bacterial multidrug abc transporter study by roger j. p. dawson and kaspar p. locher presented by...

Bacterial Multidrug ABC Transporter

Study by Roger J. P. Dawson and Kaspar P. LocherPresented by Shaun Hug

ABC Transporter Family Characteristics

- 6-Pass Transmembrane Domains (TMDs)

- ATP-Binding Cassette (ABC) Domains - Also referred to as a Nucleotide-Binding Domains (NBDs)

ABC Transporter Family Characteristics

- Functional protein contains two TMDs and two NBDs - Bacterial multidrug ABC transporter exists as a dimer;

each subunit consists of one NBD attached to one TMD - Diverse functions/substrates - Export of drugs/toxins (MDR1) - Import of nutrients (BtuCD) - Flipping of phospholipids (MsbA) - Antigen-presenting pathways (TAP1/TAP2) - Ion channels (CFTR)

Sav1866 Background

- Obtained from Staphylococcus aureus - Homologous to MDR1 and TAP1/TAP2 - Stimulated by anticancer drugs doxorubicin and

vinblastine - Transporter was observed in a nucleotide-bound,

outward-facing conformation

Experimental Methods

- Sav1866 was overexpressed in E. coli - Cells were lysed, and membranes were obtained by

centrifugation - Protein was solubilized using two nonionic detergents - Protein was purified using a molecular sieve - Protein was crystallized over two to three weeks

using the sitting drop method

Nucleotide-Binding Domains (NBDs)

- ATP binding site exists at the interface of both subunits

- One ATP molecule interacts with the P-loop (Walker-A motif) of NBD1 and the ABC signature motif of NBD2

- Forms the basis of concerted and cooperative nucleotide binding and hydrolysis in transporter activity

Nucleotide-Binding Domains (NBDs)

- Sav1866 was crystallized with ADP - At 3.0 Å resolution, ADP-bound state of Sav1866 NBD

was indistinguishable from isolated archaeal NBD (MJ0796) bound to ATP

- Observable difference existed between ADP-bound NBD of Sav1866 and NBD of BtuCD crystallized without nucleotide

- Researchers concluded that the observed conformation reflected ATP-bound state

Transmembrane Domains (TMDs)

- Six membrane-spanning helices per subunit (TM1-TM6) - Helices extend into the cytoplasm through intracellular

loops (ICLs) - TMDs of both subunits interact closely - TMDs wrap around one another - TMDs separate into two “wings,” with each wing made

up of TM1-TM2 of one subunit and TM3-TM6 of the other subunit

- TM1-TM3 may be related to TM4-TM6 through duplication

Conformational Change Transmission

- Changes in NBD conformation are transmitted to TMD through two intracellular loops of a subunit (ICL1, ICL2)

- These ICLs are termed “coupling helices” - ICL1 interacts with both NBDs - ICL2 interacts with NBD of opposite subunit only - Again, intimate interaction between subunits mediates a concerted change in conformation - Genetic data and mutational studies in other ABC

transporters corroborate importance of these ICLs

Substrate Binding

- Substrate binding in ABC transporters is poorly understood

- Multiple helices appear to be involved (at least 7 out of 12 in MDR1)

- Residues involved in substrate binding in TAP1/TAP2 have homologous residues in Sav1866 that point toward the translocation pathway

Substrate Translocation

- ATP-bound Sav1866 exposes a large, hydrophilic cavity to the exterior of the cell

- Cavity is accessible from the outer leaflet, and spans the inner leaflet and beyond

- No connection exists between this cavity and the cytoplasm in the ATP-bound state

- External-facing cavity probably serves as a low-affinity extrusion pocket for hydrophobic drugs, as opposed to a high-affinity binding site

Substrate Translocation

- Although not observed in Sav1866, translocation is expected to occur through an “alternating access and release” mechanism

- In absence of ATP, substrate-binding cavity is exposed to cytoplasm

- Binding of ATP causes a conformational change that moves substrate into extrusion cavity, where it may diffuse into outer leaflet or extracellular solution

http://www.youtube.com/watch?v=8mLonmEDk0A

Conclusions

- ABC transporter subunits interact more closely than previously thought; the dimer may be the only form of the protein to actually exist in cells

- Large extrusion pocket has implications for reaction stoichiometry; more than one substrate may fit in the transporter, so one to two ATP molecules may be hydrolyzed per substrate

- New motifs and new symmetries observed that may be conserved across ABC transporters

Implications

- Due to the similarity between Sav1866 and MDR1, the structure and function of Sav1866 may provide insight into the structure and function of MDR1

- May allow for the development of compounds that interfere with the extrusion of drugs from drug-resistant cancer cells

- Adds to the growing body of knowledge on ABC transporters

- Greater understanding of ABC transporters and their mutants with known substrates (CFTR)