Molecular mechanisms of ABC multidrug transporters transporters and - - PDF document

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H E N D R I K W . V A N V E E N D E P A R T M E N T O F P H A R M A C O L O G Y U N I V E R S I T Y O F C A M B R I D G E

Molecular mechanisms of ABC transporters and beyond

Brussels 2012

Our studies on molecular mechanisms of multidrug transporters

LmrA MsbA Sav1866 PatAB ABCG2 NorM LmrP ABC exporters Ion-coupled exporters LmrA MsbA Sav1866 MD NBD Van Veen et al. PNAS 93 (1996); Nature 391 (1998)

Initial studies on mechanisms of multidrug transporters

Conservation of function

Reuter et al. J Biol Chem 278 (2003) ; Woebking et al. Biochemistry 47 (2008) Wt SASA Control Wt SASA

23/10/2012 ¡ 2 ¡ Location of drug binding in ABCB1?

Loo and Clarke, various JBC papers ; Shilling et al. TiPS 27: 195-203 (2006)

Conservation of function between LmrA, MsbA, and human ABCB1

Reuter et al. JBC 278: 35139 (2003); Van Veen et al. Nature 391: 291-5 (1998) + ABCB1 modulator Comparison LmrA/MsbA in E. coli Comparison LmrA/ABCB1 in human fibroblast cells

Model for alternating access

Nucleotide-­‑free ¡ ¡ “inward-­‑facing” ¡ ATP-­‑bound ¡ ¡ “outward-­‑facing” ¡

drug ¡entry ¡

Mg-­‑ATP ¡

drug ¡ef:lux ¡ Martin et al. Biochemistry 39: 11901 (2000); van Veen et al. EMBO J 19: 2503 (2000)

Is this all there is to be discovered about mechanisms?

Homodimeric MD only Venter et al. Nature 426: 866–70 (2003) MD NBD

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Velamakanni et al. Plos One 4: e6137 (2010); Barrera et al. Nature Meth (2009)

Link with secondary-active transporters?

LmrA-MD inserted in proteoliposomes in ISO orientation Venter et al. Nature 426: 866–70 (2003)

Et+

DNA

H+ ΔpH (interior alkaline)

Glutamate 314 is essential for proton-coupled transport

Role of carboxylates in proton and Ca2+ transport by multidrug transporter LmrP

Schaedler et al. FASEB J 24: 3653-61 (2010); J Biol Chem 287: 27682–27690 (2012)

Link with ABC channels & fast ion transporters (Cl channels)?

Velamakanni et al. Plos One 4: e6137 (2010) proteoliposomes Wt EE mutant diptip

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Modulation of ABC transporter activity?

Seeger et al. Plos One 7: e37845 (2012) DARPin ABC transporter Heterodimeric ABC transporter LmrCD

Activity of LmrCD activity

Seeger et al. Plos One 7: e37845 (2012) Accumulation of BCECF-AM Drug sensitivity

There is more to be discovered ….

MD NBD

Acknowledgement

Markus Seeger Daniel Gutmann Saroj V elamakanni Henrietta V enter Hannah W ang Sarah V anes Ute Hellmich Jenny Y ao Shen W ei Calvin Lau Barbara W

• ebking

Tavan Janvilisri Sanjay Shahi Akanksha Bapna Edmond U Lekshmy Balakrishnan Richard Shilling Galya Reuter Anna Kleijn Rupak Doshi Kelvin Agboh Lucy Crouch Y vonne Khoo Theresia Schaedler Lisa Fagg Ihsene Salaa Wilma Shi Y

• ony Jin

Anam Ali Zhen Tong Current & former members of Van Veen lab