Molecular mechanisms of ABC multidrug transporters transporters and - PDF document

23/10/2012 ¡ Our studies on molecular mechanisms of Molecular mechanisms of ABC multidrug transporters transporters and beyond Ion-coupled ABC exporters exporters PatAB LmrP 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 LmrA U N I V E R S I T Y O F C A M B R I D G E NorM MsbA Sav1866 ABCG2 Brussels 2012 Initial studies on mechanisms of Conservation of function multidrug transporters SASA Wt MD Wt LmrA NBD SASA MsbA Control Sav1866 Van Veen et al. PNAS 93 (1996); Nature 391 (1998) Reuter et al. J Biol Chem 278 (2003) ; Woebking et al. Biochemistry 47 (2008) 1 ¡

23/10/2012 ¡ Location of drug binding Conservation of function between LmrA, MsbA, and human ABCB1 in ABCB1? Comparison LmrA/ABCB1 Comparison LmrA/MsbA in E. coli in human fibroblast cells + ABCB1 modulator Loo and Clarke, various JBC papers ; Shilling et al. TiPS 27: 195-203 (2006) Reuter et al. JBC 278: 35139 (2003); Van Veen et al. Nature 391: 291-5 (1998) Is this all there is to be discovered Model for alternating access about mechanisms? drug ¡ef:lux ¡ drug ¡entry ¡ MD Mg-­‑ATP ¡ NBD Homodimeric MD only ATP-­‑bound ¡ ¡ Nucleotide-­‑free ¡ ¡ “outward-­‑facing” ¡ “inward-­‑facing” ¡ Martin et al. Biochemistry 39: 11901 (2000); van Veen et al. EMBO J 19: 2503 (2000) Venter et al. Nature 426: 866–70 (2003) 2 ¡

23/10/2012 ¡ Link with secondary-active transporters? Δ pH (interior alkaline) Et + H + DNA LmrA-MD inserted in proteoliposomes in ISO orientation Glutamate 314 is essential for proton-coupled transport Velamakanni et al. Plos One 4: e6137 (2010); Barrera et al. Nature Meth (2009) Venter et al. Nature 426: 866–70 (2003) Link with ABC channels & fast ion Role of carboxylates in proton and Ca 2+ transporters (Cl channels)? transport by multidrug transporter LmrP diptip proteoliposomes Wt EE mutant Schaedler et al. FASEB J 24: 3653-61 (2010); J Biol Chem 287: 27682–27690 (2012 ) Velamakanni et al. Plos One 4: e6137 (2010) 3 ¡

23/10/2012 ¡ Activity of LmrCD activity Modulation of ABC transporter activity? Drug sensitivity Accumulation of BCECF-AM DARPin ABC transporter Heterodimeric ABC transporter LmrCD Seeger et al. Plos One 7: e37845 (2012) Seeger et al. Plos One 7: e37845 (2012) There is more to be discovered …. Acknowledgement Current & former members of Van Veen lab Markus Seeger � Rupak Doshi � Kelvin Agboh � Daniel Gutmann � Lucy Crouch � Saroj V elamakanni � Y vonne Khoo � Henrietta V enter � Hannah W ang � Theresia Schaedler � MD Lisa Fagg � Sarah V anes � Ihsene Salaa � Ute Hellmich � Wilma Shi � Jenny Y ao � Y oony Jin � Shen W ei � NBD Anam Ali � Calvin Lau � Zhen Tong � Barbara W oebking � Tavan Janvilisri � Sanjay Shahi � Akanksha Bapna � Edmond U � Lekshmy Balakrishnan � Richard Shilling � Galya Reuter � Anna Kleijn � 4 ¡