Bacterial Multidrug ABC Transporter Study by Roger J. P. Dawson and Kaspar P. Locher Presented 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

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)