Utilizing Thermal Shift Assay to Probe Substrate Binding to Selenoprotein O.

Defining the biological importance of proteins with unknown functions poses a significant obstacle in understanding cellular processes. Although bioinformatic and structural predictions have contributed to the study of unknown proteins, in vitro experimental validations are often hampered by the optimal conditions and cofactors required for biochemical activity. Cofactor binding is not only essential for the activity of some enzymes but may also enhance the thermal stability of the protein. One practical application of this phenomenon lies in utilizing the change in thermal stability, as measured by alterations in the protein's melting temperature, to probe ligand binding. Thermal shift assay (TSA) can be used to analyze the binding of different ligands to the protein of interest or find a stabilizing condition to perform experiments such as X-ray crystallography. Here we will describe a protocol for TSA utilizing the pseudokinase, Selenoprotein O (SelO), for a simple and high-throughput method for testing metal and nucleotide binding. In contrast to canonical kinases, SelO binds ATP in an inverted orientation to catalyze the transfer of AMP to the hydroxyl side chains of proteins in a posttranslational modification known as protein AMPylation. By leveraging the shift in the melting temperatures, we provide crucial insights into the molecular interactions underlying SelO function.

Emerging functions of pseudoenzymes.

As sequence and structural databases grow along with powerful analysis tools, the prevalence and diversity of pseudoenzymes have become increasingly evident. Pseudoenzymes are present across the tree of life in a large number of enzyme families. Pseudoenzymes are defined as proteins that lack conserved catalytic motifs based on sequence analysis. However, some pseudoenzymes may have migrated amino acids necessary for catalysis, allowing them to catalyze enzymatic reactions. Furthermore, pseudoenzymes retain several non-enzymatic functions such as allosteric regulation, signal integration, scaffolding, and competitive inhibition. In this review, we provide examples of each mode of action using the pseudokinase, pseudophosphatase, and pseudo ADP-ribosyltransferase families. We highlight the methodologies that facilitate the biochemical and functional characterization of pseudoenzymes to encourage further investigation in this burgeoning field.

Redefining pseudokinases: A look at the untapped enzymatic potential of pseudokinases.

Catalytically inactive kinases, known as pseudokinases, are conserved in all three domains of life. Due to the lack of catalytic residues, pseudokinases are considered to act as allosteric regulators and scaffolding proteins with no enzymatic function. However, since these "dead" kinases are conserved along with their active counterparts, a role for pseudokinases may have been overlooked. In this review, we will discuss the recently characterized pseudokinases Selenoprotein O, Legionella effector SidJ, and the SARS-CoV2 protein nsp12 which catalyze AMPylation, glutamylation, and RNAylation, respectively. These studies provide structural and mechanistic insight into the versatility and diversity of the kinase fold.

Identification of selenoprotein O substrates using a biotinylated ATP analog.

Selenoprotein O is one of 25 human selenoproteins that incorporate the 21st amino acid selenocysteine. Recent studies have revealed a previously undocumented mechanism of redox regulation by which SelO protects cells from oxidative damage. SelO catalyzes the covalent addition of AMP from ATP to the hydroxyl side chain of protein substrates in a post translational modification known as AMPylation. Although AMPylation was discovered over 50 years ago, methods to detect and enrich substrates are limited. Here, we describe protocols to clone, purify, and identify the substrates of bacterial SelO using a biotinylated ATP analog. Identification of SelO substrates and the functional consequences of AMPylation will illuminate the significance of this evolutionarily conserved selenoprotein.

A Legionella effector kinase is activated by host inositol hexakisphosphate.

The transfer of a phosphate from ATP to a protein substrate, a modification known as protein phosphorylation, is catalyzed by protein kinases. Protein kinases play a crucial role in virtually every cellular activity. Recent studies of atypical protein kinases have highlighted the structural similarity of the kinase superfamily despite notable differences in primary amino acid sequence. Here, using a bioinformatics screen, we searched for putative protein kinases in the intracellular bacterial pathogen Legionella pneumophila and identified the type 4 secretion system effector Lpg2603 as a remote member of the protein kinase superfamily. Employing an array of biochemical and structural biology approaches, including in vitro kinase assays and isothermal titration calorimetry, we show that Lpg2603 is an active protein kinase with several atypical structural features. Importantly, we found that the eukaryote-specific host signaling molecule inositol hexakisphosphate (IP6) is required for Lpg2603 kinase activity. Crystal structures of Lpg2603 in the apo-form and when bound to IP6 revealed an active-site rearrangement that allows for ATP binding and catalysis. Our results on the structure and activity of Lpg2603 reveal a unique mode of regulation of a protein kinase, provide the first example of a bacterial kinase that requires IP6 for its activation, and may aid future work on the function of this effector during Legionella pathogenesis.

A Bacterial Effector Mimics a Host HSP90 Client to Undermine Immunity.

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.

Protein AMPylation by an Evolutionarily Conserved Pseudokinase.

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.

Phosphorylation of spore coat proteins by a family of atypical protein kinases.

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.

The secretory pathway kinases.

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 only 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. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.

Vibrio effector protein VopQ inhibits fusion of V-ATPase-containing membranes.

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.

Inhibiting AMPylation: a novel screen to identify the first small molecule inhibitors of protein AMPylation.

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 parahemolyticus, catalyzes the transfer of AMP onto 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 Ki's ranging from 6 to 50 µM and upward of 30-fold selectivity versus HYPE, the only known human AMPylator.

The pore-forming bacterial effector, VopQ, halts autophagic turnover.

Vibrio parahemolyticus Type III effector VopQ is both necessary and sufficient to induce autophagy within one hour of infection. We demonstrated that VopQ interacts with the Vo domain of the conserved vacuolar H(+)-ATPase. Membrane-associated VopQ subsequently forms pores in the membranes of acidic compartments, resulting in immediate release of protons without concomitant release of lumenal protein contents. These studies show how a bacterial pathogen can compromise host ion potentials using a gated pore-forming effector to equilibrate levels of small molecules found in endolysosomal compartments and disrupt cellular processes such as autophagy.

Vibrio effector protein, VopQ, forms a lysosomal gated channel that disrupts host ion homeostasis and autophagic flux.

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 Vo 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 Šin 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.

Amyloidogenic peptide/single-walled carbon nanotube composites based on tau-protein-related peptides derived from AcPHF6: preparation and dispersive properties.

We investigated the abilities of a family of tau-protein-related amphiphilic peptides with predictable self-association characteristics (N-acetyl-VQIVXK-NH2 (X = F, L, V, W, Y, A, K)) to disperse single-walled carbon nanotubes (SWCNTs). The dispersion abilities of these peptides could be explained by a linear combination of their hydrophobic and amyloidogenic properties in a 60/40 ratio. Circular dichroism (CD) spectra of one of the peptides having a high propensity to form an amyloid (N-acetyl-VQIVYK-NH2 (AcPHF6)) showed that this peptide exists as a random coil in water but assumes a ß-sheet conformation when sonicated with SWCNTs. Electron microscopy results, changes in near-infrared spectra, and changes in the Raman spectra upon formation of composites suggest that AcPHF6 intercalates, coats, and exfoliates SWCNT bundles. N-terminal truncation of AcPHF6 greatly reduced its ability to disperse SWCNTs. Taken together, our results suggest that amyloidogenic peptides wrap SWCNTs, forming an extensive ß-sheet network. To date, peptides based on the AcVQIVXK framework are structurally the simplest peptides that have been found to disperse CNTs, and an understanding of those properties that determine their efficiency may be used to design even more efficient peptides for these purposes. We believe that due to the structural simplicity, this family of peptides will have clear synthetic advantages over peptides now known to disperse CNTs.

Manipulation of host membranes by bacterial effectors.

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.