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Bacterial Multidrug ABC Transporter

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Bacterial Multidrug ABC Transporter Study by Roger J. P. Dawson and Kaspar P. Locher Presented by Shaun Hug ABC Transporter Family Characteristics - 6-Pass ... – PowerPoint PPT presentation

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Title: Bacterial Multidrug ABC Transporter


1
Bacterial Multidrug ABC Transporter
  • Study by Roger J. P. Dawson and Kaspar P. Locher
  • Presented by Shaun Hug

2
ABC Transporter Family Characteristics
  • - 6-Pass Transmembrane Domains (TMDs)
  • - ATP-Binding Cassette (ABC) Domains
  • - Also referred to as a Nucleotide-Binding
    Domains (NBDs)

3
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)

4
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

5
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

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9
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

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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

12
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

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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

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17
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

18
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

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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?v8mLonmEDk0A

22
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

23
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)
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