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 A TP- B B inding C C assette”) Transporters oligopeptide histidine J A Outside B C Membrane Q M BmrA BmrA BmrCBmrD MsbAMsbA Inside D F P P Lipid A Multiple Multiple drugs drugs P-gp Membrane CFTR TAP TAP 2 1 Multiple Chloride Peptides drugs ion -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).

Structure of the homo-dimer of 2 NBDs : the transient ‘sandwich’ dimer lipoproteins Out Smith (2002) Mol Cell Mb C E ABC Walker A LolD In D D subunit Conserved motifs : motifs : Conserved NBD1 NBD2  Walker Walker A & B A & B : : ATP ATP   H H & & Q Q : : ATP ATP   ABC ABC signature signature ABC  Walker A ==> ==> ATP ATP too too The inactive mutant traps 2 ATP molecules at the interface of the 2 NBDs

Structure of the homo-dimer of 2 NBDs : the transient ‘sandwich’ dimer lipoproteins Out Smith (2002) Mol Cell Mb C E ABC Walker A LolD In D D subunit Conserved motifs : motifs : Conserved NBD1 NBD2  Walker Walker A & B A & B : : ATP ATP   H H & & Q Q : : ATP ATP   ABC ABC signature signature ABC  Walker A ==> ==> ATP ATP too too The inactive mutant traps 2 ATP molecules at the interface of the 2 NBDs

2006: 3-D structure of a bacterial ABC MDR transp transp. . 2006: 3-D structure of a bacterial ABC MDR out Sav Sav mb 1866 1866 ATP ATP in mb Multiple Drugs TMD 3 sub-domains : - NBD - TMD (6 TMH / monomer) ICD2 ICD1 - ICD1 (TMH 2 and 3) interacts in cis but ICD2 (TMH 4 and 5) interacts in trans : domain swapping of ICD2 NBD TMD TMD TMD TMD 2 ICD ICD ICD ICD 1 2 1 NBD NBD NBD NBD Dawson & Locher (2006) Nature

3 conformations of MsbA MsbA 3 conformations of out mb MsbAMsbA in Rigid body motion Ward et al (2007) PNAS Lipid A out mb in + ATP ~ 50 Å Closed ATP-bound Open apo Closed apo (or ADP-Vi) 2 2 ICD 1 ICD ICD ICD 2 1 ICD ICD ICD ICD ICD ICD ICD ICD 1 2 2 1 1 2 1

Are these open conformations physiologically relevant ? Aller et al. (2009) Science ICD ICD ICD ICD 1 2 1 2 Open Open apo EcMsbA apo P-gp … “Thus, the structure may represent a crystallization artifact or a nonfunctional conformation that has only very transient existence”.

Overexpression of BmrA and purification BmrA BmrA • Purification in a single step (Ct-polyhistidine-tagged) Multiple ~2-3 mg of BmrA/Liter drugs Orelle et al. (2003) JBC Hoechst ATP • Inside-out membrane vesicles: 100 BmrA ~ 50% of mb proteins Normalized fluorescence (%) kDa 1 2 3 75 94 67 Hoechst 50 43 33342 25 30 ADP 0 0 200 400 600 + Pi Time (sec.) ATP Steinfels et al. (2002) BBA

Behavior of BmrA in membrane or in detergent : 2 different conformations ? Membrane DDM purified Apo Time Time MW 5’ 15’ 30’ 1h 2h 3h C 119 C 5’ 15’ 30’ 5’ 15’ 30’ 79 46 31 24 19 Apo Vi-inhibited Vi-inhibited Time MW C 5’ 15’ 30’ 1h 2h 3h 119 79 The Vi-inhibited BmrA is much less sensitive to limited digestion by trypsin 46 than BmrA in the resting state. ==> BmrA switches between two very 31 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 • Different types of hydrogen in proteins D D D H : Do not exchange H : Too fast to be monitored H : Best candidates for deuterium exchange; exchange rate depends on structure and accessibility

The H/D exchange technique Exchange H D H + , 0°C • Different types of hydrogen in proteins 1- Global kinetics Proteolysis Dilution D 2 O D D D Labeling quench H/D Obtain exchange peptides to Labeled HPLC kinetics locate D region H : Do not exchange Separate MS H : Too fast to be monitored peptides Deuteration (%) H : Best candidates for deuterium exchange; Mass exchange rate depends on structure and accessibility measurement of each peptide 2- Local kinetics Time (sec)

sequence coverage 1 2 3 4 5 6 ICD1 ICD2 TMD NBD NBD TMD : poorly defined, but ICDs OK NBD : well NBD defined Total : 78% using pepsin

Portfolio of different peptides WT Apo E504A (ATPase inactive) + ATP/Mg

Few examples : Walker A motif %
H/D
Exchange 80 Open 60 Closed 40 20 0 15 60 300 600 1800 3600 Time
(s) 372-383 Protection by ATP Walker A

ABC signature peptide %
H/D
Exchange 80 60 Open Closed 40 20 0 15 60 300 600 1800 3600 Time
(s) 479-492 Protection by ATP ABC motif

Walker B motif %
H/D
Exchange 80 60 40 Open Closed 20 0 15 60 300 600 1800 3600 Time
(s) 501-509 Protection by ATP Walker B

Peptide from ICD1 ( cis with its own NBD) 104-114 ICD 1 %
H/D
Exchange 80 60 Open 40 Closed 20 0 15 60 300 600 1800 3600 Time
(s)

Peptide from ICD2 (trans with the other NBD) 203-215 ICD 2 %
H/D
Exchange 80 Open 60 Closed 40 20 0 15 60 300 600 1800 3600 Time
(s)

2nd Peptide from ICD2 (trans with the other NBD) 216-236 ICD 2 %
H/D
Exchange 80 Open 60 Closed 40 20 0 15 60 300 600 1800 3600 Time
(s)

Highly exchangeable, especially for ICDs Poorly exchangeable Different from 3D structure OK with 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 ? BmrCBmrD Multiple drugs Heterodimer with asymmetric NBDs (MRP, CFTR, TAP1/TAP2…) BmrC/BmrD model based on the TM heterodimer (Seeger’s group)

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

Many thanks to…. IBCP, Lyon Our team in IBS Vincent Chaptal Pierre Falson Shahid Mehmood Jonathan Sarwan Attilio Di Pietro Benjamin Wiseman Emilie Boncoeur Eric Forest Univ. Oxford, UK Carmen Domene