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

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 (<i>N</i>-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 (<i>N</i>-acetyl-VQIVYK-NH₂ (AcPHF6)) showed that this peptide exists as a random coil in water but assumes a β-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 β-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

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\u27s ranging from 6 to 50 muM and upward of 30-fold selectivity versus HYPE, the only known human AMPylator

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

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 <i>Vibrio parahemolyticus</i>, 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 <i>K</i>_i’s ranging from 6 to 50 μM and upward of 30-fold selectivity versus HYPE, the only known human AMPylator

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