Translocation of isoprenoid linked complex sugars across membranes

1. 8 2 Translocation of isoprenoid linked complex sugars across membranes Polyisoprenol OH 2 Dolichol OH 7

2. Gram positive (S.aureus) Gram negative (Salmonella) Cell wall Bacterial envelopes membrane

3. G M G G A A A A A A E E E E E E K K K K K K A A A A A A A A A A A A Lipid I M M MraY MurG Lipid II M M G G UDP M UDP Bacterial Cell Wall Synthesis Lipid II out in

4. G M G G A A A A A A E E E E E E K K K K K K A A A A A A A A A A A A Lipid I M M MraY MurG Lipid II M M G G UDP M UDP Sites of Action of Antibiotics penicillins Ramoplanin Mersacidin Nisin Plectacin Vancomycin Bacitracin out Moenomycin in D-cycloserine Fosfomycin Mureidomycin

5. G M G G A A A A A A E E E E E E K K K K K K A A A A A A A A A A A A Lipid I M M MraY MurG Lipid II M M G G UDP M UDP Transport of peptidoglycan precursors occurs via an unknown mechanism Lipid II out in

6. penicillins Ramoplanin Mersacidin Nisin G Vancomycin M Bacitracin Moenomycin G G A A A A A A E E E E E E K K K K K K A A A A A A A A A A A A Lipid I M M MraY MurG Lipid II M M G G UDP M UDP D-cycloserine Fosfomycin Mureidomycin Sites of Action of Antibiotics out in

7. an assay that can determine the optimal conditions for Lipid II transport a method that is able to identify the components involved in the transport process In order to identify proteins involved in the transport process we need:

8. Is it a spontaneous process?

9. Lipid structure predicts flip rate in model membrane What about polyisoprenoid based lipids? 10-1 s seconds 10 h >10 h DAG Cer Chol PC LPC ganglioside

10. Dansyl chloride G M A E A A K Pi Pyrene chloride Pi O NBD chloride + R2 R1NH CH C 8 (CH2)4 NH2 2 FITC

11. synthesis of NBD-labeled Lipid II I2-stain Fluorescence TLC NBD-Lipid II Lipid II

12. Lipid II topology in membranesDithionite assay • Based on irreversible quenching of fluorescent NBD-label by dithionite

13. NBD-based lipid flip/flop test Does Lipid II spontaneous flip/flop across the membrane? 2 NBD-fluorescence (a.u.) 1 0 0 25 50 100 75 time (s) DOPE/DOPG/NBD-Lipid II 75:25:0.2

14. Does Lipid II spontaneous flip/flop across the membrane? 2 dithionite triton X-100 NBD-fluorescence (a.u.) 50% 1 0 0 25 50 100 75 time (s) DOPE/DOPG/NBD-Lipid II 75:25:0.2

15. Lipid II Does not spontaneously flop across the membrane! Incubation at RT 100 75 % protection ? 25 0 0 1 2 3 20 time (hrs) DOPE/DOPG/NBD-Lipid II 75:25:0.2

16. Protein activity is necessary for efficient transport of Lipid II across the bacterial plasma membrane Dithionite assay failed in biological membranes……

17. G M A E A A K Pi Pi 8 2 Options?

18. Can the specific recognition of Lipid II by vancomycin be used to determine the Lipid II topology?

19. A Lipid II translocation assay based on FRET out in Pi Plasma membrane Pi M Fluorescent lipid II G

20. A Lipid II translocation assay based on FRET G M out Pi Pi in Pi Plasma membrane Pi M Fluorescent lipid II G

21. Vanco A Lipid II translocation assay based on FRET between Lipid II and vancomycin G M out Pi Pi in Pi Plasma membrane Pi M Fluorescent lipid II G

22. Vanco A Lipid II translocation assay based on FRET between Lipid II and vancomycin FRET G M out Pi Pi in Pi Plasma membrane Pi M Fluorescent lipid II G

23. FRET Criteria for choosing donor & acceptor fluorophores: – Sufficient separations in excitation spectra – Overlap between emission spectra of donor and excitation of acceptor – Reasonable separation in emission spectra

24. A FRET-based Lipid II translocation assay • Make use of: • NBD-labeledLipid II • coumarinorrhodaminelabeled vancomycin Coumarin spectrumNBD spectrum

25. Which sites to choose on vanco?

26. Preparation of fluorescent vancomycin • Label vancomycin with: Reacts with: • 7-amino-4-methylcoumarin carboxyl • Tetramethylrhodamine cadaverine (TMR) carboxyl • AMCA-X (coumarin) amine • DACITC (coumarin) amine • 7-methoxycoumarin-3-carboxylic acid amine • Lissamine Rhodamine B (LRB) amine

27. Analysis of vanco-TMR and vanco-LRB Lipid II Vanco

28. TMR-vancomycin vancomycin Tetramethyl rhodamine cadaverine Absorption max ~550 Labeling did not reduce activity

29. Vanco Are NBD-Lipid II and TMR-vancomycin able to form a complex and does that result in FRET? G M ? + Pi = Pi In buffer containing triton X-100

30. Fluorescent spectra of the individual molecules Fluorescence spectra of NBD-LII and vanco-TMR excitation 481 nm 3.0 vanco-TMR NBD-LII NBD-LII + vanco-TMR 2.5 Sum of black and red 2.0 1.5 Fluorescence (A.U.) 1.0 0.5 0.0 500 520 540 560 580 600 620 640 wavelength (nm)

31. Vanco-TMR recognizes NBD-Lipid II, resulting in FRET Fluorescence spectra of NBD-LII and vanco-TMR excitation 481 nm 3.0 vanco-TMR NBD-LII NBD-LII + vanco-TMR 2.5 Sum of black and red FRET… 2.0 1.5 Fluorescence (A.U.) 1.0 0.5 0.0 500 520 540 560 580 600 620 640 wavelength (nm)

32. Now what? how to proceed from here?

33. E. coli membrane vesicle systems ISO RSO

34. In vitro Lipid II translocation~FRET assay~ Getting NBD-UMpp inside the RSOs (Freeze-thawing versus incubation) fr/thincubcontrol LI LII LI LII LI LII

35. FRET A A E E K K A A A A 15 °C G Vanco M vanco-TMR Pi Pi G G UDP UDP M M UDP UDP In vitro Lipid II translocationFRET assay RSO • General procedure • Right side out (RSO) vesicles E. coli W3899 • NBD-UDP-MurNAc-pentapeptide + UDP-GlcNAc • Two freeze-thaw cycles • Lipid II synthesis (on ice) • Fluorescence spectra in the • presence of vanco-TMR (15 °C) Freeze-thaw Synthesis out in Membrane Pi Pi M Fluorescent lipid II G

36. The appearance of FRET demonstrates that Lipid II translocation occurred Spectra at 0, 5, 10, 20 and 30 min at 15 °C with vanco-TMR

37. Bert Rutten, Aurin Vos & Sacha Dalhuijsen