 9c, d). Mutations in both Y196 and Y247 gave rise to a mutant that had lost the ability to self-AMPylate but retained the activity to convert PR-Ub into ADPR-Ub (Fig. 5b, c). These results establish LnaB as an enzyme that catalyzes the cleavage of ATP at the α phosphate position to transfer the AMP moiety onto the phosphoryl group in PR-Ub and the side chain of tyrosine residues.

**Fig. 5: Self-AMPylation activity of members of the S-HxxxE toxin family.**

To determine the AMPylator activity of the S-HxxxE family, we purified recombinant proteins of a selection of toxins and evaluated their self-modification using ³²P-α-ATP. For the five toxins examined, self-AMPylation was readily detected for the toxin from Burkholderia ambifaria (Tba675) and a fragment of the toxin from Edwardsiella ictaluri (Tei158) (Fig. 5d). Weak but detectable self-AMPylation was detected for WP_075066242.1 (0750) from Candidatus Berkiella aquae, WP_148338824.1 (1483) from Aquicella siphonis, MAZ44397.1 (MAZ443) from a bacterium of the Legionellales. In each case, an intact S-HxxxE motif was required for the activity as mutations in the serine residue abolished self-AMPylation (Fig. 5d). Furthermore, we found that Tei158 and Tba675 were toxic to yeast in a manner that requires an intact S-HxxxE motif (Fig. 5e). The lack of toxicity by other tested toxins suggested that their cellular targets are absent in yeast or that such targets are not essential for yeast viability.

The LnaB-Actin complex reveals an AMPylator that recognizes ATP by a unique mechanism

To analyze the catalytic mechanism of LnaB, we solved the crystal structure of the LnaB_19-371-Actin complex, which had activity indistinguishable from that of full-length protein (Fig. 6). The asymmetric unit (ASU) of the structure contains five LnaB_19-371 and six Actin molecules, which form three LnaB_19-371-Actin heterodimeric complex (Supplementary Fig. 10a). In the LnaB_19-371-Actin complex, Actin mainly docks onto the carboxyl helix α14 of LnaB_19-371 (Fig. 6a) with an interface area of 1763.9 Å². The structure of LnaB contains fourteen helixes and a long loop that folds into two subdomains: The N-terminal domain (NTD) and the catalytic domain (CD). A structural homology search of LnaB_19-371 with the DaLi server did not yield any significant hits, suggesting that it is a novel folding protein. Interestingly, S261, H305, and E309, the three residues critical for catalysis form a continuous platform located in an area that has concentrated positive electrostatic potential (Fig. 6b), which may be the site for protein-protein or protein-substrate interactions.

**Fig. 6: LnaB-Actin binary complex structure reveals a unique catalytic mechanism on AMPylation.**

Two regions of LnaB_19-441 are in direct contact with Actin via extensive polar and hydrophobic interactions: a long loop consisting of a pair of antiparallel β-sheet proximal to the S-HxxxE motif and the carboxyl end helix α18, which we designated as Interface1 and Interface2, respectively. In interface 1, T225 of LnaB engages by hydrogen-bonding interaction with K113 and R116 of Actin; N220 of LnaB forms hydrogen bonds with A170 and Y169 of Actin; T209 of LnaB contacts residues E286 of Actin via a hydrogen bond (Fig. 6c). In interface 2, H359 engages in hydrogen-bonding interaction with T148 and E167 of Actin; E370 and Q363 form hydrogen bonds with R147 of Actin (Fig. 6d). Other hydrogen bonds include R365(L, LnaB):S348(A, Actin), E361(L):T351(A), L352(L):Y169(A), D347(L):R372(A) and Q355(L):Y143(A). L362 of LnaB is inserted into a hydrophobic pocket composed of I345, L346, and Y134 of Actin. Substitution of T225, E361, or L362 with alanine indeed reduced LnaB activity toward PR-Ub. Yet, single substitution mutation in other sites involved in its interaction with Actin did not detectably affect its enzymatic activity (Fig. 6e, f).

Despite extensive efforts, we were unable to obtain crystals of the LnaB-Actin complex containing ATP. We thus docked the ATP molecule into LnaB by molecular docking, which revealed that ATP may bind to a positively charged pocket neighboring the S261-H305-E309 motif of LnaB (Fig. 6g and Supplementary Fig. 10b). Alanine substitution in K199, Y299 or Y304 markedly reduced LnaB activity (Fig. 6h) and the binding of these mutants to ATP became almost undetectable (Fig. 6i and Supplementary Fig. 11), validating our hypothesis that this positively charged pocket is involved in ATP binding.

Discussion

Three mechanisms for protein AMPylation have been described²⁸, including the Cx₁₁DxD motif employed by the glutamine synthetase from E. coli and the multifunctional L. pneumophila effector SidM/DrrA^29,30,31, the Fic domain exemplified by VopS of Vibrio parahaemolyticus³² and the pseudokinase domain found in widely distributed proteins of the selenoprotein-O protein family³³. Differing from enzymes that only target hydroxyl-containing side chains of their substrate, LnaB targets the phosphate group in PR-Ub. Self-AMPylation by members of the S-HxxxE family suggests that at least a fraction of these enzymes AMPylate their substrate in host cells. The Fic domain has been shown to use CDP-choline³⁴ and UTP³⁵ as reactants, it is possible that some S-HxxxE proteins may use other nucleotides or their derivatives as substrates for protein modification. AMPylation is a reversible modification, signaling by this mechanism can be modulated by specific stimuli^36,37,38. It is of great interest to identify enzymes involved in the reversal of AMPylation catalyzed by these diverse modifiers.

The requirement of a host cell-specific co-factor allows pathogens to restrict the activity of virulence factors within target cells. For instance, both the edema factor of Bacillus anthracis and CyaA of Bordetella pertussis^39,40 use calmodulin (CaM) as the co-factor to ensure that cAMP is generated only in host cells. The requirement of CaM by the glutamylase SidJ is to prevent premature inactivation of SidEs in L. pneumophila^20,21. PR-Ub has only been found in cells infected by L. pneumophila^7,9,10 produced by DupA and DupB from proteins modified by SidEs. Our results have highlighted the importance of ubiquitin homeostasis in cells infected by L. pneumophila (Fig. 7). Yet, the reason for the requirement of Actin for LnaB activity is less clear. Actin dependence may prevent LnaB-induced ATP depletion, or active LnaB may recognize phosphoribosyl-bearing metabolites in bacterial cells. Alternatively, binding to Actin may facilitate the targeting of host proteins of relevant cellular processes such as NFκB signaling. Further investigation of the catalytic mechanism of LnaB, the function of toxins of this family, and the potential use of the S-HxxxE motif by eukaryotic cells in signaling will shed insights into not only protein biochemistry but also novel cell signaling cascades potentially important for development and disease.

Fig. 7: The cycling of ubiquitin by Dot/Icm effectors in cells infected by *L. pneumophila.*

Methods

Media, bacteria strains, plasmid construction and cell lines

Escherichia coli strains were grown on LB agar plates or in LB broth. When necessary, antibiotics were added to media at the following concentrations: ampicillin, 100 μg/mL; kanamycin, 30 μg/mL. L. pneumophila strains used in this study were derivatives of the Philadelphia 1 strain Lp02⁴¹. Lp03 is an isogenic dotA^- mutant⁴¹. All strains were grown and maintained on CYE plates or in ACES-buffered yeast extract (AYE) broth as previously described⁴¹. When needed, thymidine was added at a final concentration of 100 μg/mL. The Lp02∆dupA∆dupB mutant was described earlier⁹. Mutants lacking mavL or lnaB were constructed using strain Lp02 as previously described⁴². Complementation plasmids were constructed by inserting the gene of interest into pZL507⁴³. The plasmid for expression SdeA, mutants SdeA_E/A and SdeA_H/A were from previous studies^6,20. For ectopic expression of proteins in mammalian cells, genes were inserted into pEGFPC1 (Clontech) or p4xFlagCMV⁶. The plasmid for expressing 3xHA-Ub in mammalian cells had been described earlier⁶. Genes for purifications were cloned into pGEX-6P-1 (Amersham), pQE30 (QIANGEN), pET-28a (Novagen) or pET28a-Sumo (Novagen). The integrity of all constructs was verified by sequencing analysis. Genes coding for WP_015869719.1 from Edwardsiella ictaluri, WP_006755655.1 from Burkholderia ambifaria, and WP_075066242.1 from Candidatus Berkiella aquae, WP_148338824.1 from Aquicella siphonis, MAZ44397.1 from a species of Legionellales were synthesized by GenScript Biotech Corp (Nanjing, China) with codon optimized for E. coli. HEK293T cells purchased from the ATCC were cultured in Dulbecco’s modified minimal Eagle’s medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS). All mammalian cell lines were regularly checked for potential mycoplasma contamination by the universal mycoplasma detection kit from ATCC (Cat# 30-1012 K).

Yeast toxicity assays

Yeast strains were grown in YPD (1% yeast extract, 2% peptone, 2% glucose) or SD minimal media containing nitrogen base, glucose, and amino acid drop-out mix for selection of transformed plasmids as described⁴⁴. The genes coding for the testing toxins was individually inserted into pYES2/NTA (Invitrogen) that carries a galactose-inducible promoter⁴⁵. In each case, the sequence coding for the Flag tag was added to the amino terminal end of the gene to facilitate the detection of gene expression. The resulting plasmids were introduced into yeast strain W303⁴⁶, respectively. Ten microliters of 5-fold dilutions of saturated cultures were spotted in a dropout medium containing glucose or galactose. Plates were incubated at 30 ^oC for 3 d prior to image acquisition to assess growth. To detect protein expression, cells cultured in medium containing 2% raffinose were washed once with galactose medium and were induced in 2% galactose medium for 2 d at 30 ^oC.

Transfection, infection and immunoprecipitation

Plasmids were transfected into mammalian cells by using Lipofectamine 3000 (Invitrogen, cat# L3000150). After 24 h transfection, cells were collected and lysed with the TBS buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5) with 1% Triton X-100. When needed, immunoprecipitation was performed with lysates of transfected cells by Flag-specific antibody-coated agarose beads (Sigma, cat# F2426) or GFP-specific antibody, which was coupled to protein G beads (Cytiva, cat# 17061801) at 4 °C for 8 h. Beads were washed 3x with pre-cold lysis buffer. After that, Flag peptide solution (150 μg/mL) (Sigma, cat# F3290) or 0.1 M glycine buffer (pH 2.5) was used to elute Flag-tagged proteins or GFP-tagged proteins, respectively. All samples were resolved by SDS-PAGE and followed by immunoblotting analysis with the specific antibodies.

For intracellular bacterial growth, L. pneumophila strains grown to early post-exponential phase (OD₆₀₀ = 3.3–3.8) were used to infect bone marrow-derived macrophages or D. discoideum at an MOI of 0.05 as described earlier⁴². 2 h after adding the bacteria, infections were synchronized by washing cells with warm PBS to remove the extracellular bacteria, and IPTG was added at a final concentration of 0.05 mM to induce the expression of SdeA and its mutant. Infected cells were lysed with 0.02% saponin at the indicated time points, and the total viable bacterial cells were determined by plating appropriate dilutions on CYE plates.

To determine ADPR-Ub levels in infected cells, HEK293T cells were co-transfected with plasmids expressing the FcγII receptor and 3xHA-Ub⁶. The indicated L. pneumophila strains were grown to the post-exponential growth phase (OD₆₀₀ = 3.4–3.8) in AYET broth containing Kanamycin (20 µg/mL). Four h prior to infection, IPTG was added into the broth at a final concentration of 0.2 mM. L. pneumophila cells were opsonized by mixing with L. pneumophila-specific rabbit antibodies at a 1:500 ratio for 30 min at 37 °C. Thirty-six h post-transfection, opsonized bacteria were used to infect transfected cells at an MOI of 50. 2 h post-infection, cells were washed with cold PBS and lysed with TBS buffer containing 150 mM NaCl, 50 mM Tris-HCl, 1 mM DTT, 1% Triton X-100, following with 10% sonication for 10 s. Lysates were centrifuged twice at 20,000 × g for 15 min, and the supernatants were collected and incubated anti-HA beads at 4 °C for 8 h. Beads were washed three times with cold lysis buffer and were boiled in 1x sample buffer for 10 min.

To detect PR-Ub in cells infected with L. pneumophila strains, cell lysates obtained using an injection procedure described above were divided into two identical samples, and 10 μg His₆-LnaB was added to one sample, and 10 μL TBS buffer was added to another sample as a control. The reactions were allowed to proceed for 2 h at 37 °C and samples were then incubated with anti-HA beads to immunoprecipitated 3xHA-Ub by incubation at 4 °C for 8 h. Beads were washed three times in cold lysis buffer and were boiled in 1x sample buffer for 10 min prior to SDS-PAGE.

To determine the association of MavL and LnaB with the LCV, HEK293 cells transfected to express HA-MavL or 4Flag-LnaB were infected with the relevant L. pneumophila strains for 1 h, and the samples were stained with the appropriate antibodies. The intracellular and total bacteria were distinguished by sequential immunostaining. Samples were inspected and analyzed using an Olympus IX-83 florescence microscope.

Protein purification

For His₆- or GST-tagged recombinant protein production for in vitro assays, 20 mL saturated E. coli cultures were transferred to 400 mL LB medium supplemented with 30 μg/mL kanamycin or 100 μg/mL ampicillin, the cultures were grown to OD_600nm of 0.6–0.8 at 37 °C. Protein expression was induced with 0.2 mM IPTG at 18 °C for 16–18 h on a shaker (200 rpm). Bacterial cells were collected by centrifugation and lysed by sonication. The soluble lysates were cleared by spinning at 15,000 × g at 4 °C for 30 min. Supernatant containing recombinant proteins were purified by Ni²⁺-NTA beads (QIAGEN) or Glutathione agarose beads (Pierce), and were eluted from beads by using PBS buffer containing 300 mM imidazole or 10 mM reduced glutathione in a Tris buffer (50 mM Tris-HCl, pH 8.0). Purified proteins were dialyzed against PBS buffer containing 10% glycerol and 1 mM DTT at 4 °C for 8 h.

To purify proteins for structural study, the coding regions or the truncation mutants of MavL or LnaB were inserted into pET28a-sumo and the resulting plasmids each were transformed into E. coli strain BL21(DE3). The bacterial strains were cultured at 37 °C in LB broth on a shaker (220 rpm) and then induced with 0.5 mM IPTG when the bacteria grew to a density OD600 = 0.8. The bacteria were then cultured for 16 h at 18 °C before collecting the cells by centrifugation (5000 × g, 15 min). Cells resuspended in a cold lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl) were lysed by ultrasonication. Lysates were centrifuged at 35,000 × g for 30 min at 4 °C to obtain supernatant, which was used to purify His₆-tagged proteins by affinity chromatography (Ni²⁺ resin). The SUMO tag was removed by the SUMO protease ULP1. Proteins were further purified by size-exclusion chromatography using a Superdex 200 Increase column (GE Healthcare) equilibrated with a buffer containing 25 mM HEPES, pH 7.5, 150 mM NaCl, and 2 mM DTT. The best fractions of protein peak were pooled, concentrated to 10 mg/mL with an Amicon Centrifugal filter (Millipore), flash-frozen in liquid nitrogen, and stored at − 80 °C for crystallization and activity assay. Protein concentration was measured at A280 and calculated using their theoretical extinction coefficients.

Isothermal titration calorimetry

Isothermal titration calorimetry ITC experiments were carried out using a Low Volume Nano ITC (TA instrument) set at 20 °C. The protein of MavL or its mutants and ADPR were dissolved in the buffer containing 50 mM Tris-HCl, pH 7.5, and 150 mM NaCl. The concentration of ADPR in the syringe was 1 mM and the concentrations of proteins in the sample cell were 0.1 mM. Twenty-five consecutive 2 µl injections of ADPR were titrated into a 350 µl sample cell with a 200 s interval between injections using a stirring rate of 120 rpm. A single‐site binding model was used for nonlinear curve fitting using the Launch NanoAnalyze software provided by the manufacturer.

LnaB-Actin binding was performed using a microcalorimeter Affinity ITC (Waters^TM, USA) at 20 °C. LnaB and its mutants purified as above was diluted into a buffer of 50 mM Tris-HCl and 150 mM NaCl to a final concentration of 0.2 mM. Actin (Sangon Biotech, A001041) was prepared in the same buffer at a concentration of 22 μM. To measure LanB-ATP binding, Concentrations of LnaB or its mutants and ATP were 0.1 mM and 1 mM, respectively. Titrations were set for 20 injections each of 2 µL with 200 s intervals except from the first injection of 0.2 μL. Baseline subtraction and data analysis were performed using NanoAnayze (WatersTM, USA). Heat spikes were integrated and fitted with a 1:1 binding model. The first injection was excluded from the analysis.

Analytical ultracentrifugation (AUC)

Sedimentation velocity (SV) experiments were performed in a buffer containing 50 mM Tris-HCl,pH 8.0, and 150 mM NaCl using Proteomelab XL-I Analytical Ultracentrifuge (Beckman-Coulter). In AUC-SV analysis,runs were carried out at 50,000 r.p.m. and at a temperature of 10.0 °C using 12 mm charcoal-epon double sector centerpieces and An60 Ti analytical rotor. The evolution of the resulting concentration gradient was monitored with absorbance detection optics at 280 nm, All AUC-SV raw data were analyzed by the continuous C(s) distribution model implemented in the program SEDFIT⁴⁷. Partial specific volume and extinction coefficient of the protein as well as buffer density and viscosity, were calculated from amino acid and buffer composition, respectively, by the program SEDNTERP⁴⁸ and and were used to calculate protein concentration and correct experimental s-values to s_20,w.

Ni²⁺-agarose affinity pull-down assays

This assay was carried out in the binding buffer 150 mM NaCl, 20 mM Tris pH 8.0, 20 mM imidazole, and 0.02% Triton X-100. 1 mL of the reaction mixture, including 70 μg His-tagged LnaB or its mutants and 20 μg Actin, was incubated at 4 °C. After 4 h, 20 μL of nickel sepharose beads were added and incubated for another 1 h. The bound beads were added with 1.25 × SDS-loading buffer and boiled at 95 °C for 10 min after washing three times with the binding buffer, then separating by SDS-PAGE and detecting by an anti-Actin antibody.

Biochemical AMPylation assays

In a 20 μL reaction, 1 μg His₆-LnaB or its mutants, 1 μg Actin (Cytoskeleton, cat# APHL99), and 1.5 μg PR-Ub were used in a solution containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂ and 1 mM ATP, and the reaction was allowed to proceed for 1 h at 37 °C. To measure the activity of LnaB using ATP-α-³²P, 1 μg His₆-LnaB, 1 μg Actin (Cytoskeleton, cat# APHL99) and 1.5 μg PR-Ub were incubated in a 20 μL reaction system containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂ and 5 μCi ATP-α-³²P (Perkin Elmer, cat# BLU003H250UC) for 1 h at 37 °C. Samples were resolved by SDS-PAGE, and gels were stained with Coomassie brilliant blue. Gels were then dried and the signals were detected with x-ray films.

For the AMPylation activity of the selected members of the S-HxxxE family proteins, 1 μg His₆-LnaB or homologous proteins and 0.5 μg actin were incubated in a 20 μL reaction system containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂ and 5 µCi ATP-α-³²P for 2 h at 37 °C. Products were resolved by 12% SDS-PAGE at 100 V for 2 hr. Gels were stained with Coomassie brilliant blue (CBB) for 1 h, de-stained twice for 2 h and then dried for 8 h. Signals were detected with X-ray films using a BioMax TranScreen LE (Kodak) for 4 h at RT.

Biochemical de-ADP-ribosylation assays

To examine the activity of MavL on different modified ubiquitin, 2 μM MavL was incubated with 100 μM ADPR-Ub, PR-Ub or Ub for 30 min at 37 °C in a solution containing 50 mM Tris-HCl (pH 7.5). Proteins in reactions resolved by native PAGE or SDS-PAGE were detected by Coomassie blue stain or by immunoblotting with the anti-ADPR antibody. To determine the activity of MavL, DupA, and DupB against ADPR-Ub, 2 μM MavL, DupA, or DupB was incubated with 100 μM ADPR-Ub for 30 min at 37 °C in a solution containing 50 mM Tris-HCl (pH 7.5). Samples resolved by native PAGE or SDS-PAGE were detected by Coomassie blue stain, phosphoprotein stain (ABP Biosciences), or immunoblotting with the anti-ADPR antibody.

To examine the specificity of the de-ADP-ribosylation activity of MavL, we prepared several ADP-ribosylated proteins including ADPR-Actin catalyzed by SpvB of Salmonella enterica¹⁴, ADPR-ANT1 by Ceg3 of L. pneumophila¹⁵, ADPR-PARP1 by Sirt6¹⁶. In each case, the substrate protein and the enzyme were co-expressed in 293HEK cells by transfection, and the ADP-ribosylated proteins were isolated by immunoprecipitation using beads coated with antibody specific for the Flag or HA tag. ADPR-T66-Ub was generated by incubating His₆-ubiquitin with GST-CteC of C. violaceum¹³. Each of the ADP-ribosylated proteins was incubated with MavL or MavL_D323A for 30 min at 37 °C and de-ADP-ribosylation effects were probed by immunoblotting using the anti-ADPR antibody.

Biochemical ubiquitination assays

For the SdeA-mediated ubiquitination reaction, 2 μg His₆-LnaB, 1.5 μg Actin (Cytoskeleton, cat# APHL99) and 6 μg PR-Ub were preincubated in a 25 μL reaction system containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂ and 1 mM ATP for 1 h at 37 °C. After preincubation, a cocktail containing 0.1 μg His₆-SdeA_E/A and 0.6 μg His₆−4xFlag-Rab33b were supplemented into reactions, and the reaction was allowed to proceed for another 2 h at 37 °C.

For SidC-mediated ubiquitination reaction⁴⁹, 0.3 μg GST-E1, 1 μg His₆-UbcH7, 3 μg GST-SidC_1-542 and 4 μg Ub were incubated in a 20 μL reaction system containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 1 mM DTT and 2 mM ATP for 1 h at 37 °C.

To test the ability of LnaB and MavL to convert PR-Ub into active ubiquitin, 1.5 μg His₆-LnaB, 1.5 μg Actin, 1.5 μg GST-MavL and 4 μg PR-Ub were preincubated in a 25 μL reaction system containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂ and 1 mM ATP for 1 h at 37 °C. After preincubation, reactions were boiled for 5 min at 95 °C, then a cocktail containing 0.3 μg GST-E1, 1 μg His₆-UbcH7, 3 μg GST-SidC_1-542, 1 mM DTT, and 2 mM ATP were supplemented into these boiled reactions and the reaction was allowed to proceed for another 1 h at 37 °C.

Antibodies and immunoblotting

Purified His₆-GFP and His₆-GST proteins were used to raise rabbit-specific antibodies using a standard protocol (Pocono Rabbit Farm & Laboratory). These antibodies were affinity purified as described. Antibodies specific for SdeA had been described⁶. For immunoblotting, samples resolved by SDS-PAGE were transferred onto 0.2 μm nitrocellulose membranes (Bio-Rad, cat# 1620112), which were blocked with 5% non-fat milk or 3% BSA at R.T. for 1 h prior to being incubated with the appropriate primary antibodies: anti-Flag (Sigma, cat# F1804), 1:5000; anti-Actin (MP Biochemicals, cat# 0869100), 1:5000; anti-tubulin (DSHB, E7) 1:10,000; anti-ADPR (Sigma, cat# MABE1016), 1:1000, anti-His (Sigma, cat# H1029), 1:5000; anti-Ub (Santa Cruz, P4D1, cat# sc-8017), 1:1000. Membranes were then incubated with appropriate IRDye infrared secondary antibodies and scanned by an Odyssey infrared imaging system (Li-Cor’s Biosciences).

LC-MS/MS analysis

Protein bands were digested in-gel with trypsin as previously described⁵⁰. Digested peptides were analyzed by LC-ESI-MS/MS using the Dionex UltiMate 3000 RSLC nano System coupled to the Q Exactive™ HF Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific, Waltham, MA). The reverse phase peptide separation was accomplished using a trap column (300 μm ID × 5 mm) packed with 5 μm 100 Å PepMap C18 medium, and then separated on a reverse phase column (50 cm long × 75 µm ID) packed with 2 µm 100 Å PepMap C18 silica (Thermo Fisher Scientific, Waltham, MA). The column temperature was maintained at 50 °C.

Mobile phase solvent A was 0.1% FA in water, and solvent B was 0.1% FA in 80% ACN. Loading buffer was 98% water/2% ACN/0.1% FA. Peptides were separated by loading into the trap column in a loading buffer for 5 min at a 5 µL/min flow rate and eluted from the analytical column at a flow rate of 150 µL/min using a 130 min LC gradient as follows: linear gradient of 5% to 27% of solvent B in 80 min, 27–45% in next 20 min, 45–100% of B in next 5 min at which point the gradient was held at 100% of B for 7 min before reverting back to 2% of B at 112 min, and held at 2% of B for next 18 min for equilibration. The mass spectrometer was operated in positive ion and standard data-dependent acquisition mode with the Advanced Peak Detection function activated for the top 20n. The fragmentation of precursor ions was accomplished by a stepped normalized collision energy setting of 27%. The resolution of the Orbitrap mass analyzer was set to 120,000 and 15,000 for MS1 and MS2, respectively. The full scan MS1 spectra were collected in the mass range of 350–1600 m/z, with an isolation window of 1.2 m/z and a fixed first mass of 100 m/z for MS2. The spray voltage was set at 2, and the Automatic Gain Control (AGC) target of 4e5 for MS1 and 5e4 for MS2, respectively.

For protein identification, the raw data were processed with the software MaxQuant (version 1.6.3.3) against the Homo sapiens database (Uniprot, UP000005640) or L. pneumophila database (Uniprot, UP000000609). MaxQuant was set to search with the following parameters: peptide tolerance at 10 ppm, MS/MS tolerance at 0.02 Da, carbamidomethyl (C) as a fixed modification, oxidation (M) as a variable modification, and a maximum of two missed cleavages. The false-discovery rates (FDR) were controlled at <1%. To identify the Phosphoribosylation or ADP-ribosylation modification peptides, raw data were analyzed manually in Xcalibur QualBrowser.

Crystallization, data collection, and structural determination

Crystallization of the complexes of MavL-ADPR, MavL_{40-404(D315A)}-ADPR-Ub, and LnaB-Actin were conducted using the hanging-drop vapor diffusion method at 16 °C, with drops containing 0.5 µl of the protein solution mixed with 0.5 µl of reservoir solution. Diffraction quality of the MavL-ADPR complex crystals was obtained in 0.25 M potassium citrate tribasic monohydrate,18% PEG3350, and MavL_{40-404(D315A)}-ADPR-Ub complex crystals was obtained in 0.1 M HEPES/sodium hydroxide pH7.5, 20% polyethylene glycol 10,000, respectively. LnaB-Actin complex crystals were observed in 0.15 M Ammonium sulfate, 0.1 M Sodium HEPES pH 7.0, 20% (w/v) polyethylene glycol (PEG) 4000 after one week. After optimization, the best crystals of LnaB-Actin were obtained in 0.15 M Ammonium sulfate, 0.1 M Sodium HEPES pH 7.0, 22% (w/v) PEG 4000. Crystals were harvested and flash-frozen in liquid nitrogen with 20% glycerol as a cryoprotectant. Complete X-ray diffraction data sets were collected at the BL02U1 beamline of the Shanghai Synchrotron Radiation Facility (SSRF). Diffraction images were processed with the HKL-2000 program. Molecular Replacement was then performed with the model of Apo MavL (PDB:6OMI) and Ub (PDB:6K11) as a template to determine the structure of the MavL-ADPR (PDB:8IPW) and MavL_{40-404(D315A)}-ADPR-Ub (PDB:8IPJ) complexes, respectively. The Actin and LnaB structures (1–361 region) predicted with Alphafold2⁵¹ were employed as a template. Model building and crystallographic refinement were carried out in Coot and PHENIX⁵². Detailed data collection and refinement statistics are listed in Supplementary Table 1. The interactions were analyzed with PyMOL (http://www.pymol.org/), and PDBsum and figures were generated with PyMOL.

Bioinformatic identification of members of the S-HxxxE family

To obtain LnaB orthologous sequences from other genera (we excluded the Legionella genus from the blast search), we utilized PSI-BLAST²⁵ (Position-Specific Iterative Basic Local Alignment Search Tool) searches at the NCBI (National Center for Biotechnology Information; http://blast.ncbi.nlm.nih.gov/) against the nr (non-redundant) protein database. We limited the PSI-BLAST search to three rounds to minimize the effect of possible convergent evolution while still being able to detect all the genera that contain LnaB orthologs. For the purpose of phylogenetic analysis and tree construction, we first removed duplicate sequences from the same species and selected closely related protein sequences for analysis. Sequence alignment was generated by MAFFT⁵³, and the resulting alignment was used to infer a phylogenetic tree using IQ-TREE⁵⁴. VT + F + R4 model was selected by the ModelFinder⁵⁵ with 1000 nonparametric replicated bootstrap analyses. The obtained phylogenetic tree was visualized with iTOL web-server⁵⁶.

Molecular docking

The configuration of ATP was optimized at the B3LYP/6-31 G* level using the Gaussian 09 package⁵⁷. The structure of LnaB was extracted from the crystal structure of the LnaB/Actin complex resolved in this work. We carried out 300 independent docking runs with the AutoDock 4.2 program⁵⁸ using the Lamarckian genetic algorithm (LGA)⁵⁹ as a search engine. The size of the grid box was 90 × 90 × 90 with a grid spacing of 0.375 Å. The grid center was set at the center of mass of LnaB. ATP was treated as a flexible molecule whereas LnaB was rigid. The results of 300 docking runs were grouped into clusters according to the ligand binding conformation with default parameters implemented in AutoDockTools 1.5⁵⁸. Among these cluster, there was one dominant cluster accounting for 68.7% of total docking conformations (Supplementary Fig. 10b). Furthermore, this cluster held the lowest mean binding energy (− 6.53 ± 0.41 kcal/mol) as calculated by AutoDockTools 1.5 (Supplementary Fig.10b), indicating a robust structural stability. Thus, the conformation with the lowest binding energy from this cluster was selected as the putative complex model. The residues in the ATP binding site were defined by an atomic distance-based cutoff: the residues contained at least one atom within 4.5 Å of any atom in the bound ATP. Accordingly, the binding site residues of ATP included residues Y196, K199, R201, P235, I237, A258-G262, and G303-H305.

Statistics and reproducibility

The Student’s t test was used to compare the mean levels between two groups, each with at least three independent samples. All immunoblotting results shown are representative of three independent experiments.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Structure factors and atomic coordinates have been deposited in the Protein Data Bank (PDB) under accessions: MavL-ADPR (PDB:8IPW), MavL40-404(D315A)-ADPR-Ub (PDB:8IPJ), LnaB-actin (PDB:8J9B). The data that support the conclusions of this study are included in this published article along with its Supplementary Information files and are also available from the corresponding author upon request. LnaB interacting proteins identified by IP-MS are included in Supplementary Dataset 1, including full gels and blots provided in the Source Data file. Source data are provided as a Source Data file. Source data are provided with this paper.

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Acknowledgements

This work was supported in part by the National Key Research and Development Program of China 2021YFC2301403 (S.O.) and 2022YFA1304500 (X.L.), the National Natural Science Foundation of China grants 32370185 (J.F.), 32270185 (L.S.), 82225028 and 82172287 (S.O.), 31900879 and 32171265 (H.G.), 21974002 and 22174003 (X.L.), and the High-level personnel introduction grant of Fujian Normal University (Z0210509) (S.O.), and by National Institutes of Health grants R01AI127465 and R01GM126296. Mass spectrometric analysis was performed in the core facility of the First Hospital of Jilin University, the authors thank Dr. Naicui Zhai for assistance.

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These authors contributed equally: Jiaqi Fu, Siying Li, Hongxin Guan, Chuang Li, Yan-Bo Zhao.

Authors and Affiliations

Department of Respiratory Medicine, Center for Infectious Diseases and Pathogen Biology, Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, The First Hospital of Jilin University, Changchun, China
Jiaqi Fu, Siying Li, Qingtian Guan & Lei Song
Key Laboratory of Microbial Pathogenesis and Interventions of Fujian Province University, the Key Laboratory of Innate Immune Biology of Fujian Province, Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, China
Hongxin Guan, Yan-Bo Zhao, Tao-Tao Chen, Jingting Wang, Qiuhua Lu, Lina Kang, Si-Ru Zheng & Songying Ouyang
Department of Biological Sciences, Purdue University, West Lafayette, IN, USA
Chuang Li, Yao Liu & Zhao-Qing Luo
Department of Microbiology, NHC Key Laboratory of Medical Immunology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China
Wei Xian & Xiaoyun Liu
Department of Chemistry, Purdue University, West Lafayette, IN, USA
Zhengrui Zhang & Chittaranjan Das
College of Chemistry, Fuzhou University, Fuzhou, Fujian, China
Jinyu Li & Shoujing Cao

Authors

Jiaqi Fu
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Siying Li
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Hongxin Guan
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Chuang Li
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Yan-Bo Zhao
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Tao-Tao Chen
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Wei Xian
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Zhengrui Zhang
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Yao Liu
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Qingtian Guan
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Jingting Wang
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Qiuhua Lu
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Lina Kang
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Si-Ru Zheng
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Jinyu Li
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Shoujing Cao
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Chittaranjan Das
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Xiaoyun Liu
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Lei Song
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Songying Ouyang
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Zhao-Qing Luo
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Contributions

Z.Q.L., J.F., X.L., S.O., and L.S. conceived the ideas for this work. Unless otherwise specified, J.F., S.L., and C.L. performed the biochemical experiments and infection experiments. L.S. performed the yeast experiments; H.G. and Z.Z. performed the experiments with MavL, Y.L., and Q.G. performed the bioinformatics analysis, J.F., W.X., and X.L. performed mass spectrometric analyses. H.G., Y.Z., T.T.C., J.W., Q.L., L.K., S.Z., and S.O. performed structural studies and analyzed protein binding using biophysical tools. J.L. and S.C. performed the molecular docking. Z.Q.L., J.F., X.L., L.S., S.O., and C.D. interpreted the results. Z.Q.L. wrote the manuscript and all authors provided editorial input.

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Correspondence to Xiaoyun Liu, Lei Song, Songying Ouyang or Zhao-Qing Luo.

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Fu, J., Li, S., Guan, H. et al. Legionella maintains host cell ubiquitin homeostasis by effectors with unique catalytic mechanisms. Nat Commun 15, 5953 (2024). https://doi.org/10.1038/s41467-024-50311-2

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Received: 01 April 2024
Accepted: 05 July 2024
Published: 15 July 2024
DOI: https://doi.org/10.1038/s41467-024-50311-2
Springer Nature Limited

Legionella maintains host cell ubiquitin homeostasis by effectors with unique catalytic mechanisms

Abstract

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Introduction

Results

The macro domain protein MavL is an (ADP-ribosyl)hydrolase against ADPR-Ub

The effector LnaB is an adenylyltransferase that converts PR-Ub into ADPR-Ub

LnaB utilizes an S-HxxxE motif to catalyze the conversion of PR-Ub into ADPR-Ub

LnaB and tested members of the S-HxxxE family catalyze protein AMPylation

The LnaB-Actin complex reveals an AMPylator that recognizes ATP by a unique mechanism

Discussion

Methods

Media, bacteria strains, plasmid construction and cell lines

Yeast toxicity assays

Transfection, infection and immunoprecipitation

Protein purification

Isothermal titration calorimetry

Analytical ultracentrifugation (AUC)

Ni2+-agarose affinity pull-down assays

Biochemical AMPylation assays

Biochemical de-ADP-ribosylation assays

Biochemical ubiquitination assays

Antibodies and immunoblotting

LC-MS/MS analysis

Crystallization, data collection, and structural determination

Bioinformatic identification of members of the S-HxxxE family

Molecular docking

Statistics and reproducibility

Reporting summary

Data availability

References

Acknowledgements

Author information

Authors and Affiliations

Contributions

Corresponding authors

Ethics declarations

Competing interests

Peer review

Peer review information

Additional information

Supplementary information

Source data

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Ni²⁺-agarose affinity pull-down assays