Within bacterial populations, a small fraction of persister cells is transiently capable of surviving exposure to lethal doses of antibiotics. As a bet-hedging strategy, persistence levels are determined both by stochastic induction and by environmental stimuli called responsive diversification. Little is known about the mechanisms that link the low frequency of persisters to environmental signals. Our results support a central role for the conserved GTPase Obg in determining persistence in Escherichia coli in response to nutrient starvation. Obg-mediated persistence requires the stringent response alarmone (p)ppGpp and proceeds through transcriptional control of the hokB-sokB type I toxin-antitoxin module. In individual cells, increased Obg levels induce HokB expression, which in turn results in a collapse of the membrane potential, leading to dormancy. Obg also controls persistence in Pseudomonas aeruginosa and thus constitutes a conserved regulator of antibiotic tolerance. Combined, our findings signify an important step toward unraveling shared genetic mechanisms underlying persistence.

SUMMARY Members of the large superclass of P-loop GTPases share a core domain with a conserved three-dimensional structure. In eukaryotes, these proteins are implicated in various crucial cellular processes, including translation, membrane trafficking, cell cycle progression, and membrane signaling. As targets of mutation and toxins, GTPases are involved in the pathogenesis of cancer and infectious diseases. In prokaryotes also, it is hard to overestimate the importance of GTPases in cell physiology. Numerous papers have shed new light on the role of bacterial GTPases in cell cycle regulation, ribosome assembly, the stress response, and other cellular processes. Moreover, bacterial GTPases have been identified as high-potential drug targets. A key paper published over 2 decades ago stated that, “It may never again be possible to capture [GTPases] in a family portrait” (H. R. Bourne, D. A. Sanders, and F. McCormick, Nature 348 :125-132, 1990) and indeed, the last 20 years have seen a tremendous increase in publications on the subject. Sequence analysis identified 13 bacterial GTPases that are conserved in at least 75% of all bacterial species. We here provide an overview of these 13 protein subfamilies, covering their cellular functions as well as cellular localization and expression levels, three-dimensional structures, biochemical properties, and gene organization. Conserved roles in eukaryotic homologs will be discussed as well. A comprehensive overview summarizing current knowledge on prokaryotic GTPases will aid in further elucidating the function of these important proteins.

Triblock copolymeric nanoreactors are introduced as an alternative for liposomes as encapsulating carrier for prodrug activating enzymes. Inosine-adenosine-guanosine preferring nucleoside hydrolase of Trypanosoma vivax, a potential prodrug activating enzyme, was encapsulated in nanometer-sized vesicles constructed of poly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-(2-methyloxazoline) triblock copolymers. The nanoreactor is functionalized by incorporation of bacterial porins, OmpF or Tsx, in the reactor wall. Efficient cleavage of three natural substrates and one prodrug, 2-fluoroadenosine, by the nanoreactors was demonstrated.

Yeast and cancer cells share the unusual characteristic of favoring fermentation of sugar over respiration. We now reveal an evolutionary conserved mechanism linking fermentation to activation of Ras, a major regulator of cell proliferation in yeast and mammalian cells, and prime proto-oncogene product. A yeast mutant (tps1∆) with overactive influx of glucose into glycolysis and hyperaccumulation of Fru1,6bisP, shows hyperactivation of Ras, which causes its glucose growth defect by triggering apoptosis. Fru1,6bisP is a potent activator of Ras in permeabilized yeast cells, likely acting through Cdc25. As in yeast, glucose triggers activation of Ras and its downstream targets MEK and ERK in mammalian cells. Biolayer interferometry measurements show that physiological concentrations of Fru1,6bisP stimulate dissociation of the pure Sos1/H-Ras complex. Thermal shift assay confirms direct binding to Sos1, the mammalian ortholog of Cdc25. Our results suggest that the Warburg effect creates a vicious cycle through Fru1,6bisP activation of Ras, by which enhanced fermentation stimulates oncogenic potency.

Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein containing two catalytic domains: a Ras of complex proteins (Roc) G-domain and a kinase domain. Mutations associated with familial and sporadic Parkinson's disease (PD) have been identified in both catalytic domains, as well as in several of its multiple putative regulatory domains. Several of these mutations have been linked to increased kinase activity. Despite the role of LRRK2 in the pathogenesis of PD, little is known about its overall architecture and how PD-linked mutations alter its function and enzymatic activities. Here, we have modeled the 3D structure of dimeric, full-length LRRK2 by combining domain-based homology models with multiple experimental constraints provided by chemical cross-linking combined with mass spectrometry, negative-stain EM, and small-angle X-ray scattering. Our model reveals dimeric LRRK2 has a compact overall architecture with a tight, multidomain organization. Close contacts between the N-terminal ankyrin and C-terminal WD40 domains, and their proximity-together with the LRR domain-to the kinase domain suggest an intramolecular mechanism for LRRK2 kinase activity regulation. Overall, our studies provide, to our knowledge, the first structural framework for understanding the role of the different domains of full-length LRRK2 in the pathogenesis of PD.LRRK2 | Parkinson's disease | structural modeling | EM | CL-MS

Nucleoside hydrolases cleave the N-glycosidic bond of ribonucleosides. Because of their vital role in the protozoan purine salvage pathway, nucleoside hydrolases from parasitic protozoa in particular have been studied extensively by X-ray crystallography, kinetic methods and site-directed mutagenesis. An elaborate network of conserved interactions between the metalloenzyme and the ribose enables steric and electrostatic stabilisation of the oxocarbenium-ion-like transition state. Activation of the leaving group by protonation before the formation of the transition state is a recurring catalytic strategy of enzymes that cleave N-glycosidic bonds. However, the mechanisms underlying leaving group activation are still the subject of debate for the nucleoside hydrolases.

The purine salvage pathway of parasitic protozoa is currently considered as a target for drug development because these organisms cannot synthesize purines de novo. Insight into the structure and mechanism of the involved enzymes can aid in the development of potent inhibitors, leading to new curative drugs. Nucleoside hydrolases are key enzymes in the purine salvage pathway of Trypanosomatidae, and they are especially attractive because they have no equivalent in mammalian cells. We cloned, expressed and purified a nucleoside hydrolase from Trypanosoma vivax. The substrate activity profile establishes the enzyme to be a member of the inosine-adenosine-guanosine-preferring nucleoside hydrolases (IAG-NH). We solved the crystal structure of the enzyme at 1.6 A resolution using MAD techniques. The complex of the enzyme with the substrate analogue 3-deaza-adenosine is presented. These are the first structures of an IAG-NH reported in the literature. The T. vivax IAG-NH is a homodimer, with each subunit consisting of ten beta-strands, 12 alpha-helices and three small 3(10)-helices. Six of the eight strands of the central beta-sheet form a motif resembling the Rossmann fold. Superposition of the active sites of this IAG-NH and the inosine-uridine-preferring nucleoside hydrolase (IU-NH) of Crithidia fasciculata shows the molecular basis of the different substrate specificity distinguishing these two classes of nucleoside hydrolases. An "aromatic stacking network" in the active site of the IAG-NH, absent from the IU-NH, imposes the purine specificity. Asp10 is the proposed general base in the reaction mechanism, abstracting a proton from a nucleophilic water molecule. Asp40 (replaced by Asn39 in the IU-NH) is positioned appropriately to act as a general acid and to protonate the purine leaving group. The second general acid, needed for full enzymatic activity, is probably part of a flexible loop located in the vicinity of the active site.

Nucleoside hydrolases are key enzymes in the purine salvage pathway of Trypanosomatidae and are considered as targets for drug design. We previously reported the first x-ray structure of an inosine-adenosineguanosine preferring nucleoside hydrolase (IAG-NH) from Trypanosoma vivax (1). Here we report the 2.0-Å crystal structure of the slow D10A mutant in complex with the inhibitor 3-deaza-adenosine and the 1.6-Å crystal structure of the same enzyme in complex with a genuine substrate inosine. The enzyme-substrate complex shows the substrate bound to the enzyme in a different conformation from 3-deaza-adenosine and provides a snapshot along the reaction coordinate of the enzyme-catalyzed reaction. The chemical groups on the substrate important for binding and catalysis are mapped. The 2-OH, 3-OH, and 5-OH contribute 4.6, 7.5, and 5.4 kcal/mol to k cat /K m , respectively. Specific interactions with the exocyclic groups on the purine ring are not required for catalysis. Site-directed mutagenesis indicates that the purine specificity of the IAG-NHs is imposed by a parallel aromatic stacking interaction involving Trp 83 and Trp 260. The pH profiles of k cat and k cat /K m indicate the existence of one or more proton donors, possibly involved in leaving group activation. However, mutagenesis of the active site residues around the nucleoside base and an alanine scan of a flexible loop near the active site fail to identify this general acid. The parallel aromatic stacking seems to provide the most likely alternative mechanism for leaving group activation.