5. ABC transporters

primary active transport

Movement of molecules/ions across cell membrane against concentration gradient (from region of low conc to region of high conc) using ATP.

A simple allosteric model for  membrane pumps- Jardetzky (1966)

principle of active transport

·Interior cavity transports molecule.

· Alternating access – protein has 2 conformations. Access to one side or another.

· Binding site has different affinities in the 2 conformations.

· Energy input required to drive conformational and binding affinity change.

· Energy input can be light (bacteriorhodopsin) or conc grad or proton grads in secondary transporters.

ABC transporters

primary active transporters.

·  Superfamily

· Can be importers or exporters.

·  2 NBD – similar across all ABC transporters.

NBD forms ATP sandwich in closed conformation.

 ATPase Subdomain contains walker A & B motifs – hydrolyse ATP.   

·  2 TMD – subunits normally separate in prokaryotes and fused in higher organisms.

TMD forms cavity where molecule sits.

· bacterial Importers will have soluble substrate binding proteins.

·  Coupling helices  - helix held by salt bridge interactions in cavity (like ball in socket joint.) communicate structural changes in NBD due to binding of nucleotide to TBD.  Helix swivels within cavity – rotates TBD to give large movement. Coincides with opening and closing of NBD interface. Couples ATP hydrolysis to transport.

E.coli Maltose transporter

__X-ray structures show 3 conformational states__

Co (outward facing)

·       Structure trapping

·       E159Q mutation prevents ATP hydrolysis and traps structure in 1 conformation (Co).

·       WT structures with ADP+Pi analogues look similar 

·       Makes crystallisation easier.

Ci (inward facing)

·       TM1 of malF removed

·       Doesn’t affect transport or ATPase activity.

·       Makes crystallisation possible.

Sav1866

·       Multi-drug transporter (exporter).

·       2 polypeptide chains form homodimer.

·       TMD intertwined. Constraint on movement – don’t act independently.

·       Large internal cavity lacks defined drug binding site (typical of MDRs)

·       Cavity lined by hydrophilic residues. Low affinity for hydrophobic drugs.

·       Biochemical evidence suggests Ci conformation protien has high affinity for drugs.

·       Each TMD has pseudo symmetry.

·       Coupling helcies interact with NBDs – one contacts both and one contacts opposite NBD.

·       Whole structure intertwined –suggests coupling between NBDs and TMDs differ from importers.

·       Homolog of human P-glycoprotien  

P-gylcoprotien

·       Human exporter.

·       Involved in drug resistance in cancer cells.

·       One polypeptide with 2 homologous wings – each with 2 coupling helices.

·       4 coupling helices in total

·       Binds drugs from cytoplasm or hydrophobic drugs from membrane.