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)