Pronucleotide Probes Reveal a Diverging Specificity for AMPylation vs UMPylation of Human and Bacterial Nucleotide Transferases.

AMPylation is a post-translational modification utilized by human and bacterial cells to modulate the activity and function of specific proteins. Major AMPylators such as human FICD and bacterial VopS have been studied extensively for their substrate and target scope in vitro. Recently, an AMP pronucleotide probe also facilitated the in situ analysis of AMPylation in living cells. Based on this technology, we here introduce a novel UMP pronucleotide probe and utilize it to profile uninfected and Vibrio parahaemolyticus infected human cells. Mass spectrometric analysis of labeled protein targets reveals an unexpected promiscuity of human nucleotide transferases with an almost identical target set of AMP- and UMPylated proteins. Vice versa, studies in cells infected by V. parahaemolyticus and its effector VopS revealed solely AMPylation of host enzymes, highlighting a so far unknown specificity of this transferase for ATP. Taken together, pronucleotide probes provide an unprecedented insight into the in situ activity profile of crucial nucleotide transferases, which can largely differ from their in vitro activity.

The DNA-binding induced (de)AMPylation activity of a Coxiella burnetii Fic enzyme targets Histone H3.

The intracellular bacterial pathogen Coxiella burnetii evades the host response by secreting effector proteins that aid in establishing a replication-friendly niche. Bacterial filamentation induced by cyclic AMP (Fic) enzymes can act as effectors by covalently modifying target proteins with the posttranslational AMPylation by transferring adenosine monophosphate (AMP) from adenosine triphosphate (ATP) to a hydroxyl-containing side chain. Here we identify the gene product of C. burnetii CBU_0822, termed C. burnetii Fic 2 (CbFic2), to AMPylate host cell histone H3 at serine 10 and serine 28. We show that CbFic2 acts as a bifunctional enzyme, both capable of AMPylation as well as deAMPylation, and is regulated by the binding of DNA via a C-terminal helix-turn-helix domain. We propose that CbFic2 performs AMPylation in its monomeric state, switching to a deAMPylating dimer upon DNA binding. This study unveils reversible histone modification by a specific enzyme of a pathogenic bacterium.

Dephosphocholination by Legionella effector Lem3 functions through remodelling of the switch II region of Rab1b.

Bacterial pathogens often make use of post-translational modifications to manipulate host cells. Legionella pneumophila, the causative agent of Legionnaires disease, secretes the enzyme AnkX that uses cytidine diphosphate-choline to post-translationally modify the human small G-Protein Rab1 with a phosphocholine moiety at Ser76. Later in the infection, the Legionella enzyme Lem3 acts as a dephosphocholinase, hydrolytically removing the phosphocholine. While the molecular mechanism for Rab1 phosphocholination by AnkX has recently been resolved, structural insights into the activity of Lem3 remained elusive. Here, we stabilise the transient Lem3:Rab1b complex by substrate mediated covalent capture. Through crystal structures of Lem3 in the apo form and in complex with Rab1b, we reveal Lem3's catalytic mechanism, showing that it acts on Rab1 by locally unfolding it. Since Lem3 shares high structural similarity with metal-dependent protein phosphatases, our Lem3:Rab1b complex structure also sheds light on how these phosphatases recognise protein substrates.

The Alarmone Diadenosine Tetraphosphate as a Cosubstrate for Protein AMPylation.

Diadenosine polyphosphates (Apn As) are non-canonical nucleotides whose cellular concentrations increase during stress and are therefore termed alarmones, signaling homeostatic imbalance. Their cellular role is poorly understood. In this work, we assessed Apn As for their usage as cosubstrates for protein AMPylation, a post-translational modification in which adenosine monophosphate (AMP) is transferred to proteins. In humans, AMPylation mediated by the AMPylator FICD with ATP as a cosubstrate is a response to ER stress. Herein, we demonstrate that Ap4 A is proficiently consumed for AMPylation by FICD. By chemical proteomics using a new chemical probe, we identified new potential AMPylation targets. Interestingly, we found that AMPylation targets of FICD may differ depending on the nucleotide cosubstrate. These results may suggest that signaling at elevated Ap4 A levels during cellular stress differs from when Ap4 A is present at low concentrations, allowing response to extracellular cues.

Revisiting AMPylation through the lens of Fic enzymes.

AMPylation, a post-translational modification (PTM) first discovered in the late 1960s, is catalyzed by adenosine monophosphate (AMP)-transferring enzymes. The observation that filamentation-induced-by-cyclic-AMP (fic) enzymes are associated with this unique PTM revealed that AMPylation plays a major role in hijacking of host signaling by pathogenic bacteria during infection. Studies over the past decade showed that AMPylation is conserved across all kingdoms of life and, outside their role in infection, also modulates cellular functions. Many aspects of AMPylation are yet to be uncovered. In this review we present the advancement in research on AMPylation and Fic enzymes as well as other distinct classes of enzymes that catalyze AMPylation.

Erratum: Monoclonal Anti-AMP Antibodies Are Sensitive and Valuable Tools for Detecting Patterns of AMPylation.

[This corrects the article DOI: 10.1016/j.isci.2020.101800.].

Specificity of AMPylation of the human chaperone BiP is mediated by TPR motifs of FICD.

To adapt to fluctuating protein folding loads in the endoplasmic reticulum (ER), the Hsp70 chaperone BiP is reversibly modified with adenosine monophosphate (AMP) by the ER-resident Fic-enzyme FICD/HYPE. The structural basis for BiP binding and AMPylation by FICD has remained elusive due to the transient nature of the enzyme-substrate-complex. Here, we use thiol-reactive derivatives of the cosubstrate adenosine triphosphate (ATP) to covalently stabilize the transient FICD:BiP complex and determine its crystal structure. The complex reveals that the TPR-motifs of FICD bind specifically to the conserved hydrophobic linker of BiP and thus mediate specificity for the domain-docked conformation of BiP. Furthermore, we show that both AMPylation and deAMPylation of BiP are not directly regulated by the presence of unfolded proteins. Together, combining chemical biology, crystallography and biochemistry, our study provides structural insights into a key regulatory mechanism that safeguards ER homeostasis.

SopD from Salmonella specifically inactivates Rab8.

Salmonella outer protein D (SopD) is secreted into a host during the first stages of the Salmonella infection and contributes to the systemic virulence of the bacterium. SopD2 is a SopD homolog and possesses GTPase activating protein (GAP) activity towards Rab32. Here, we identified Rab-proteins as putative SopD-targets using a yeast two-hybrid approach. In vitro investigations subsequently revealed Rab8a as an exclusive SopD substrate in contrast to SopD2, which has a broader specificity targeting Rab29, Rab32 and Rab38 in vitro. Additionally, we determined the catalytic efficiencies of SopD and SopD2 towards their physiologically relevant substrates. Moreover, mutagenesis studies provided insights into possible key residues of the Rab-protein and the GAP involved in the conversion of active to inactive GTPase. In conclusion, we demonstrate that Salmonella SopD and SopD2 act as RabGAPs and can inactivate Rab signaling.

Current Advances in Covalent Stabilization of Macromolecular Complexes for Structural Biology.

Structural characterization of macromolecular assemblies is often limited by the transient nature of the interactions. The development of specific chemical tools to covalently tether interacting proteins to each other has played a major role in various fundamental discoveries in recent years. To this end, protein engineering techniques such as mutagenesis, incorporation of unnatural amino acids, and methods using synthetic substrate/cosubstrate derivatives were employed. In this review, we give an overview of both commonly used and recently developed biochemical methodologies for covalent stabilization of macromolecular complexes enabling structural investigation via crystallography, nuclear magnetic resonance, and cryo-electron microscopy. We divided the strategies into nonenzymatic- and enzymatic-driven cross-linking and further categorized them in either naturally occurring or engineered covalent linkage. This review offers a compilation of recent advances in diverse scientific fields where the structural characterization of macromolecular complexes was achieved by the aid of intermolecular covalent linkage.

Rab1-AMPylation by Legionella DrrA is allosterically activated by Rab1.

Legionella pneumophila infects eukaryotic cells by forming a replicative organelle - the Legionella containing vacuole. During this process, the bacterial protein DrrA/SidM is secreted and manipulates the activity and post-translational modification (PTM) states of the vesicular trafficking regulator Rab1. As a result, Rab1 is modified with an adenosine monophosphate (AMP), and this process is referred to as AMPylation. Here, we use a chemical approach to stabilise low-affinity Rab:DrrA complexes in a site-specific manner to gain insight into the molecular basis of the interaction between the Rab protein and the AMPylation domain of DrrA. The crystal structure of the Rab:DrrA complex reveals a previously unknown non-conventional Rab-binding site (NC-RBS). Biochemical characterisation demonstrates allosteric stimulation of the AMPylation activity of DrrA via Rab binding to the NC-RBS. We speculate that allosteric control of DrrA could in principle prevent random and potentially cytotoxic AMPylation in the host, thereby perhaps ensuring efficient infection by Legionella.

Proteolysis of Rab32 by Salmonella GtgE induces an inactive GTPase conformation.

Rab GTPases are central regulators of intracellular vesicular trafficking. They are frequently targeted by bacterial pathogens through post-translational modifications. Salmonella typhimurium secretes the cysteine protease GtgE during infection, leading to a regioselective proteolytic cleavage of the regulatory switch I loop in the small GTPases of the Rab32 subfamily. Here, using a combination of biochemical methods, molecular dynamics simulations, NMR spectroscopy, and single-pair Förster resonance energy transfer, we demonstrate that the cleavage of Rab32 causes a local increase of conformational flexibility in both switch regions. Cleaved Rab32 maintains its ability to interact with the GDP dissociation inhibitor (GDI). Interestingly, the Rab32 cleavage enables GDI binding also with an active GTP-bound Rab32 in vitro. Furthermore, the Rab32 proteolysis provokes disturbance in the interaction with its downstream effector VARP. Thus, the proteolysis of Rab32 is not a globally degradative mechanism but affects various biochemical and structural properties of the GTPase in a diverse manner.

Monoclonal Anti-AMP Antibodies Are Sensitive and Valuable Tools for Detecting Patterns of AMPylation.

AMPylation is a post-translational modification that modifies amino acid side chains with adenosine monophosphate (AMP). Recently, a role of AMPylation as a universal regulatory mechanism in infection and cellular homeostasis has emerged, driving the demand for universal tools to study this modification. Here, we describe three monoclonal anti-AMP antibodies (mAbs) from mouse that are capable of protein backbone-independent recognition of AMPylation, in denatured (western blot) as well as native (ELISA, IP) applications, thereby outperforming previously reported tools. These antibodies are highly sensitive and specific for AMP modifications, highlighting their potential as tools for new target identification, as well as for validation of known targets. Interestingly, applying the anti-AMP mAbs to various cancer cell lines reveals a previously undescribed broad and diverse AMPylation pattern. In conclusion, these anti-AMP mABs will further advance the current understanding of AMPylation and the spectrum of modified targets.

Identification of targets of AMPylating Fic enzymes by co-substrate-mediated covalent capture.

Various pathogenic bacteria use post-translational modifications to manipulate the central components of host cell functions. Many of the enzymes released by these bacteria belong to the large Fic family, which modify targets with nucleotide monophosphates. The lack of a generic method for identifying the cellular targets of Fic family enzymes hinders investigation of their role and the effect of the post-translational modification. Here, we establish an approach that uses reactive co-substrate-linked enzymes for proteome profiling. We combine synthetic thiol-reactive nucleotide derivatives with recombinantly produced Fic enzymes containing strategically placed cysteines in their active sites to yield reactive binary probes for covalent substrate capture. The binary complexes capture their targets from cell lysates and permit subsequent identification. Furthermore, we determined the structures of low-affinity ternary enzyme-nucleotide-substrate complexes by applying a covalent-linking strategy. This approach thus allows target identification of the Fic enzymes from both bacteria and eukarya.

The trimer to monomer transition of Tumor Necrosis Factor-Alpha is a dynamic process that is significantly altered by therapeutic antibodies.

The cytokine tumor necrosis factor-alpha (TNF-α) readily forms homotrimers at sub-nM concentrations to promote inflammation. For the treatment of inflammatory diseases with upregulated levels of TNF-α, a number of therapeutic antibodies are currently used as scavengers to reduce the active TNF-α concentration in patients. Despite their clinical success, the mode-of-action of different antibody formats with regard to a stabilization of the trimeric state is not entirely understood. Here, we use a biosensor with dynamic nanolevers to analyze the monomeric and trimeric states of TNF-α together with the binding kinetics of therapeutic biologics. The intrinsic trimer-to-monomer decay rate k = 1.7 × 10-3 s-1 could be measured directly using a microfluidic system, and antibody binding affinities were analyzed in the pM range. Trimer stabilization effects are quantified for Adalimumab, Infliximab, Etanercept, Certolizumab, Golimumab for bivalent and monovalent binding formats. Clear differences in trimer stabilization are observed, which may provide a deeper insight into the mode-of-action of TNF-α scavengers.

Legionella effector AnkX displaces the switch II region for Rab1b phosphocholination.

The causative agent of Legionnaires disease, Legionella pneumophila, translocates the phosphocholine transferase AnkX during infection and thereby posttranslationally modifies the small guanosine triphosphatase (GTPase) Rab1 with a phosphocholine moiety at S76 using cytidine diphosphate (CDP)-choline as a cosubstrate. The molecular basis for Rab1 binding and enzymatic modification have remained elusive because of lack of structural information of the low-affinity complex with AnkX. We combined thiol-reactive CDP-choline derivatives with recombinantly introduced cysteines in the AnkX active site to covalently capture the heterocomplex. The resulting crystal structure revealed that AnkX induces displacement of important regulatory elements of Rab1 by placing a ß sheet into a conserved hydrophobic pocket, thereby permitting phosphocholine transfer to the active and inactive states of the GTPase. Together, the combination of chemical biology and structural analysis reveals the enzymatic mechanism of AnkX and the family of filamentation induced by cyclic adenosine monophosphate (FIC) proteins.

PINK1-dependent phosphorylation of Serine111 within the SF3 motif of Rab GTPases impairs effector interactions and LRRK2-mediated phosphorylation at Threonine72.

Loss of function mutations in the PTEN-induced kinase 1 (PINK1) kinase are causal for autosomal recessive Parkinson's disease (PD) whilst gain of function mutations in the LRRK2 kinase cause autosomal dominant PD. PINK1 indirectly regulates the phosphorylation of a subset of Rab GTPases at a conserved Serine111 (Ser111) residue within the SF3 motif. Using genetic code expansion technologies, we have produced stoichiometric Ser111-phosphorylated Rab8A revealing impaired interactions with its cognate guanine nucleotide exchange factor and GTPase activating protein. In a screen for Rab8A kinases we identify TAK1 and MST3 kinases that can efficiently phosphorylate the Switch II residue Threonine72 (Thr72) in a similar manner as LRRK2 in vitro. Strikingly, we demonstrate that Ser111 phosphorylation negatively regulates the ability of LRRK2 but not MST3 or TAK1 to phosphorylate Thr72 of recombinant nucleotide-bound Rab8A in vitro and demonstrate an interplay of PINK1- and LRRK2-mediated phosphorylation of Rab8A in transfected HEK293 cells. Finally, we present the crystal structure of Ser111-phosphorylated Rab8A and nuclear magnetic resonance structure of Ser111-phosphorylated Rab1B. The structures reveal that the phosphorylated SF3 motif does not induce any major changes, but may interfere with effector-Switch II interactions through intramolecular H-bond formation and/or charge effects with Arg79. Overall, we demonstrate antagonistic regulation between PINK1-dependent Ser111 phosphorylation and LRRK2-mediated Thr72 phosphorylation of Rab8A indicating a potential cross-talk between PINK1-regulated mitochondrial homeostasis and LRRK2 signalling that requires further investigation in vivo.

Divergent Evolution of Legionella RCC1 Repeat Effectors Defines the Range of Ran GTPase Cycle Targets.

Legionella pneumophila governs its interactions with host cells by secreting >300 different "effector" proteins. Some of these effectors contain eukaryotic domains such as the RCC1 (regulator of chromosome condensation 1) repeats promoting the activation of the small GTPase Ran. In this report, we reveal a conserved pattern of L. pneumophila RCC1 repeat genes, which are distributed in two main clusters of strains. Accordingly, strain Philadelphia-1 contains two RCC1 genes implicated in bacterial virulence, legG1 (Legionella eukaryotic gene 1), and ppgA, while strain Paris contains only one, pieG The RCC1 repeat effectors localize to different cellular compartments and bind distinct components of the Ran GTPase cycle, including Ran modulators and the small GTPase itself, and yet they all promote the activation of Ran. The pieG gene spans the corresponding open reading frames of legG1 and a separate adjacent upstream gene, lpg1975legG1 and lpg1975 are fused upon addition of a single nucleotide to encode a protein that adopts the binding specificity of PieG. Thus, a point mutation in pieG splits the gene, altering the effector target. These results indicate that divergent evolution of RCC1 repeat effectors defines the Ran GTPase cycle targets and that modulation of different components of the cycle might fine-tune Ran activation during Legionella infection.IMPORTANCELegionella pneumophila is a ubiquitous environmental bacterium which, upon inhalation, causes a life-threatening pneumonia termed Legionnaires' disease. The opportunistic pathogen grows in amoebae and macrophages by employing a "type IV" secretion system, which secretes more than 300 different "effector" proteins into the host cell, where they subvert pivotal processes. The function of many of these effector proteins is unknown, and their evolution has not been studied. L. pneumophila RCC1 repeat effectors target the small GTPase Ran, a molecular switch implicated in different cellular processes such as nucleocytoplasmic transport and microtubule cytoskeleton dynamics. We provide evidence that one or more RCC1 repeat genes are distributed in two main clusters of L. pneumophila strains and have divergently evolved to target different components of the Ran GTPase activation cycle at different subcellular sites. Thus, L. pneumophila employs a sophisticated strategy to subvert host cell Ran GTPase during infection.

Conformational control of small GTPases by AMPylation.

Posttranslational modifications (PTMs) are important physiological means to regulate the activities and structures of central regulatory proteins in health and disease. Small GTPases have been recognized as important molecules that are targeted by PTMs during infections of mammalian cells by bacterial pathogens. The enzymes DrrA/SidM and AnkX from Legionella pneumophila AMPylate and phosphocholinate Rab1b during infection, respectively. Cdc42 is AMPylated by IbpA from Histophilus somni at tyrosine 32 or by VopS from Vibrio parahaemolyticus at threonine 35. These modifications take place in the important regulatory switch I or switch II regions of the GTPases. Since Rab1b and Cdc42 are central regulators of intracellular vesicular trafficking and of the actin cytoskeleton, their modifications by bacterial pathogens have a profound impact on the course of infection. Here, we addressed the biochemical and structural consequences of GTPase AMPylation and phosphocholination. By combining biochemical experiments and NMR analysis, we demonstrate that AMPylation can overrule the activity state of Rab1b that is commonly dictated by binding to guanosine diphosphate or guanosine triphosphate. Thus, PTMs may exert conformational control over small GTPases and may add another previously unrecognized layer of activity control to this important regulatory protein family.

Validation of the Slow Off-Kinetics of Sirtuin-Rearranging Ligands (SirReals) by Means of Label-Free Electrically Switchable Nanolever Technology.

We have discovered the sirtuin-rearranging ligands (SirReals) to be highly potent and selective inhibitors of the NAD+ -dependent lysine deacetylase Sirt2. Using a biotinylated SirReal in combination with biolayer interferometry, we previously observed a slow dissociation rate of the inhibitor-enzyme complex; this had been postulated to be the key to the high affinity and selectivity of SirReals. However, to attach biotin to the SirReal core, we introduced a triazole as a linking moiety; this was shown by X-ray co-crystallography to interact with Arg97 of the cofactor binding loop. Herein, we aim to elucidate whether the observed long residence time of the SirReals is induced mainly by triazole incorporation or is an inherent characteristic of the SirReal inhibitor core. We used the novel label-free switchSENSE® technology, which is based on electrically switchable DNA nanolevers, to prove that the long residence time of the SirReals is indeed caused by the core scaffold.

Phosphorylation of Ser111 in Rab8a Modulates Rabin8-Dependent Activation by Perturbation of Side Chain Interaction Networks.

GTPases are key players during cellular signaling. Phosphorylation of Rab proteins, which belong to the Ras superfamily of small GTPases regulating intracellular transport, has been implicated in the pathogenesis of Parkinson's disease. For Rab8a, it was shown that serine 111 phosphorylation (pS111) is dependent on the protein kinase PINK1 and that mimicking the phosphorylation at S111 by a serine/glutamate substitution (S111E) impaired Rab8a activation by its cognate nucleotide exchange factor (GEF) Rabin8. However, Ser111 is not part of the interface of the Rab8a:Rabin8 complex. Here, we performed comparative molecular dynamics and free energy simulations on Rab8a and Rab8a:Rabin8 complexes to elucidate the molecular details of how pS111 and S111E may influence the interaction with Rabin8. The simulations indicate that S111E and pS111 establish an intramolecular interaction with arginine 79 (R79). The interaction persists in the complex and perturbs a favorable intermolecular salt-bridge contact between R79 in Rab8a and aspartate 187 in Rabin8. Binding free energy analysis reveals that S111E and pS111, as well as the R79A mutation, drastically decrease the binding affinity for Rabin8. Combining the R79A mutation with S111E or pS111 nearly diminishes Rab8a-Rabin8 binding. In vitro experiments confirm our computational results showing a >80% decrease in the nucleotide exchange rate of the respective Rab8a mutants in the presence of Rabin8 compared to that of the wild type. In addition to insights into how S111 phosphorylation of Rab8a influences GEF-mediated activation, the simulations demonstrate how side chain modifications in general can allosterically influence the surface side chain interaction network between binding partners.