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ATP-grasp superfamily enzymes contain a hand-like ATP-binding fold and catalyze a variety of reactions using a similar catalytic mechanism. More than 30 protein families are categorized in this superfamily, and they are involved in a plethora of cellular processes and human diseases. Here, we identify C12orf29 (RLIG1) as an atypical ATP-grasp enzyme that ligates RNA. Human RLIG1 and its homologs autoadenylate on an active site Lys residue as part of a reaction intermediate that specifically ligates RNA halves containing a 5'-phosphate and a 3'-hydroxyl. RLIG1 binds tRNA in cells and can ligate tRNA within the anticodon loop in vitro. Transcriptomic analyses of Rlig1 knockout mice revealed significant alterations in global tRNA levels in the brains of female mice, but not in those of male mice. Furthermore, crystal structures of a RLIG1 homolog from Yasminevirus bound to nucleotides revealed a minimal and atypical RNA ligase fold with a conserved active site architecture that participates in catalysis. Collectively, our results identify RLIG1 as an RNA ligase and suggest its involvement in tRNA biology., Competing Interests: Competing interests statement:The authors declare no competing interest.

During homeostasis, the endoplasmic reticulum (ER) maintains productive transmembrane and secretory protein folding that is vital for proper cellular function. The ER-resident HSP70 chaperone, binding immunoglobulin protein (BiP), plays a pivotal role in sensing ER stress to activate the unfolded protein response (UPR). BiP function is regulated by the bifunctional enzyme filamentation induced by cyclic-AMP domain protein (FicD) that mediates AMPylation and deAMPylation of BiP in response to changes in ER stress. AMPylated BiP acts as a molecular rheostat to regulate UPR signaling, yet little is known about the molecular consequences of FicD loss. In this study, we investigate the role of FicD in mouse embryonic fibroblast (MEF) response to pharmacologically and metabolically induced ER stress. We find differential BiP AMPylation signatures when comparing robust chemical ER stress inducers to physiological glucose starvation stress and recovery. Wildtype MEFs respond to pharmacological ER stress by down-regulating BiP AMPylation. Conversely, BiP AMPylation in wildtype MEFs increases upon metabolic stress induced by glucose starvation. Deletion of FicD results in widespread gene expression changes under baseline growth conditions. In addition, FicD null MEFs exhibit dampened UPR signaling, altered cell stress recovery response, and unconstrained protein secretion. Taken together, our findings indicate that FicD is important for tampering UPR signaling, stress recovery, and the maintenance of secretory protein homeostasis., Competing Interests: Competing interests statement:The authors declare no competing interest.

The plant pathogen Pseudomonas syringae encodes a type III secretion system avirulence effector protein, AvrB, that induces a form of programmed cell death called the hypersensitive response in plants as a defense mechanism against systemic infection. Despite the well-documented catalytic activities observed in other Fido ( Fi c, Do c, and AvrB) proteins, the enzymatic activity and target substrates of AvrB have remained elusive. Here, we show that AvrB is an unprecedented glycosyltransferase that transfers rhamnose from UDP-rhamnose to a threonine residue of the Arabidopsis guardee protein RIN4. We report structures of various enzymatic states of the AvrB-catalyzed rhamnosylation reaction of RIN4, which reveal the structural and mechanistic basis for rhamnosylation by a Fido protein. Collectively, our results uncover an unexpected reaction performed by a prototypical member of the Fido superfamily while providing important insights into the plant hypersensitive response pathway and foreshadowing more diverse chemistry used by Fido proteins and their substrates.

Previous study has demonstrated that the WNK kinases 1 and 3 are direct osmosensors consistent with their established role in cell-volume control. WNK kinases may also be regulated by hydrostatic pressure. Hydrostatic pressure applied to cells in culture with N 2 gas or to Drosophila Malpighian tubules by centrifugation induces phosphorylation of downstream effectors of endogenous WNKs. In vitro, the autophosphorylation and activity of the unphosphorylated kinase domain of WNK3 (uWNK3) is enhanced to a lesser extent than in cells by 190 kPa applied with N 2 gas. Hydrostatic pressure measurably alters the structure of uWNK3. Data from size exclusion chromatography in line with multi-angle light scattering (SEC-MALS), SEC alone at different back pressures, analytical ultracentrifugation (AUC), NMR, and chemical crosslinking indicate a change in oligomeric structure in the presence of hydrostatic pressure from a WNK3 dimer to a monomer. The effects on the structure are related to those seen with osmolytes. Potential mechanisms of hydrostatic pressure activation of uWNK3 and the relationships of pressure activation to WNK osmosensing are discussed.

The proper balance of synthesis, folding, modification, and degradation of proteins, also known as protein homeostasis, is vital to cellular health and function. The unfolded protein response (UPR) is activated when the mechanisms maintaining protein homeostasis in the endoplasmic reticulum become overwhelmed. However, prolonged or strong UPR responses can result in elevated inflammation and cellular damage. Previously, we discovered that the enzyme filamentation induced by cyclic-AMP (Fic) can modulate the UPR response via posttranslational modification of binding immunoglobulin protein (BiP) by AMPylation during homeostasis and deAMPylation during stress. Loss of fic in Drosophila leads to vision defects and altered UPR activation in the fly eye. To investigate the importance of Fic-mediated AMPylation in a mammalian system, we generated a conditional null allele of Fic in mice and characterized the effect of Fic loss on the exocrine pancreas. Compared to controls, Fic -/- mice exhibit elevated serum markers for pancreatic dysfunction and display enhanced UPR signaling in the exocrine pancreas in response to physiological and pharmacological stress. In addition, both fic -/- flies and Fic -/- mice show reduced capacity to recover from damage by stress that triggers the UPR. These findings show that Fic-mediated AMPylation acts as a molecular rheostat that is required to temper the UPR response in the mammalian pancreas during physiological stress. Based on these findings, we propose that repeated physiological stress in differentiated tissues requires this rheostat for tissue resilience and continued function over the lifetime of an animal.

The mammalian target of rapamycin complex 1 (mTORC1) senses multiple stimuli to regulate anabolic and catabolic processes. mTORC1 is typically hyperactivated in multiple human diseases such as cancer and type 2 diabetes. Extensive research has focused on signaling pathways that can activate mTORC1 such as growth factors and amino acids. However, less is known about signaling cues that can directly inhibit mTORC1 activity. Here, we identify A-kinase anchoring protein 13 (AKAP13) as an mTORC1 binding protein, and a crucial regulator of mTORC1 inhibition by G-protein coupled receptor (GPCR) signaling. GPCRs paired to Gαs proteins increase cyclic adenosine 3'5' monophosphate (cAMP) to activate protein kinase A (PKA). Mechanistically, AKAP13 acts as a scaffold for PKA and mTORC1, where PKA inhibits mTORC1 through the phosphorylation of Raptor on Ser 791. Importantly, AKAP13 mediates mTORC1-induced cell proliferation, cell size, and colony formation. AKAP13 expression correlates with mTORC1 activation and overall lung adenocarcinoma patient survival, as well as lung cancer tumor growth in vivo. Our study identifies AKAP13 as an important player in mTORC1 inhibition by GPCRs, and targeting this pathway may be beneficial for human diseases with hyperactivated mTORC1., Competing Interests: The authors have declared that no competing interests exist.

ADP-ribosyltransferases (ARTs) are a widespread superfamily of enzymes frequently employed in pathogenic strategies of bacteria. Legionella pneumophila, the causative agent of a severe form of pneumonia known as Legionnaire's disease, has acquired over 330 translocated effectors that showcase remarkable biochemical and structural diversity. However, the ART effectors that influence L. pneumophila have not been well defined. Here, we took a bioinformatic approach to search the Legionella effector repertoire for additional divergent members of the ART superfamily and identified an ART domain in Legionella pneumophila gene0181, which we hereafter refer to as Legionella ADP-Ribosyltransferase 1 (Lart1) (Legionella ART 1). We show that L. pneumophila Lart1 targets a specific class of 120-kDa NAD+-dependent glutamate dehydrogenase (GDH) enzymes found in fungi and protists, including many natural hosts of Legionella. Lart1 targets a conserved arginine residue in the NAD+-binding pocket of GDH, thereby blocking oxidative deamination of glutamate. Therefore, Lart1 could be the first example of a Legionella effector which directly targets a host metabolic enzyme during infection., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The Vibrio parahaemolyticus T3SS effector VopQ targets host-cell V-ATPase, resulting in blockage of autophagic flux and neutralization of acidic compartments. Here, we report the cryo-EM structure of VopQ bound to the V o subcomplex of the V-ATPase. VopQ inserts into membranes and forms an unconventional pore while binding directly to subunit c of the V-ATPase membrane-embedded subcomplex V o . We show that VopQ arrests yeast growth in vivo by targeting the immature V o subcomplex in the endoplasmic reticulum (ER), thus providing insight into the observation that VopQ kills cells in the absence of a functional V-ATPase. VopQ is a bacterial effector that has been discovered to inhibit a host-membrane megadalton complex by coincidentally binding its target, inserting into a membrane and disrupting membrane potential. Collectively, our results reveal a mechanism by which bacterial effectors modulate host cell biology and provide an invaluable tool for future studies on V-ATPase-mediated membrane fusion and autophagy.

Extracellular vesicles secreted from tumor cells are functional vehicles capable of contributing to intercellular communication and metastasis. A growing number of studies have focused on elucidating the role that tumor-derived extracellular vesicles play in spreading pancreatic cancer to other organs, due to the highly metastatic nature of the disease. We recently showed that small extracellular vesicles secreted from pancreatic cancer cells could initiate malignant transformation of healthy cells. Here, we analyzed the protein cargo contained within these vesicles using mass spectrometry-based proteomics to better understand their makeup and biological characteristics. Three different human pancreatic cancer cell lines were compared to normal pancreatic epithelial cells revealing distinct differences in protein cargo between cancer and normal vesicles. Vesicles from cancer cells contain an enrichment of proteins that function in the endosomal compartment of cells responsible for vesicle formation and secretion in addition to proteins that have been shown to contribute to oncogenic cell transformation. Conversely, vesicles from normal pancreatic cells were shown to be enriched for immune response proteins. Collectively, results contribute to what we know about the cargo contained within or excluded from cancer cell-derived extracellular vesicles, supporting their role in biological processes including metastasis and cancer progression.

The molecular chaperone HSP90 facilitates the folding of several client proteins, including innate immune receptors and protein kinases. HSP90 is an essential component of plant and animal immunity, yet pathogenic strategies that directly target the chaperone have not been described. Here, we identify the HopBF1 family of bacterial effectors as eukaryotic-specific HSP90 protein kinases. HopBF1 adopts a minimal protein kinase fold that is recognized by HSP90 as a host client. As a result, HopBF1 phosphorylates HSP90 to completely inhibit the chaperone's ATPase activity. We demonstrate that phosphorylation of HSP90 prevents activation of immune receptors that trigger the hypersensitive response in plants. Consequently, HopBF1-dependent phosphorylation of HSP90 is sufficient to induce severe disease symptoms in plants infected with the bacterial pathogen, Pseudomonas syringae. Collectively, our results uncover a family of bacterial effector kinases with toxin-like properties and reveal a previously unrecognized betrayal mechanism by which bacterial pathogens modulate host immunity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Enzymes with a protein kinase fold transfer phosphate from adenosine 5'-triphosphate (ATP) to substrates in a process known as phosphorylation. Here, we show that the Legionella meta-effector SidJ adopts a protein kinase fold, yet unexpectedly catalyzes protein polyglutamylation. SidJ is activated by host-cell calmodulin to polyglutamylate the SidE family of ubiquitin (Ub) ligases. Crystal structures of the SidJ-calmodulin complex reveal a protein kinase fold that catalyzes ATP-dependent isopeptide bond formation between the amino group of free glutamate and the γ-carboxyl group of an active-site glutamate in SidE. We show that SidJ polyglutamylation of SidE, and the consequent inactivation of Ub ligase activity, is required for successful Legionella replication in a viable eukaryotic host cell., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Approximately 10% of human protein kinases are believed to be inactive and named pseudokinases because they lack residues required for catalysis. Here, we show that the highly conserved pseudokinase selenoprotein-O (SelO) transfers AMP from ATP to Ser, Thr, and Tyr residues on protein substrates (AMPylation), uncovering a previously unrecognized activity for a member of the protein kinase superfamily. The crystal structure of a SelO homolog reveals a protein kinase-like fold with ATP flipped in the active site, thus providing a structural basis for catalysis. SelO pseudokinases localize to the mitochondria and AMPylate proteins involved in redox homeostasis. Consequently, SelO activity is necessary for the proper cellular response to oxidative stress. Our results suggest that AMPylation may be a more widespread post-translational modification than previously appreciated and that pseudokinases should be analyzed for alternative transferase activities., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Protein chaperones play a critical role in proteostasis. The activity of the major endoplasmic reticulum chaperone BiP (GRP78) is regulated by Fic-mediated AMPylation during resting states. By contrast, during times of stress, BiP is deAMPylated. Here, we show that excessive AMPylation by a constitutively active Fic E247G mutant is lethal in Drosophila This lethality is cell-autonomous, as directed expression of the mutant Fic E247G to the fly eye does not kill the fly but rather results in a rough and reduced eye. Lethality and eye phenotypes are rescued by the deAMPylation activity of wild-type Fic. Consistent with Fic acting as a deAMPylation enzyme, its activity was both time- and concentration-dependent. Furthermore, Fic deAMPylation activity was sufficient to suppress the AMPylation activity mediated by the constitutively active Fic E247G mutant in Drosophila S2 lysates. Further, we show that the dual enzymatic activity of Fic is, in part, regulated by Fic dimerization, as loss of this dimerization increases AMPylation and reduces deAMPylation of BiP., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Electrospray ionization (ESI) combined with ion mobility-mass spectrometry (IM-MS) is adding new dimensions, that is, structure and dynamics, to the field of biological mass spectrometry. There is increasing evidence that gas-phase ions produced by ESI can closely resemble their solution-phase structures, but correlating these structures can be complicated owing to the number of competing effects contributing to structural preferences, including both inter- and intramolecular interactions. Ions encounter unique hydration environments during the transition from solution to the gas phase that will likely affect their structure(s), but many of these structural changes will go undetected because ESI-IM-MS analysis is typically performed on solvent-free ions. Cryogenic ion mobility-mass spectrometry (cryo-IM-MS) takes advantage of the freeze-drying capabilities of ESI and a cryogenically cooled IM drift cell (80 K) to preserve extensively solvated ions of the type [M + xH](x+)(H2O)n, where n can vary from zero to several hundred. This affords an experimental approach for tracking the structural evolution of hydrated biomolecules en route to forming solvent-free gas-phase ions. The studies highlighted in this Account illustrate the varying extent to which dehydration can alter ion structure and the overall impact of cryo-IM-MS on structural studies of hydrated biomolecules. Studies of small ions, including protonated water clusters and alkyl diammonium cations, reveal structural transitions associated with the development of the H-bond network of water molecules surrounding the charge carrier(s). For peptide ions, results show that water networks are highly dependent on the charge-carrying species within the cluster. Specifically, hydrated peptide ions containing lysine display specific hydration behavior around the ammonium ion, that is, magic number clusters with enhanced stability, whereas peptides containing arginine do not display specific hydration around the guanidinium ion. Studies on the neuropeptide substance P illustrate the ability of cryo-IM-MS to elucidate information about heterogeneous ion populations. Results show that a kinetically trapped conformer is stabilized by a combination of hydration and specific intramolecular interactions, but upon dehydration, this conformer rearranges to form a thermodynamically favored gas-phase ion conformation. Finally, recent studies on hydration of the protein ubiquitin reveal water-mediated dimerization, thereby illustrating the extension of this approach to studies of large biomolecules. Collectively, these studies illustrate a new dimension to studies of biomolecules, resulting from the ability to monitor snapshots of the structural evolution of ions during the transition from solution to gas phase and provide unparalleled insights into the intricate interplay between competing effects that dictate conformational preferences.

When the all-cis polyproline-I helix (PPI, favored in 1-propanol) of polyproline-13 is introduced into water, it folds into the all-trans polyproline-II (PPII) helix through at least six intermediates [Shi, L., Holliday, A.E., Shi, H., Zhu, F., Ewing, M.A., Russell, D.H., Clemmer, D.E.: Characterizing intermediates along the transition from PPI to PPII using ion mobility-mass spectrometry. J. Am. Chem. Soc. 136, 12702-12711 (2014)]. Here, we show that the solvent-free intermediates refold into the all-cis PPI helix with high (>90%) efficiency. Moreover, in the absence of solvent, each intermediate appears to utilize the same small set of pathways observed for the solution-phase PPII → PPI transition upon immersion of PPIIaq in 1-propanol. That folding in solution (under conditions where water is displaced by propanol) and folding in vacuo (where energy required for folding is provided by collisional activation) occur along the same pathway is remarkable. Implicit in this statement is that 1-propanol mimics a "dry" environment, similar to the gas phase. We note that intermediates with structures that are similar to PPIIaq can form PPII under the most gentle activation conditions-indicating that some transitions observed in water (i.e., "wet" folding, are accessible (albeit inefficient) in vacuo. Lastly, these "dry" folding experiments show that PPI (all cis) is favored under "dry" conditions, which underscores the role of water as the major factor promoting preference for trans proline. Graphical Abstract ᅟ.

The dynamics, structures, and functions of most biological molecules are strongly influenced by the nature of the peptide's or protein's interaction with water. Here, cryogenic ion mobility-mass spectrometry studies of ubiquitin have directly captured a water-mediated protein-protein binding event involving hydrated, noncovalently bound dimer ions in solution, and this interaction has potential relevance to one of the most important protein-protein interactions in nature. As solvent is removed, dimer ions, viz. [2 M + 14H](14+), can be stabilized by only a few attached water molecules prior to dissociation into individual monomeric ions. The hydrophobic patch of ubiquitin formed by the side chains of Leu-8, Ile-44, and Val-70 meet all the necessary conditions for a protein-protein binding "hot spot," including the requirement for occlusion of water to nearby hydrophilic sites, and it is suggested that this interaction is responsible for formation of the hydrated noncovalent ubiquitin dimer.

Hydration of the ammonium ion plays a key role in determining the biomolecular structure as well as local structure of water in aqueous environments. Experimental data obtained by cryogenic ion mobility-mass spectrometry (cryo-IM-MS) show that dehydration of alkyl diammonium cations induces a distinct unfolding transition at a critical number of water molecules, n = 21 to 23, n = 24 to 26, and n = 27 to 29, for 1,7-diaminoheptane, 1,8-diaminooctane, and 1,10-diaminodecane, respectively. Results are also presented that reveal compelling evidence for unique structural transitions of hydrated ammonium ions associated with the development of the hydrogen-bond network around individual charged groups. The ability to track the evolution of structure upon stepwise dehydration provides direct insight into the intricate interplay between solvent-molecule interactions that are responsible for defining conformations. Such insights are potentially valuable in understanding how ammonium ion solvation influences conformation(s) of larger biomolecules.

Substance P (RPKPQQFFGLM-NH2) [M + 3H](3+) ions have been shown to occupy two distinct conformer states, a compact population of conformers that is formed by evaporation of hydrated ions, and an elongated population of conformers that is formed by collisional heating of the compact conformer. Molecular dynamics (MD) simulations and amino acid mutations revealed that the compact conformer is stabilized by intramolecular interactions between the localized charge-carrying sites, specifically the N-terminus, R(1), and K(3), with the side chains of glutamine and phenylalanine residues present in the peptide. Here, we employ amino acid mutations and cryogenic ion mobility-mass spectrometry (cryo-IM-MS) in an effort to understand how eliminating specific intramolecular interactions alters ion hydration, as well as the dehydration dynamics of substance P during the final stages of the electrospray process. The results clearly illustrate a direct link between the stabilizing effects of intramolecular self-solvation and the formation of substance P [M + 3H](3+) ions. Most notably, removal of these stabilizing interactions leads to a reduction in the abundances of [M + 3H](3+) ions induced by charge reduction reactions, i.e., loss of H(+)(H2O)n ions to form [M + 2H](2+) ions during the final stages of the electrospray process.

Substance P (RPKPQQFFGLM-NH2) [M + 3H](3+) ions have been shown to exist as two conformers: one that is kinetically trapped and one that is thermodynamically more stable and therefore energetically preferred. Molecular dynamics (MD) simulations suggested that the kinetically trapped population is stabilized by interactions between the charge sites and the polar side chains of glutamine (Q) located at positions 5 and 6 and phenylalanine (F) located at positions 7 and 8. Here, the individual contributions of these specific intramolecular interactions are systematically probed through site-directed alanine mutations of the native amino acid sequence. Ion mobility spectrometry data for the mutant peptide ions confirm that interactions between the charge sites and glutamine/phenylalanine (Q/F) side chains afford stabilization of the kinetically trapped ion population. In addition, experimental data for proline-to-alanine mutations at positions 2 and 4 clearly show that interactions involving the charge sites and the Q/F side chains are altered by the cis/trans orientations of the proline residues and that mutation of glycine to proline at position 9 supports results from MD simulations suggesting that the C-terminus also provides stabilization of the kinetically trapped conformation.

Cryogenic (80 K) ion mobility-mass spectrometry (cryo-IM-MS) is employed to study structural transitions of protonated water clusters in both the small, H(+)(H2O)n (n = 1 to 30), and large, (n = 31 to ∼120), size regions. In agreement with previous studies, we find compelling evidence of regions of uniform cluster decay in the small size region, accompanied by sharp transition points whereby the loss of a single water monomer induces a different H-bonding motif. The investigation of the isomeric distribution of each species at 80 K reveals experimental evidence supporting the notion that H(+)(H2O)n (n = 6) is the smallest system to possess both Eigen- (H3O(+)) and Zundel- (H5O2(+)) centered structures. Cryo-IM-MS is particularly well-suited for studying clusters in the large size region, for which previous spectroscopic experimental studies are scarce.

Past experimental results and molecular dynamics simulations provide evidence that, under some conditions, electrospray ionization (ESI) of biomolecules produces ions that retain elements of solution phase structures. However, there is a dearth of information regarding the question raised by Breuker and McLafferty, "for how long, under what conditions, and to what extent, can solution structure be retained without solvent?" (Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 18145). Here, we use cryogenic ion mobility-mass spectrometry to experimentally probe the structural evolution of the undecapeptide substance P (SP) during the final stages of ESI. The results reveal that anhydrous SP conformers originate from evaporation of cluster ions, specifically, [SP + 2H](2+) (H2O)n (n = 0 to ∼50) and [SP + 3H](3+) (H2O)n (n = 0 to ∼30), and that major structural changes do not occur during the evaporative process. In the case of [SP + 3H](3+), the results demonstrate that a compact dehydrated conformer population can be kinetically trapped on the time scale of several milliseconds, even when an extended gas phase conformation is energetically favorable.

Evaporation of water from extensively hydrated protons and peptides formed by electrospray ionization (ESI) has been examined for the first time by cryogenic ion mobility-mass spectrometry (IM-MS). The extent of hydration was controlled using a heated capillary inlet operated between 340 and 391 K. Cold cluster ions formed in the source region were transported into a low temperature (∼80 K) IM drift tube using an electrostatic ion guide where they were separated on the basis of size-to-charge via low-energy collisions with helium gas. The eluting IM profile was subsequently pulsed into an orthogonal time-of-flight (TOF) mass spectrometer for mass-to-charge (m/z) identification of the cluster ion species. Key parameters that influence the cluster distributions were critically examined including the inlet temperature, drift tube temperature, and IM field strength. In agreement with previous studies, our findings indicate that water evaporation is largely dependent upon the particular charge-carrying species within the cluster. IM-MS results for protonated water clusters suggest that the special stability of H(+)(H(2)O)(n) (n = 21) is attributed to the presence of a compact isomer (assigned to a clathrate cage) that falls below the trendline produced by adjacent clusters in the n = 15 to 35 size range. Peptide studies are also presented in which specific and nonspecific solvation is observed for gramicidin S [GS + 2H](2+)(H(2)O)(n) (n = 0 to ∼26) and bradykinin [BK + 2H](2+)(H(2)O)(n) (n = 0 to ∼73), respectively.