Bacteriophage CEV1 was isolated from sheep resistant to Escherichia coli O157:H7 colonization. In vitro, CEV1 efficiently infected E. coli O157:H7 grown both aerobically and anaerobically. In vivo, sheep receiving a single oral dose of CEV1 showed a 2-log-unit reduction in intestinal E. coli O157:H7 levels within 2 days compared to levels in the controls.

Vacuolar ATPases (V-ATPases) are essential proton pumps that acidify the lumen of subcellular organelles in all eukaryotic cells and the extracellular space in some tissues. V-ATPase activity is regulated by a unique mechanism referred to as reversible disassembly, wherein the soluble catalytic sector, V 1 , is released from the membrane and its MgATPase activity silenced. The crystal structure of yeast V 1 presented here shows that activity silencing involves a large conformational change of subunit H, with its C-terminal domain rotating~150°from a position near the membrane in holo V-ATPase to a position at the bottom of V 1 near an open catalytic site. Together with biochemical data, the structure supports a mechanistic model wherein subunit H inhibits ATPase activity by stabilizing an open catalytic site that results in tight binding of inhibitory ADP at another site.

SUMMARY Vacuolar ATPases (V-ATPases) are multisubunit rotary motor proton pumps that function to acidify subcellular organelles in all eukaryotic organisms. V-ATPase is regulated by a unique mechanism that involves reversible dissociation into V1-ATPase and Vo proton channel, a process that involves breaking of protein interactions mediated by subunit C, the cytoplasmic domain of subunit 'a' and three 'peripheral stalks', each made of a heterodimer of E and G subunits. Here we present crystal structures of a yeast V-ATPase heterotrimeric complex composed of EG heterodimer and the head domain of subunit C (Chead). The structures show EG heterodimer folded in a non-canonical coiled coil that is stabilized at its N-terminal ends by binding to Chead. The coiled coil is disrupted by a bulge of partially unfolded secondary structure in subunit G and we speculate that this unique feature in the eukaryotic V-ATPase peripheral stalk may play an important role in enzyme structure and regulation by reversible dissociation.

To determine which subunit C domain binds EG with high affinity, we have generated C head and C foot and characterized their interaction with subunit EG heterodimer. Our findings indicate that the high affinity site for EGC interaction is C head . In addition, we provide evidence that the EGC head interaction greatly stabilizes EG heterodimer.2 is a ubiquitous multisubunit enzyme that couples the free energy of ATP hydrolysis to active proton transport. In all eukaryotic cells, V-ATPase function sets up an electrochemical potential and acidifies intracellular compartments (1-5). These functions make the V-ATPase a crucial enzyme involved in intracellular traffic, vesicular transport, endo/exocytosis, and pH homeostasis. In the specialized cells of higher eukaryotes, plasma membrane-associated V-ATPases serve to acidify the extracellular space. The malfunction of the V-ATPase in these cells has been linked to a range of diseases including osteoporosis, diabetes, renal tubular acidosis, and cancer (6 -9). As shown in EM images and reconstructions, the V-ATPase is bilobular in overall structure, typical of the rotary ATPases (10 -13). These two lobes are composed of distinct functional domains, a soluble catalytic sector, V 1 , and a membrane integral proton pore, V o . The V 1 sector contains subunits A 3 B 3 (C)DE 3 FG 3 H, and the V o is made of ac 3-4 cЈcЉde. Most V-ATPase subunits have functional and structural counterparts in the A-and F-type motors; however, only the nucleotide-binding and proteolipid subunits share significant sequence identity. Much like in the F-ATP synthase, the AB subunits come together in an alternating arrangement to form a catalytic hexamer with nucleotide-binding sites located at the AB interfaces (14, 15). ATP hydrolysis-driven rotation of a central rotor domain, composed of subunits DFd and a ring of the proteolipid subunits, c 3-4 cЈcЉ, is coupled to proton translocation along the interface of the proteolipid ring and the C-terminal domain of the a subunit. During catalysis, three peripheral stalks (each formed by a subunit EG heterodimer) in conjunction with subunits C and H and the N-terminal domain of subunit a (a NT ), resist the torque of rotation, thus keeping the A 3 B 3 hexamer static and allowing for rotation to be productive.Unlike F-ATPase, the activity of eukaryotic V-ATPase is regulated by a unique mechanism of reversible dissociation, a process first described for the enzymes from yeast and insect (16, 17) but more recently also found in cells of higher animals (18 -20). In yeast under conditions of nutrient deprivation, the V 1 sector dissociates from the V o sector, and both are functionally silenced (21,22). Although dissociated, a single V 1 subunit, C, is released into the cytosol and is reincorporated into the enzyme upon nutrient readdition (16). Because the C subunit has been proposed to be a molecular switch for controlled enzyme dissociation, the interactions between the C subunit and its intraenzyme binding partners are of particular interest.The crystal ...

The vacuolar ATPase (V-ATPase; V 1 V o -ATPase) is a large multisubunit proton pump found in the endomembrane system of all eukaryotic cells where it acidifies the lumen of subcellular organelles including lysosomes, endosomes, the Golgi apparatus, and clathrin-coated vesicles. V-ATPase function is essential for pH and ion homeostasis, protein trafficking, endocytosis, mechanistic target of rapamycin (mTOR), and Notch signaling, as well as hormone secretion and neurotransmitter release. V-ATPase can also be found in the plasma membrane of polarized animal cells where its proton pumping function is involved in bone remodeling, urine acidification, and sperm maturation. Aberrant (hypo or hyper) activity has been associated with numerous human diseases and the V-ATPase has therefore been recognized as a potential drug target. Recent progress with moderate to high-resolution structure determination by cryo electron microscopy and X-ray crystallography together with sophisticated single-molecule and biochemical experiments have provided a detailed picture of the structure and unique mode of regulation of the V-ATPase. This review summarizes the recent advances, focusing on the structural and biophysical aspects of the field.