Updated mechanism of multidrug ABC transporters from Bacillus - - PowerPoint PPT Presentation

10th French-Belgian ABC meeting Brussels, Belgium, October 19-20, 2012

‘Updated’ mechanism of multidrug ABC transporters from Bacillus subtilis

Jean-Michel Jault (Institut de Biologie Structurale, Grenoble, France)

ABC (“A ATP-B Binding C Cassette”) Transporters

• Family of ubiquitous transporters, importers + exporters: ˜ 25 000 / 71 659

(Transporter DataBase) Ren et al (2007) NAR

• Modular Structure : 4 core domains ( + 1 substrate binding protein for importers).

Membrane

B C D F P P Q M A J

Lipid A

MsbAMsbA

Outside Inside

• ligopeptide

histidine Multiple drugs Chloride ion

Membrane P-gp CFTR TAP 1 TAP 2

Peptides Multiple drugs

BmrA BmrA

Multiple drugs

BmrCBmrD

Structure of the homo-dimer of 2 NBDs : the transient ‘sandwich’ dimer

The inactive mutant traps 2 ATP molecules at the interface of the 2 NBDs

Walker A Walker A ABC ABC

D D C E

lipoproteins

Mb Out In

LolD subunit

Conserved Conserved motifs : motifs :

  Walker

Walker A & B A & B: : ATP ATP

  H

H & & Q Q: : ATP ATP

  ABC

ABC signature signature ==> ==> ATP ATP too too

NBD1 NBD2

Smith (2002) Mol Cell

Structure of the homo-dimer of 2 NBDs : the transient ‘sandwich’ dimer

The inactive mutant traps 2 ATP molecules at the interface of the 2 NBDs

Walker A Walker A ABC ABC

D D C E

lipoproteins

Mb Out In

LolD subunit

Conserved Conserved motifs : motifs :

  Walker

Walker A & B A & B: : ATP ATP

  H

H & & Q Q: : ATP ATP

  ABC

ABC signature signature ==> ==> ATP ATP too too

NBD1 NBD2

Smith (2002) Mol Cell

2006: 3-D structure of a bacterial ABC MDR 2006: 3-D structure of a bacterial ABC MDR transp transp. .

ICD1 ICD2 3 sub-domains :

• NBD
• TMD (6 TMH / monomer)
• ICD1 (TMH 2 and 3) interacts in cis but

ICD2 (TMH 4 and 5) interacts in trans :

• ut

in

Multiple Drugs

Sav 1866 Sav 1866

mb

ATP ATP

mb

NBD

Dawson & Locher (2006) Nature

TMD TMD TMD NBD NBD NBD NBD ICD ICD ICD ICD

1 2 1 2

TMD TMD domain swapping of ICD2

3 conformations of 3 conformations of MsbA MsbA

Open apo Closed apo

Ward et al (2007) PNAS

~ 50 Å

• ut

in mb

Lipid A

MsbAMsbA

mb

• ut

in

+ ATP ICD ICD ICD ICD

1 2 1 2

ICD ICD ICD ICD

1 2 1 2

ICD ICD ICD ICD 1

2 1 2

Closed ATP-bound (or ADP-Vi)

Rigid body motion

Are these open conformations physiologically relevant ?

Open apo P-gp Open apo EcMsbA … “Thus, the structure may represent a crystallization artifact or a nonfunctional conformation that has only very transient existence”. ICD ICD ICD ICD

1 2 1 2 Aller et al. (2009) Science

Overexpression of BmrA and purification

• Purification in a single step

(Ct-polyhistidine-tagged) ~2-3 mg of BmrA/Liter

Orelle et al. (2003) JBC

Hoechst 33342 ATP ADP + Pi

25 50 75 100 200 400 600 Time (sec.) Normalized fluorescence (%) 1 2 3 kDa 94 67 43 30

ATP

Hoechst

• Inside-out membrane vesicles:

BmrA ~ 50% of mb proteins

Steinfels et al. (2002) BBA

Multiple drugs

BmrA BmrA

Time

MW

Time

119 79 46 31

5’ 15’ 30’ 1h 2h 3h C

C

5’ 15’ 30’ 1h 2h 3h

Membrane Apo Vi-inhibited

Apo Vi-inhibited

Time C

MW

24 19 119 79 46 31

5’ 15’ 30’ 5’ 15’ 30’

DDM purified

Behavior of BmrA in membrane or in detergent : 2 different conformations ?

The Vi-inhibited BmrA is much less sensitive to limited digestion by trypsin than BmrA in the resting state. ==> BmrA switches between two very different conformations in mb or in detergent

Are the two BmrA conformations really ‘stable’ ? Rigid body motion ?

interac(on
between
TMD
(ICD)
&
NBD
similar
in
open
or
closed
state

The H/D exchange technique

H : Do not exchange H : Too fast to be monitored H : Best candidates for deuterium exchange; exchange rate depends on structure and accessibility

• Different types of hydrogen in proteins

D D D

The H/D exchange technique

H : Do not exchange H : Too fast to be monitored H : Best candidates for deuterium exchange; exchange rate depends on structure and accessibility

• Different types of hydrogen in proteins

D D D

Labeling quench

H+, 0°C

Obtain peptides to locate D Proteolysis H/D exchange kinetics Labeled region

Exchange H D Dilution D2O 1- Global kinetics HPLC MS

Separate peptides Mass measurement of each peptide 2- Local kinetics Time (sec) Deuteration (%)

TMD : poorly defined, but ICDs OK NBD : well defined

sequence coverage

Total : 78% using pepsin

NBD 1 2 3 4 5 6 NBD

ICD1 ICD2

TMD NBD

Portfolio of different peptides

WT Apo E504A (ATPase inactive) + ATP/Mg

20 40 60 80 15 60 300 600 1800 3600 Time
(s)

%
H/D
Exchange

Open Closed

372-383 Walker A

Few examples : Walker A motif

Protection by ATP

20 40 60 80 15 60 300 600 1800 3600 Time
(s)

%
H/D
Exchange

Open Closed

479-492 ABC motif Protection by ATP

ABC signature peptide

20 40 60 80 15 60 300 600 1800 3600 Time
(s)

%
H/D
Exchange

Open Closed

501-509 Walker B

Walker B motif

Protection by ATP

20 40 60 80 15 60 300 600 1800 3600

Time
(s) %
H/D
Exchange

Open Closed

104-114 ICD 1

Peptide from ICD1 (cis with its own NBD)

20 40 60 80 15 60 300 600 1800 3600 Time
(s)

%
H/D
Exchange

Open Closed

203-215 ICD 2

Peptide from ICD2 (trans with the other NBD)

20 40 60 80 15 60 300 600 1800 3600 Time
(s)

%
H/D
Exchange

Open Closed

216-236 ICD 2

2nd Peptide from ICD2 (trans with the other NBD)

Poorly exchangeable OK with 3D structure Highly exchangeable, especially for ICDs Different from 3D structure Greater flexibility of ICDs Reorientation of the NBDs and/or unfolding

Conclusions from H/D exchange

• Similar behavior of BmrA in membrane and purified in DDM
• BmrA switches between two conformations :
• Resting state : open conformation (high H/D exchange)
• ATP-bound state : closed conformation (low H/D exchange)
• In the resting state, the ICDs and in particular ICD2, are

highly flexible ==> ICDs disengaged from the NBD ==> different orientation and/or unfolding of the NBD ==> ≠ 3D structure of EcMsbA (rigid body motion) ==> Can this flexibility help to recognize many substrates ?

Mehmood et al (2012) PNAS

Is this flexibility of the ICDs restricted to BmrA ?

Multiple drugs

BmrCBmrD

BmrC/BmrD model based on the TM heterodimer (Seeger’s group)

Heterodimer with asymmetric NBDs (MRP, CFTR, TAP1/TAP2…)

..even in the resting state Multidrug ABC transporters need to…

IBCP, Lyon

Vincent Chaptal Pierre Falson Attilio Di Pietro

Many thanks to….

Our team in IBS

Shahid Mehmood Jonathan Sarwan Benjamin Wiseman Emilie Boncoeur Eric Forest

• Univ. Oxford, UK

Carmen Domene