The crystal structure of the c-ring from the proton-coupled F1Fo ATP synthase from Spirulina platensis is shown at 2.1-A resolution. The ring includes 15 membrane-embedded c subunits forming an hourglass-shaped assembly. The structure demonstrates that proton translocation across the membrane entails protonation of a conserved glutamate located near the membrane center in the c subunit outer helix. The proton is locked in this site by a precise hydrogen bond network reminiscent of that in Na+-dependent ATP synthases. However, the structure suggests that the different coordination chemistry of the bound proton and the smaller curvature of the outer helix drastically enhance the selectivity of the H+ site against other cations, including H3O+. We propose a model for proton translocation whereby the c subunits remain in this proton-locked state when facing the membrane lipid. Proton exchange would occur in a more hydrophilic and electrostatically distinct environment upon contact with the a subunit interface.

The microscopic mechanism of coupled c-ring rotation and ion translocation in F(1)F(o)-ATP synthases is unknown. Here we present conclusive evidence supporting the notion that the ability of c-rings to rotate within the F(o) complex derives from the interplay between the ion-binding sites and their nonhomogenous microenvironment. This evidence rests on three atomic structures of the c(15) rotor from crystals grown at low pH, soaked at high pH and, after N,N'-dicyclohexylcarbodiimide (DCCD) modification, resolved at 1.8, 3.0 and 2.2 Å, respectively. Alongside a quantitative DCCD-labeling assay and free-energy molecular dynamics calculations, these data demonstrate how the thermodynamic stability of the so-called proton-locked state is maximized by the lipid membrane. By contrast, a hydrophilic environment at the a-subunit-c-ring interface appears to unlock the binding-site conformation and promotes proton exchange with the surrounding solution. Rotation thus occurs as c-subunits stochastically alternate between these environments, directionally biased by the electrochemical transmembrane gradient.

antibiotic resistance • fluorescence-based assay • metallo-β-lactamase (MBL) • NDM-1 • thiols.ABSTRACT: Resistance to β-lactam antibiotics can be mediated by metallo-β-lactamase enzymes (MBLs). An MBL inhibitor could restore the effectiveness of β-lactams. We report on the evaluation of approved thiol-containing drugs as inhibitors of NDM-1, VIM-1 and IMP-7. Drugs were assessed by a novel assay using a purchasable fluorescent substrate and thermal shift. Best compounds were tested in antimicrobial susceptibility assay. Using these orthogonal screening methods we identified drugs which restored the activity of Imipenem.INTRODUCTION:

The membrane-embedded rotors of Na(+)-dependent F-ATP synthases comprise 11 c-subunits that form a ring, with 11 Na(+) binding sites in between adjacent subunits. Following an updated crystallographic analysis of the c-ring from Ilyobacter tartaricus, we report the complete ion-coordination structure of the Na(+) sites. In addition to the four residues previously identified, there exists a fifth ligand, namely, a buried structural water molecule. This water is itself coordinated by Thr67, which, sequence analysis reveals, is the only residue involved in binding that distinguishes Na(+) synthases from H(+)-ATP synthases known to date. Molecular dynamics simulations and free-energy calculations of the c-ring in a lipid membrane lend clear support to the notion that this fifth ligand is a water molecule, and illustrate its influence on the selectivity of the binding sites. Given the evolutionary ascendancy of sodium over proton bioenergetics, this structure uncovers an ancient strategy for selective ion coupling in ATP synthases.

The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m1acp3Ψ) located next to the P-site tRNA on the small subunit 18S rRNA. While S-adenosylmethionine was identified as the source of the aminocarboxypropyl (acp) group more than 40 years ago the enzyme catalyzing the acp transfer remained elusive. Here we identify the cytoplasmic ribosome biogenesis protein Tsr3 as the responsible enzyme in yeast and human cells. In functionally impaired Tsr3-mutants, a reduced level of acp modification directly correlates with increased 20S pre-rRNA accumulation. The crystal structure of archaeal Tsr3 homologs revealed the same fold as in SPOUT-class RNA-methyltransferases but a distinct SAM binding mode. This unique SAM binding mode explains why Tsr3 transfers the acp and not the methyl group of SAM to its substrate. Structurally, Tsr3 therefore represents a novel class of acp transferase enzymes.