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A novel TB drug targets the ATP synthase rotorring of mycobacteriaLaura Preiss1, Luise Eckhardt-Strelau1, Julian Langer2,Anil Koul3, Thomas Meier11Department of Structural Biology, Max-von-Laue-Str. 3,60438 Frankfurt, Germany2Department of Molecular Membrane Biology Max-Planck-Institute ofBiophysics, Max-von-Laue-Str. 3, 60438 Frankfurt, Germany3Janssen R&D (Johnson&Johnson Pharmaceuticals), Beerse, BelgiumE-mail: [email protected]
The increasing number of tuberculosis (TB) infections with multi-drug resistant (MDR) Mycobacterium tuberculosis strains causes asevere worldwide health problem, necessitating the development ofnew antibiotics. A novel drug, the diarylquinoline TMC207, was foundto be highly effective in the treatment of MDR-TB. It was reported thatthe drug targets the rotor ring of the energy converting enzyme F1Fo-ATP synthase, which is essential for the survival of mycobacteria.However, structural and biochemical information about the precisebinding site of TMC207 and its mechanism of action are not knownat the level of the isolated ATP synthase or its rotor ring complex.We performed ATP synthesis experiments on a mycobacterial ATPsynthase, including inhibition studies using two different antibiotics,TMC207 and mefloquine. TMC207 inhibited 50% of ATP synthesis at0.03 μM. In contrast, mefloquine was less active, but nevertheless stillan efficient inhibitor of the mycobacterial ATP synthase at 15 μM(IC50). We then purified a mycobacterial ATP synthase rotor (c-) ringand established a drug competition assay using the well known ATPsynthesis inhibitor dicyclohexylcarbodiimide (DCCD). The resultsshow that TMC207 competes with DCCD at the proton binding site ofthe c-ring. This is direct biochemical proof that the c-ring's ionbinding site is the actual target of the drug, to which it binds withhigh affinity and specificity. Similar results were found for mefloquine,although we detected lower affinities for the proton binding site. Tofurther explore the highly specific drug-target interaction of TMC207with the mycobacterial c-ring, we aim to solve its structure in complexwith TMC207. X-ray diffracting 3D crystals of the rotor ring-drug complexare available. This work provides insights into the specific interaction ofthe isolated mycobacterial ATP synthase rotor ring with a novel andhighly potent drug, in the perspective to treat infections in patients withMDRM. tuberculosis strains. Based on these results, an optimisation ofTMC207 derivatives and further development of drugs, which targetthe ATP synthase to treat TB becomes conceivable.
doi:10.1016/j.bbabio.2012.06.068
1P36
The effect of mutations γM23K and βL249Q on ADP-inhibitionof H+-FOF1-ATP-synthase in Escherichia coliA. Prikhodko, A. Lapashina, R. Zinovkin, M. Vitushkina, B. FenioukFaculty of bioengeneering and bioinformatics, Moscow State University,Leninskiye gory, 1\73, 119991, Moscow, Russia; Faculty of biology,Moscow State University, Leninskiye gory, 1/12, 119234, Moscow, RussiaE-mail: [email protected]
H+-FOF1-ATP-synthase catalyzes generation of ATP from ADP andinorganic phosphate using the energy of transmembrane electro-chemical potential difference of protons. This enzyme can function inopposite direction as ATP-driven proton pump. ADP-inhibition isknown to reduce this ATPase activity: ADP without phosphate binds
empty catalytic site and this binding induces transition of enzyme toADP-inhibited state.
We studied ADP-inhibition on inverted membranes from Escherichiacoli. ATP hydrolysis is accompanied by stoichiometric proton releaseat pHN7.2. This acidification was measured by pH indicator phenolred absorption changes.
We studied wild-type enzyme at room temperature (24 °C ) and37 °C. At room temperature ADP-inhibition was weaker than at 37 °C.Also we studied the influence of inorganic phosphate on the ADP-inhibition of the enzyme. In the presence of phosphate ADP-inhibition increased in the wild-type enzyme.
It was shown previously [1] that mutation Met23Lys in the γsubunit of the enzyme enhanced ADP-inhibition compared to thewild-type enzyme in purple bacteria Rhodobacter capsulatus. Ourresults indicate that mutation showed the same effect on membranesfrom E.coli at 37 °C, but at room temperature this effect was reversedas in the wild type. Inorganic phosphate enhanced ADP-inhibition ofthe enzyme carrying this mutation.
We also found that enzyme with mutation βLeu249Gln situatedon the interface between α and β subunits had weak ADP-inhibitionat 24 °C, but at 37 °C the inhibition was stronger. In contrast toγMet23Lys and wild-type, addition of phosphate reduced ADP-inhibition of the βL249Q mutant. Our results allow us to supposethat this position is important for modulation of ADP-inhibition byphosphate.
Reference[1] B.A. Feniouk, A. Rebecchi, D. Giovannini, S. Anefors, A.Y.
Mulkidjanian, W. Junge, P. Turina, B.A. Melandri, Biochim.Biophys. Acta 1767 (2007) 1319–1330.
doi:10.1016/j.bbabio.2012.06.069
1P37
Cholesterol-dependent translocation of the F1F0-ATP synthaseinhibitor factor 1 (IF1) to cell surfaceAmit Kumar Rai1, Federica Dabbeni-Sala1,Giovanna Lippe2, Michelangelo Campanella31Department of Pharmacology, University of Padua2Department of Food Science and Technology, University of Udine, Italy3Royal Veterinary College and UCL Consortium for MitochondrialResearch London, UKE-mail: [email protected]
The mitochondrial F1F0-ATP synthase is one of the most ubiqui-tously studied enzymes. In physiological conditions it carries out ATPsynthesis, utilizing the H+ gradient generated by the ElectronRespiratory Chain. When mitochondrial function is compromised,the enzyme acts as ATP consumer depleting cells from ATP which isinhibited by IF1-the natural endogenous inhibitor of ATP synthase [1].Recent reports, including ours [2], have suggested the presence ofsubunits or thewhole complex on the cell surface of various cell types [3].
Both by immunofluorescent and western blotting analyses, wefound that IF1 co-localizes with the ATP synthase (β-subunit) andflotillin, a lipid raft marker, on the cell surface of HeLa cells. The OSCP,a subunit of the ATP synthase assembled at very late stage, was alsofound to pair the β-subunit and IF1 in non-permeabilized cells,suggesting the presence of a small functional mitochondria-likeorganization. Notably, cholesterol loading, performed at differenttime intervals, reduced the mitochondrial content of IF1, with aparallel increase of the ectopic expression.
In conclusion, our data report that IF1 (re)localizes in the lipid raftsof plasma membranes in association with the F1F0-ATPase depending
AbstractsS22
on the cholesterol level to which cells were exposed. The highersurface expression of IF1 could therefore represent a mechanism topreserve ATP level on the extra cellular space sparing this from theF1F0-ATPase mediated hydrolysis.
AKR is supported by a fellowship from Fondazione Cassa diRisparmio di Padova e Rovigo.
References[1] M. Campanella, N. Parker, C.H. Tan, A.M. Hall, M.R. Duchen, IF1:
setting the pace of the F1F0-ATP synthase, Trends Biochem. Sci. 34(2009) 343–350.
[2] V. Giorgio, E. Bisetto, R. Franca, D.A. Harris, S. Passamonti, G.Lippe, The ectopic F0F1 ATP synthase of rat liver is modulated inacute cholestasis by the inhibitor protein IF1, J. Bioenerg.Biomembr. (2010) 1–7.
[3] S.L. Chi, S.V. Pizzo, Cell surface F1F0 ATP synthase: A new para-digm? Ann. Med. 38 (2006) 429–438.
doi:10.1016/j.bbabio.2012.06.070
1P38
High-resolution structure of a Na+-driven ATP synthaserotor ring with a two-carboxylate ion-binding motifSarah Schulz1, Alexander Krah2, Özkan Yildiz1,José D. Faraldo-Gómez2, Thomas Meier11Department of Structural Biology2Theoretical Molecular Biophysics Group Max-Planck-Instituteof BiophysicsE-mail: [email protected]
In the bacterium Fusobacterium nucleatum, a Na+ motive force isgenerated by the fermentation of glutamate via the hydroxyglutaratepathway, which involves a Na+ pump, the glutaconyl-CoA-decarbox-ylase. We hypothesized that F. nucleatum contains a Na+-dependentATP synthase, which directly uses this smf to produce ATP. Using aheterologous hybrid ATP synthase expression system, we producedand isolated the F. nucleatum c-ring and performed a biochemicallabeling study using a fluorescent analogue of the ATP synthaseinhibitor dicyclohexylcarbodiimide. The study demonstrates that thismembrane rotor is coupled to Na+ at neutral pH and in the presenceof 10 mM Na+. We created a homology model of this c-ring andcarried out molecular simulations to assess the characteristics of itsion-binding sites. The calculations support the notion that this rotor iscoupled to Na+ under physiological conditions, as a result of theamino-acid make-up of the ion-coordination shell, which includes theuniversally conserved carboxylate side-chain as well as other polarmoieties from the protein and a water molecule. Interestingly, amongthese there is a second glutamate side chain, which our calculationssuggest is constitutively protonated, effectively replacing a glutaminein homologous c-rings. Hence it confers neither H+ coupling nor asignificantly more pronounced Na+ specificity. We succeeded tocrystallize this c-ring and to solve its structure by X-ray diffraction at2.2 Å resolution. The experimental structure shows that the ringcontains 11 c-subunits, which form an hourglass-shaped cylinderwith a central pore. Each c-subunit consists of an a-helical hairpinwith the N- and C-termini on the periplasmic side. The structure alsoshows the location of the 11 Na+ binding sites, formed betweenneighboring c-subunits on the outer surface of the ring, at themembrane middle. Importantly, the Na+ coordination networkrevealed by the crystal structure is entirely consistent with thepresence of an additional H+, shared between the two carboxylateside-chains in the binding site. Such a constellation is feasibleconsidering that F. nucleatum faces temporal chemical changes of its
natural habitat, e.g. the human mouth flora. As this c-ring structuredeviates significantly from the structure of a eukaryotic c-ring, thisworkprovides the structural basis for screening of novel drugs against theATP synthase of the opportunistic pathogenic bacterium F. nucleatum.
ReferenceBeatrix et al, 1990 Beatrix, et al., Arch. Microbiol. 154 (1990) 362–369.
doi:10.1016/j.bbabio.2012.06.071
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H+/ATP ratio of FoF1-ATP synthase from the thermophilicBacillus PS3N. Soga, K. Kimura, Y. Kasuya, T. Suzuki, M. Yoshida, K. Kinosita Jr.Waseda University, Department of Physics, 3-4-1 Okubo, Shinjuku-ku,Tokyo, Japan, Kyoto Sangyo University, Department of MolecularBioscience, Kamigamo-Motoyama, Kyoto, JapanE-mail: [email protected]
FoF1-ATP synthase converts the energy of transmembrane protonflow into the high-energy bond between ADP and phosphate. Theproton motive force that drives this reaction consists of twocomponents, the pH difference (ΔpH) across the membrane andtransmembrane electrical potential (Δψ). The H+/ATP ratio and thestandard Gibbs free energy of ATP synthase are important parametersof coupling between proton translocation and ATP synthesis.Previously, we have developed the highly reproducible, simpleprocedures for the preparation of active proteoliposomes and forkinetic analysis of ATP synthesis which was driven by acid-basetransition and valinomycin-mediated K+ diffusion potential, andprecisely shown the kinetic equivalence between ΔpH and Δψ[1].Here, to determine the H+/ATP ratio and the free energy of ATPsynthesis for the thermophilic Bacillus PS3 ATP synthase, weexamined how the proton motive force is balanced by counteractingATP hydrolysis [2]. We measured the rates of ATP synthesis andhydrolysis with luciferin-lucifease system as a function of the protonmotive force. For various nucleotide conditions we determined theequilibrium proton motive force at which FoF1 neither synthesizednor hydrolyzed ATP. The shift of the equilibrium proton motiveforce as a function of [ATP]/[ADP][Pi] indicated the H+/ATP ratio of3.0±0.4 and the standard Gibbs free energy of ATP synthesis of36±4 kJ/mol for this thermophilic enzyme.
References[1] N. Soga, K. Kinosita Jr., M. Yoshida, T. Suzuki, J. Biol. Chem. 287
(2012) 9633–9639.[2] S. Steigmiller, P. Turina, P. Gräber, Proc. Natl. Acad. Sci. U.S.A. 105
(2008) 3745–3750.
doi:10.1016/j.bbabio.2012.06.072
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The H+-ATPsynthase from Yeast: Construction andCharacterisation of an EGFP-MF0F1 Fusion Proteinwith Cysteine Mutations for FRET InvestigationsIlka Starke1, Sarah Osterwald1, Peter Gräber1, Jan Petersen2
1Institut für Physikalische Chemie, Albert-Ludwigs Universität Freiburg,Germany2Department of Biochemistry and Molecular Biology, Monash University,Clayton, VIC 3800, AustraliaE-mail: [email protected]
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