translocation of isoprenoid linked complex sugars across membranes

Translocation of isoprenoid linked complex sugars

across membranes

8

2 OH Polyisoprenol

7

2

OH Dolichol

Bacterial envelopes

Gram negative (Salmonella)

Gram positive (S.aureus)

Cell wall

membrane

out

inA AA E KMUDP

A AA E KM

Lipid I

MraY

UDP G

Lipid IIMurG

G

A AA E KM

AA AEK M

G

Lipid II

Bacterial Cell Wall SynthesisBacterial Cell Wall Synthesis

A AA E KM

G

A AA E KM

G

out

in

A AA E KM

G

A AA E KMUDP

A AA E KM

Lipid I

MraY

UDP G

Lipid IIMurG

G

A AA E KM

AA AEK M

G

A AA E KM

G

Vancomycin

RamoplaninMersacidin NisinPlectacin

D-cycloserineFosfomycin

Bacitracin

penicillins

Mureidomycin

Moenomycin

Sites of Action of AntibioticsSites of Action of Antibiotics

out

inA AA E KMUDP

A AA E KM

Lipid I

MraY

UDP G

Lipid IIMurG

G

A AA E KM

AA AEK M

G

Lipid II

Transport of peptidoglycan precursors Transport of peptidoglycan precursors occurs via an unknown mechanismoccurs via an unknown mechanism

A AA E KM

G

A AA E KM

G

out

in

A AA E KM

G

A AA E KMUDP

A AA E KM

Lipid I

MraY

UDP G

Lipid IIMurG

G

A AA E KM

AA AEK M

G

A AA E KM

G

Vancomycin

RamoplaninMersacidin Nisin

D-cycloserineFosfomycin

Bacitracin

penicillins

Mureidomycin

Moenomycin

Sites of Action of AntibioticsSites of Action of Antibiotics

In order to identify proteins involved in the In order to identify proteins involved in the transport process we need:transport process we need:

• an assay that can determine the optimal an assay that can determine the optimal conditions for Lipid II transportconditions for Lipid II transport

• a method that is able to identify the a method that is able to identify the components involved in the transport processcomponents involved in the transport process

Is it a spontaneous process?Is it a spontaneous process?

DAGDAG CerCerCholChol

LPCLPCPCPC gangliosideganglioside

1010-1 -1 s seconds 10 h >10 hs seconds 10 h >10 h

Lipid structure predicts flip rate in model membraneLipid structure predicts flip rate in model membrane

What about polyisoprenoid based lipids?

CHR1NH

(CH2)4

NH2

R2C

O

+

Pi

Pi

G

MK A A

8

2

Dansyl chloride

Pyrene chloride

NBD chloride

FITC

A E

synthesis of NBD-labeled Lipid II

Lipid II

NBD-Lipid II

I2-stain FluorescenceTLC

Lipid II topology in membranesLipid II topology in membranesDithionite assayDithionite assay

•Based on irreversible quenching of fluorescent NBD-label by dithionite

Does Lipid II spontaneous flip/flop across the membrane?

DOPE/DOPG/NBD-Lipid II 75:25:0.2

0 25 50 10075

time (s)

NB

D-f

luo

resc

ence

(a.

u.)

0

1

2

NBD-based lipid flip/flop testNBD-based lipid flip/flop test

Does Lipid II spontaneous flip/flop across the membrane?

DOPE/DOPG/NBD-Lipid II 75:25:0.2

0 25 50 10075

time (s)

NB

D-f

luo

resc

ence

(a.

u.)

0

1

2

dithionite

triton X-100

50%

Lipid II Does not spontaneously flop across the membrane!

DOPE/DOPG/NBD-Lipid II 75:25:0.2

?

Incubation at RT

time (hrs)

% p

rote

ctio

n

75

25

100

00 1 202 3

Protein activity is necessary for efficient transport of Lipid II across the bacterial plasma membrane

Dithionite assay failed in biological membranes……

out

inA AA E KMUDP

A AA E KM

Lipid I

MraY

UDP G

Lipid IIMurG

G

A AA E KM

AA AEK M

G

Lipid II

Transport of peptidoglycan precursors Transport of peptidoglycan precursors occurs via an unknown mechanismoccurs via an unknown mechanism

A AA E KM

G

A AA E KM

G

Pi

Pi

G

MK A A

8

2

A E

out

in

A AA E KM

G

A AA E KMUDP

A AA E KM

Lipid I

MraY

UDP G

Lipid IIMurG

G

A AA E KM

AA AEK M

G

A AA E KM

G

Vancomycin

RamoplaninMersacidin Nisin

D-cycloserineFosfomycin

Bacitracin

penicillins

Mureidomycin

Moenomycin

Sites of Action of AntibioticsSites of Action of Antibiotics

Options?Options?

Can the specific recognition of Can the specific recognition of Lipid II by vancomycin be used Lipid II by vancomycin be used

to determine the Lipid II to determine the Lipid II topology?topology?

A Lipid II translocation assay based on A Lipid II translocation assay based on FRETFRET

Pi

Pi

G

M

Plasma membrane

Fluorescent lipid II

in

out

Pi

Pi

G

M

Pi

Pi

G

M

Plasma membrane

Fluorescent lipid II

in

out

A Lipid II translocation assay based on FRET

A Lipid II translocation assay based on FRET between Lipid II and vancomycin

Pi

Pi

G

M

Pi

Pi

G

M

Plasma membrane

Vanco

Fluorescent lipid II

in

out

Pi

Pi

G

M

Pi

Pi

G

M

Plasma membrane

Vanco

Fluorescent lipid II

FRET

in

out

A Lipid II translocation assay based on FRET between Lipid II and vancomycin

FRETFRET

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

A FRET-based Lipid II translocation assayA FRET-based Lipid II translocation assay

Make use of:

-NBD-labeled Lipid II

-coumarin or rhodamine labeled vancomycin

Coumarin spectrum NBD spectrum

Which sites to choose on Which sites to choose on vanco?vanco?

Preparation of fluorescent vancomycinPreparation 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

Analysis of vanco-TMR and vanco-LRBAnalysis of vanco-TMR and vanco-LRB

Excitation and emission spectra

wavelength (nm)

400 450 500 550 600 650

Abs

/Flu

(A

.U.)

0.0

0.5

1.0

1.5

2.0

2.5

3.0excit. NBD-LII emis. NBD-LII excit. vanco-TMR emis. vanco-TMR excit. vanco-LRB emis. vanco-LRB

Lipid II Vanco

vancomycin

Tetramethyl rhodamine cadaverine

Absorption max ~550

Labeling did not reduce activity

TMR-vancomycinTMR-vancomycin

Pi

Pi

G

M

Are NBD-Lipid II and TMR-vancomycin able Are NBD-Lipid II and TMR-vancomycin able to form a complex and does that result in to form a complex and does that result in

FRET?FRET?

Vanco+ ?=

In buffer containing triton X-100

Fluorescence spectra of NBD-LII and vanco-TMRexcitation 481 nm

wavelength (nm)

500 520 540 560 580 600 620 640

Flu

ore

scen

ce (A

.U.)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

vanco-TMR NBD-LII NBD-LII + vanco-TMR Sum of black and red

Fluorescent spectra of the individual moleculesFluorescent spectra of the individual molecules

Fluorescence spectra of NBD-LII and vanco-TMRexcitation 481 nm

wavelength (nm)

500 520 540 560 580 600 620 6400.0

0.5

1.0

1.5

2.0

2.5

3.0

vanco-TMR NBD-LII NBD-LII + vanco-TMR Sum of black and red

Vanco-TMR recognizes NBD-Lipid II, resulting Vanco-TMR recognizes NBD-Lipid II, resulting in FRETin FRET

FRET…

Flu

ore

scen

ce (A

.U.)

Now what?Now what?

how to proceed from here?how to proceed from here?

E. coli membrane vesicle E. coli membrane vesicle systemssystems

out

inA AA E KMUDP

A AA E KM

Lipid I

MraY

UDP G

Lipid IIMurG

G

A AA E KM

AA AEK M

G

Lipid II

Transport of peptidoglycan precursors Transport of peptidoglycan precursors occurs via an unknown mechanismoccurs via an unknown mechanism

A AA E KM

G

A AA E KM

G

ISO RSO

In vitro Lipid II translocationIn vitro Lipid II translocation~FRET assay~~FRET assay~

Getting NBD-UMpp inside the RSOs (Freeze-thawing versus incubation)LI/LII

freeze-thaw or incubation

Wavelength (nm)

500 520 540 560 580 600 620

Flu

ore

sce

nce

(A.U

.)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4LII freeze-thawLII incubationbackground freeze-thawbackground incubationLI freeze-thawLI incubation fr/th incub control

LI LII LI LII LI LII

In vitro Lipid II translocationIn vitro Lipid II translocationFRET assayFRET assay

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

Pi

Pi

G

M

Membrane

Fluorescent lipid II

in

outPi

Pi

G

M

15 °C

Vanco

FRET

vanco-TMR

A AA E KMUDP

UDP G

A AA E KMUDP

UDP G

RSO

The appearance of FRET demonstrates The appearance of FRET demonstrates that Lipid II translocation occurredthat Lipid II translocation occurred

Lipid II + vanco-TMR

Wavelength (nm)

500 520 540 560 580 600 620

Flu

ore

sce

nc

e (A

.U.)

0.0

0.2

0.4

0.6

0.8

1.0

t=0t=5t=10t=20t=30

Spectra at 0, 5, 10, 20 and 30 min at 15 °C with vanco-TMR

Bert Rutten, Aurin Vos & Sacha Dalhuijsen