Summary 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.

Bacterial pathogens interact with host membranes to trigger a wide range of cellular processes during the course of infection. These processes include alterations to the dynamics between the plasma membrane and the actin cytoskeleton, and subversion of the membrane-associated pathways involved in vesicle trafficking. Such changes facilitate the entry and replication of the pathogen, and prevent its phagocytosis and degradation. In this Review, we describe the manipulation of host membranes by numerous bacterial effectors that target phosphoinositide metabolism, GTPase signalling and autophagy.

The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis; however, the mechanism by which CotH affects germination is unclear. Here, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.

Defects in normal autophagic pathways are implicated in numerous human diseases-such as neurodegenerative diseases, cancer, and cardiomyopathy-highlighting the importance of autophagy and its proper regulation. Herein we show that Vibrio parahaemolyticus uses the type III effector VopQ (Vibrio outer protein Q) to alter autophagic flux by manipulating the partitioning of small molecules and ions in the lysosome. This effector binds to the conserved V o domain of the vacuolar-type H + -ATPase and causes deacidification of the lysosomes within minutes of entering the host cell. VopQ forms a gated channel ∼18 Å in diameter that facilitates outward flux of ions across lipid bilayers. The electrostatic interactions of this type 3 secretion system effector with target membranes dictate its preference for host vacuolar-type H + -ATPase-containing membranes, indicating that its pore-forming activity is specific and not promiscuous. As seen with other effectors, VopQ is exploiting a eukaryotic mechanism, in this case manipulating lysosomal homeostasis and autophagic flux through transmembrane permeation.utophagy is a cellular process by which cells degrade and recycle cytoplasmic contents by encapsulating them within a distinctive double bilayer membrane vesicle for delivery to the degradative lysosome (1). Disruption of normal autophagic pathways is implicated in numerous human diseases, stressing the importance of autophagy and its proper regulation (2). Vibrio parahaemolyticus, a Gram-negative marine bacterium and a major cause of gastroenteritis due to the consumption of contaminated raw or undercooked seafood, induces autophagy during infection (3). V. parahaemolyticus harbors two type 3 secretion systems (T3SSs), molecular syringes that enable the translocation of bacterial proteins, known as effectors, into the eukaryotic host (3). The first T3SS (T3SS1) orchestrates a temporally regulated cell death mediated by the induction of autophagy, followed by cell rounding and resulting in lysis of the host cell (4). T3SS1 effector VopQ, also known as VepA (vp1680), is both necessary and sufficient for the rapid induction of autophagy, even in the presence of known chemical inhibitors of autophagy (5).VopQ is a 53-kDa protein with no apparent homology to any proteins outside of the Vibrio species. Vibrio homologs of VopQ have no known function or conserved structural domain. Previous work from our laboratory has shown that VopQ is a cytotoxic effector that accelerates host cell death and is essential in protecting V. parahaemolyticus from phagocytic uptake during infection (5). Based on microbial genetic studies, VopQ was shown to be necessary for the formation of an extensive network of autophagic vesicles in host cells within an hour of V. parahaemolyticus infection (5). Strikingly, recombinant VopQ alone is sufficient to induce this massive accumulation of autophagic vesicles, observed within minutes of microinjection of recombinant VopQ (picomolar concentrations) into eukaryotic cells (5). A recent report shows that Vo...

Highlights d The bacterial effector HopBF1 adopts a minimal protein kinase fold d HopBF1 phosphorylates and inactivates eukaryotic HSP90 d HopBF1 mimics an HSP90 client to achieve specificity d HopBF1 is sufficient to induce disease symptoms in plants

Protein phosphorylation is a nearly universal post-translation modification involved in a plethora of cellular events. Even though phosphorylation of extracellular proteins had been observed, the identity of the kinases that phosphorylate secreted proteins remained a mystery until recently. Advances in genome sequencing and genetic studies have paved the way for the discovery of a new class of kinases that localize within the endoplasmic reticulum, Golgi apparatus and the extracellular space. These novel kinases phosphorylate proteins and proteoglycans in the secretory pathway and appear to regulate various extracellular processes. Mutations in these kinases cause human disease, thus underscoring the biological importance of phosphorylation within the secretory pathway.

Vesicle fusion governs many important biological processes, and imbalances in the regulation of membrane fusion can lead to a variety of diseases such as diabetes and neurological disorders. Here we show that the Vibrio parahaemolyticus effector protein VopQ is a potent inhibitor of membrane fusion based on an in vitro yeast vacuole fusion model. Previously, we demonstrated that VopQ binds to the V o domain of the conserved V-type H + -ATPase (V-ATPase) found on acidic compartments such as the yeast vacuole. VopQ forms a nonspecific, voltage-gated membrane channel of 18 Å resulting in neutralization of these compartments. We now present data showing that VopQ inhibits yeast vacuole fusion. Furthermore, we identified a unique mutation in VopQ that delineates its two functions, deacidification and inhibition of membrane fusion. The use of VopQ as a membrane fusion inhibitor in this manner now provides convincing evidence that vacuole fusion occurs independently of luminal acidification in vitro.V esicle fusion governs many important physiological processes including neurotransmitter release and exocytosis. As such, many studies have focused on understanding this process and the proteins involved in fusion using various models such as yeast vacuoles and Drosophila synaptic vesicles (1, 2). Yeast vacuoles are an established and elegant model to study eukaryotic membrane fusion because of the ease of their isolation and the conserved nature of the fusion machinery required for their homotypic fusion (3). Although the core SNARE and Rab GTPase fusion machinery alone can drive the physiologically relevant fusion of liposomes in vitro (2), genetic and biochemical experiments have identified a number of additional regulators of vacuole fusion, including the membrane sector of the highly conserved V-type H + -ATPase (V-ATPase) (4, 5). The eukaryotic V-ATPase is the main electrogenic proton pump involved in the acidification of many intracellular organelles such as endosomes, lysosomes, and the yeast vacuole (6). The V-ATPase consists of two conserved, multisubunit domains: the cytoplasmic V 1 domain and the membrane bound V o domain. The V 1 domain hydrolyzes ATP, providing the energy for proton translocation through the membrane-bound V o proteolipid proton channel, thus acidifying the lumen of the vesicle. The loss of V-ATPase subunits is lethal in higher eukaryotes, highlighting the importance of this vital protein complex for normal eukaryotic physiology. However, yeast that lack subunits of the V-ATPase exhibit conditional lethality that is rescued by growth on acidic media, thus providing a unique and powerful system for the study of V-ATPase functions in vivo. In addition to its acidification function, the V-ATPase has been implicated in a broad range of biological processes, including the proper trafficking of secreted and endocytosed cargos (7), viral fusion (8), exocytosis (1, 9, 10), and the SNARE-dependent membrane fusion of yeast vacuoles (4,5,11,12). Even though the role of V-ATPase in fusion has been demons...

Enzymatic transfer of the AMP portion of ATP to substrate proteins has recently been described as an essential mechanism of bacterial infection for several pathogens. The first AMPylator to be discovered, VopS from Vibrio parahaemolyticus, catalyzes the transfer of AMP on to the host GTPases Cdc42 and Rac1. Modification of these proteins disrupts downstream signaling events, contributing to cell rounding and apoptosis, and recent studies have suggested that blocking AMPylation may be an effective route to stop infection. To date, however, no small molecule inhibitors have been discovered for any of the AMPylators. Therefore, we developed a fluorescence-polarization based high-throughput-screening assay and used it to discover the first inhibitors of protein AMPylation. Herein we report the discovery of the first small molecule VopS inhibitors (e.g. calmidazolium, GW7647 and MK886) with Kis ranging from 6–50 µM and upwards of 30-fold selectivity versus HYPE, the only known human AMPylator.